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The generation of monoclonal antibodies to neural cell type-specific antigenic determinants Smyrnis, Elie Mario 1988

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T H E G E N E R A T I O N O F M O N O C L O N A L A N T I B O D I E S T O N E U R A L C E L L TYPE-SPECIFIC A N T I G E N I C D E T E R M I N A N T S By E L I E M A R I O S M Y R N I S B . S c , T h e U n i v e r s i t y of B r i t i s h C o l u m b i a , 1982 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R ' S O F S C I E N C E i n T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Depar tment of A c a d e m i c Pa tho logy) W e accept this thesis as c o n f o r m i n g to the r e q u i r e d s t anda rd T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A A p r i l , 1988 © E L I E M A R I O S M Y R N I S , 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia 1956 Main Mal l Vancouver, Canada V6T 1Y3 Apri l , 1988 DE-60/81) ia to Dad (Bapa) i b "Orav EKJCCI EVTIHOS kai Zcocrros Tlavia Kparccs To Meramo Yi^cc" EGSavyye^os ELiipvr|s A B S T R A C T i i Somatic cell hybridization techniques were used to produce a panel of anti-neural cell monoclonal antibody-secreting murine hybridomas. Whole l iving cultures of bovine oligodendrocytes were used to inject (intra-splenically and without adjuvants) Ba lb /C mice with a total of 2.0 to 2.5 x 10 oligodendrocytes. Polyethylene glycol was subsequently used to fuse these sensitized splenic lymphocytes to the murine NS-1 myeloma cell line. After fusion, supernatants were screened immunocytochemically using murine neural cell cultures. Initial screening identified a total of 17 clones, each of which primarily labelled a surface antigen specific to galactocerebroside-positive oligodendrocytes; as demonstrated by double-immunostaining characterization. The monoclonal antibodies were determined to be of either the IgM class or IgG2b subclass. Moreover, these antibodies recognized protein bands possessing an apparent molecular weight of 29,000 and/or 59,000 Da on Western blots of homogenized bovine oligodendrocytes. This may, therefore, represent immunolabelling of a previously unrecognized surface constituent of oligodendrocytes. Culture supernatants from a second separate fusion experiment involving an intrasplenic injection with 20 u.g of SDS-PAGE subfractionated human newborn brain particulate fraction were tested using first, an ELISA, and subsequently, indirect immunofluorescence microscopy. Screening demonstrated 2 clones which produced antibodies immunoreactive to an astrocyte subclass (glial fibrillary acidic protein-positive cells) found only in cultures of newborn murine brain. These antibodies were determined to be of an IgM class and were shown to specifically reactivity with electroblots of a protein constituent of whole newborn brain particulate fraction having an apparent molecular weight of 50,000 Da. LIST OF TABLES iii Table 1.1: Characteristics of hybridomas generated against bovine oligodendrocytes 137-139 iv L I S T O F F I G U R E S Fig. 1.1: The main biosynthetic pathways for purine and pyrimidine biosynthesis 7 Fig. 1.2: The conventional procedures involved with the generation of monoclonal antibodies in the murine system 9 Fig. 1.3: Schematic representation of the major constituents of the mammalian central nervous system 22 Fig. 2.1: Preparation of a Human axolemme-enriched (HAx) fraction form post-mortem material 66 Fig. 2.2: Preparation of Human newborn brain particulate fratcion 70 Fig. 2.3: The methods used in intrasplenic injection and the subsequent isolation of sensitized splenocytes.... 80 Fig. 2.4: The use of methyl cellulose for cloning 92 Fig. 2.5: Bulk-isolation of mammalian oligodendrocytes using Percoll density gradient centrifugation 103 Fig. 2.6: Schematic representation for the preparation ofwhole brain cultures 105 Fig 2.7: Preparation of mammalian D R G cultures 107 Fig. 3.1: Phase microscopy of human newborn brain particulate fraction 110 Fig. 3.2: SDS-Polyacrylamide gel electrophoresis of human newborn brain particulate fraction 112 Fig. 3.3: Electrophoretic mobility of subfractionated particulate sample proteins 112 Fig. 3.4: Phase microscopy of freshly isolated bovine oligo-dendrocytes 115 Fig. 3.5: Phase microscopy of bovine oligodendrocytes maintained in culture for 14DIV 115 Fig. 3.6: Phase microscopy of a typical "oligodendroglial" cell culture (10 DIV) displaying the presence of a (presumably) bipolar astrocyte 117 Fig. 3.7: Adult mixed primary cultures of murine C N S 119 Fig. 3.8: Phase microscopy of newborn murine D R G in culture (5 DIV) 120 Fig. 3.9: Phase contrast characteristics of a typical field of the myeloma cell line: NS-1 in batch culture prior to fusion 122 Fig. 3.10: Phase contrast microscopy of hybridomas prior and subsequent to H A T selection 124 Fig. 3.11: Phase contrast microscopy of a newly forming hybrid colony (cloned) and growing in methyl cellulose (2-3 DIV) 126 Fig. 3.12: Cell type-specificity of PF(b)-C3A8 129 Fig. 3.13: Western blot analysis of the recognized PF(b)-C3A8 epitope 130 Fig. 3.14: Positive immunofluorescence fo Percoll-enriched bovine oligo-endrocytes used for the initial screening of the presently generated hybridomas 132 Fig. 3.15: Typical phase contrast appearance of undisturbed hybridomas growing in selective media 133 Fig. 3.16: Subclass determination of the panel of anti-BOL monoclonal antibodies using a dot-immunobinding technique 135 v i F i g . 3.17: C e l l type-spec i f ic i ty as d e t e r m i n e d t h r o u g h i m m u n o c y t o -chemis t ry 139 F i g . 3.18: W e s t e r n b lo t ana lys i s of r ecogn ized B O L specifici t ies 141 ACKNOWLEDGMENTS The present study would not have been possible had it not been for the substantial guidance, confidence, and support provided to me by a number of individuals. I am deeply indebted to my supervisor, Dr. S. Kim (Medicine/Neurology) not only for his expert instruction in innovative approaches to neurobiology, but for also displaying patience and understanding when times were hard. I gratefully acknowledge the time and effort invested by Dr. N. Auersperg (Anatomy), Dr. K. Berry (Pathology), and Dr. D. Paty (Medicine/Neurology) in helping to oversee the progress and successful completion of the present study. My sincere appreciation is extended to Dr. F. Takei (Pathology), Mr. C. Smith, and Dr. A. Buchan ( Medicine/Physiology) not only for their expert instruction in the methods for producing monoclonal antibodies, but also for allowing me free access to their facilities. I would also like to acknowledge Dr. H . Kamo (Psychiatry/Neurology) for instructing me in the techniques of intrasplenic injection; Dr. S. Sekeguchi (Medicine/Biochemistry) for advice and instruction in biochemical methodologies; Dr. C. Lee (Medicine) and Mrs. M. Kim for their excellent advice and assistance in the lab; and to Dr. J. Oger (Medicine/Neurology) and Dr. J. Chantler (Pathology/Medical Microbiology) for reading and offering their valuable suggestions in the preparation of the final text. Finally, I wish to express a special thanks to my sister, Vivian, for taking a month out of her life to type the bulk of this manuscript. TABLE OF CONTENTS ABSTRACT LIST OF TABLES ' LIST OF FIGURES ' A C K N O W L E D G M E N T S vu 1 INTRODUCTION 1.1 B a c k g r o u n d I m m u n o l o g y a n d E a r l y I m m u n o c y t o c h e m i s t r y 2 1.2 A d v e n t of Somat ic C e l l H y b r i d i z a t i o n Techniques 4 1.3 C o m p a r i s o n B e t w e e n H e t e r o c l o n a l a n d M o n o c l o n a l A n t i b o d i e s 10 1.4 A p p l i c a t i o n s of M o n o c l o n a l A n t i b o d i e s 12 1.4.1 D i a g n o s t i c A p p l i c a t i o n of M o n o c l o n a l A n t i b o d i e s 13 1.4.2 M o n o c l o n a l A n t i b o d i e s as I m m u n o t h e r a p e u t i c A g e n t s 18 1.4.3 M o n o c l o n a l A n t i b o d i e s i n Bas i c Resea rch 20 1.5 I m m u n o s p e c i f i c Iden t i f i ca t ion i n N e u r o b i o l o g y 20 1.5.1 T h e A v a i l a b i l i t y of C o n v e n t i o n a l C e l l Type -Spec i f i c A n t i b o d i e s to N e u r a l A n t i g e n s 21 H e t e r o c l o n a l A n t i s e r u m to O l i g o d e n d r o g l i a 23 H e t e r o c l o n a l A n t i s e r a to As t rocy te s 27 H e t e r o c l o n a l A n t i s e r a to N e u r o n s 29 H e t e r o c l o n a l A n t i s e r a to C o m p l e x N e u r a l A n t i g e n s 32 1.5.2 M o n o c l o n a l A n t i b o d i e s i n N e u r o b i o l o g y 35 M o n o c l o n a l A n t i b o d i e s to As t rocy tes 38 i x M o n o c l o n a l A n t i b o d i e s to N e u r o n s 41 M o n o c l o n a l A n t i b o d i e s to O l i g o d e n d r o g l i a 49 1.6 T i s sue C u l t u r e i n N e u r o b i o l o g y 55 1.6.1 P r e p a r a t i o n a n d M a i n t e n a n c e of As t rocy te s 58 1.6.2 P repa ra t ion a n d M a i n t e n a n c e of O l i g o d e n d r o c y t e s 60 1.6.3 P r e p a r a t i o n a n d M a i n t e n a n c e of N e u r o n s f r o m D o r s a l R o o t G a n g l i a ( D R G ) 61 2 M A T E R I A L S A N D M E T H O D S 2.1 Selec t ion of a Sui table M y e l o m a C e l l L i n e .63 2.1.1 M y e l o m a Selec t iv i ty to H A T 64 2.1.2 Se lec t iv i ty to A z a g u a n i n e 65 2.2 P r e p a r a t i o n a n d C h a r a c t e r i z a t i o n of the I m m u n o g e n 65 2.2.1 P r e p a r a t i o n of the H u m a n A x o l e m m a - E n r i c h e d F r a c t i o n 65 2.2.2 P r e p a r a t i o n of H u m a n N e w b o r n B r a i n Par t i cu la te F r a c t i o n .70 2.2.3 P r e p a r a t i o n of B o v i n e O l i g o d e n d r o c y t e s ( B O L ) 71 2.2.4 S D S - P o l y a c r y l a m i d e E lec t rophores i s Subf rac t iona t ion a n d Charac te r i za t ion 71 R e c o v e r y o f P r o t e i n F r o m G e l E x c i s e d Subtrac t ions -73 2.2.5 D e t e r m i n a t i o n of P r o t e i n C o n c e n t r a t i o n 75 U s e of the L o w r y M e t h o d 75 U s e of the P ro t e in D y e B i n d i n g M e t h o d 76 2.3 I m m u n i z a t i o n P r o t o c o l 76 2.3.1 C o n c e n t r a t i o n of I m m u n o g e n P r i o r to C h a r a c t e r i z a t i o n a n d I m m u n i z a t i o n 76 2.3.2 In t rasp len ic Inject ion 77 2.4 F u s i o n P r o t o c o l 78 2.4.1 P repa ra t i on o f Sens i t i zed M u r i n e Splenocytes 78 D i s s e c t i o n of the Sp leen 78 Separa t ion o f a P l a s t i c - A d h e r e n t C e l l P o p u l a t i o n 80 2.4.2 L y m p h o c y t e to M y e l o m a R a t i o Si 2.4.3 T h e P repa ra t i on a n d U s e o f Po lye thy l ene g l y c o l ( P E G ) SI 2.4.4 F u s i o n Frequenc ies a n d P l a t i n g Dens i t i es 82 2.5 C e l l C u l t u r e R e q u i r e m e n t s for H y b r i d o m a s 82 2.5.1 Bas i c C u l t u r e R e q u i r e m e n t s 82 Fe ta l C a l f S e r u m 82 T h e U s e of H A T M e d i a S3 U s e of H T M e d i a 83 2.6 In i t i a l Sc reen ing of H y b r i d o m a C u l t u r e Superna tan ts .84 2.6.1 I m m u n o c h e m i c a l d e t e r m i n a t i o n o f a n t i b o d y a c t i v i t y .84 C o n c e n t r a t i o n o f C u l t u r e Superna tan ts b y A m m o n i a Su lpha t e P r e c i p i t a t i o n 84 Indirect I m m u n o c y t o c h e m i s t r y S5 E n z y m e - l i n k e d I m m u n o s o r b a n t A s s a y ( E L I S A ) 86 A n t i s e r a 87 2.7 H a n d l i n g of P r o d u c t i v e F u s i o n P roduc t s 87 2.7.1 In V i t r o E x p a n s i o n of H y b r i d o m a C o l o n i e s 87 2.7.2 P l a t i n g , C e l l - F r e e z i n g a n d R e c o v e r y of H y b r i d o m a s 88 2.7.3 C l o n i n g of A n t i b o d y P r o d u c i n g H y b r i d o m a s 88 M a n u a l M i c r o s c o p i c Iso la t ion o f H y b r i d o m a s 89 C l o n i n g i n M e t h y l c e l l u l o s e 90 C l o n i n g b y L i m i t i n g D i l u t i o n .93 2.7.4 P r o p a g a t i o n of P o s i t i v e C l o n e s 93 2.8 H a n d l i n g of M o n o c l o n a l A n t i b o d y - C o n t a i n i n g C u l t u r e Superna tan ts 94 xi 2.8.1 L a r g e Scale P r o d u c t i o n of A n t i b o d y 94 2.8.2 Storage of M o n o c l o n a l A n t i b o d y .94 2.9 C h a r a c t e r i z a t i o n of M o n o c l o n a l A n t i b o d y 94 2.9.1 C la s s D e t e r m i n a t i o n of M o n o c l o n a l A n t i b o d y 95 U s e o f E L I S A .95 U s e of a D O T I m m u n o b i n d i n g A s s a y .95 2.10 Cha rac t e r i za t i on of the A n a l y t e b y W e s t e r n B l o t .97 2.10.1 D e t e c t i o n of E lec t rob lo t t ed P r o t e i n U s i n g N o n - I m m u n e M e t h o d s 58 2.10.2 D e t e c t i o n of P r o t e i n Transfer i n I m m u n o b l o t s U s i n g I m m u n o r e a g e n t s .98 C o l o u r D e v e l o p m e n t U s i n g D i a m i n o b e n z a d i n e ( D A B ) 99 U s e of D A B - N i c k e l D i s u l p h a t e M o d i f i e d S o l u t i o n 99 U s e of 4 - C h l o r o 1 -Nap tho l 100 2.10.3 D e t e r m i n a t i o n of Transfer Ef f icacy U s i n g S i l v e r S t a i n i n g T e c h n i q u e s 100 2.11 D i s s o c i a t e d C e l l C u l t u r e s Techn iques 101 2.11.1 P repa ra t i on a n d M a i n t e n a n c e of G l i a l C u l t u r e s 101 2.11.2 P r e p a r a t i o n a n d M a i n t e n a n c e of W h o l e B r a i n C u l t u r e s 104 2.11.3 P repa ra t i on a n d M a i n t e n a n c e of D o r s a l R o o t G a n g l i a ( D R G ) 106 3 RESULTS. 3.1 P repa ra t ion of N e u r a l A n t i g e n s 109 3.1.1 R e c o v e r y of H u m a n A x o l e m m a - E n r i c h e d F r a c t i o n 109 3.1.2 H u m a n N e w b o r n B r a i n Par t icu la te F rac t ion 109 3.2 Charac te r i s t ics a n d G r o w t h of N e u r a l C u l t u r e s 113 3.2.1 I so la t ion a n d C u l t u r e of A d u l t As t rocy t e s a n d Ol igodendrocy te s 113 x i i Character is t ics of T h e O l i g o d e n d r o c y t e s 113 Character is t ics of the As t rocy tes 116 3.2.2 N e w b o r n a n d A d u l t M i x e d P r i m a r y C N S C u l t u r e s 116 3.2.3 N e w b o r n D o r s a l R o o t G a n g l i a ( D R G ) .118 3.3 Charac ter i s t ics a n d G r o w t h of the M y e l o m a C e l l L i n e 121 3.3.1 C u l t u r e a n d Se lec t iv i ty of N S - 1 to H A T a n d A z a g u a n i n e 121 3.4 Iso la t ion of Sens i t i zed M u r i n e Splenocytes 123 3.5 Character is t ics a n d G r o w t h of F u s i o n P roduc t s 123 3.5.1 Es t ab l i shmen t of a n A n t i - H u m a n N e w b o r n B r a i n Par t i cu la te Sub t r ac t i on M o n o c l o n a l A n t i b o d y - S e c r e t i n g H y b r i d o m a 125 P r i m a r y Screen ing ( E L I S A ) of H y b r i d o m a C u l t u r e Supe rna t an t s 125 C l o n i n g i n M e t h y l c e l l u l o s e 125 I m m u n o g l o b u l i n Subclass D e t e r m i n a t i o n 127 C e l l T y p e Spec i f ic i ty as D e t e r m i n e d w i t h Indirect Immunof luoresence 127 A n a l y s i s of R e c o g n i z e d E p i t o p e 128 3.5.2 E s t a b l i s h m e n t of A n t i - B o v i n e O l i g o d e n d r o c y t e M o n o c l o n a l A n t i b o d y - S e c r e t i n g H y b r i d o m a s 128 C l o n i n g U s i n g M i c r o - m a n i p u l a t i v e T e c h n i q u e s a n d L i m i t i n g D i l u t i o n 131 Subc lass D e t e r m i n a t i o n of A n t i B O L M o n o c l o n a l A n t i b o d y 131 C e l l type specif ic i ty of a n t i - B O L a n t i b o d y 134 A n a l y s i s of B O L E p i t o p e .134 4 D I S C U S S I O N 4.1 T h e N e e d for C e l l Type-Spec i f i c Immunoreagen t s i n N e u r o b i o l o g y 142 4.1.1 A d v a n t a g e s offered w i t h the U s e of M o n o c l o n a l A n t i b o d i e s 143 x i i i 4.2 C h o i c e of the M u r i n e S y s t e m i n the G e n e r a t i o n of N e u r a l C e l l T y p e -Speci f ic M o n o c l o n a l A n t i b o d i e s 144 4.2.1 T h e U s e of B a l b / C Splenocytes 145 4.2.2 T h e U s e of the M y e l o m a C e l l L i n e N S - 1 145 4.3 C h o i c e of N e u r a l C e l l H o m o g e n a t e a n d Subt rac t ions i n the P r o d u c t i o n of M o n o c l o n a l A n t i b o d i e s to N e u r a l C e l l T y p e s 146 4.3.1 T h e C h o i c e of N e w b o r n B r a i n Par t icu la te Subfrac t ion as a n A l t e r n a t i v e I m m u n o g e n to H u m a n A x o l e m m a - E n r i c h e d F r a c t i o n 146 4.3.2 T h e U s e of W h o l e C u l t u r e s of L i v i n g B o v i n e O l i g o d e n d r o g l i a as a n I m m u n i z i n g A g e n t 149 4.4 T h e I m p l e m e n t a t i o n of A l t e r n a t i v e M e t h o d s ove r C o n v e n t i o n a l I m m u n i z a t i o n Reg imes 150 4.4.1 In V i t r o I m m u n i z a t i o n 150 4.4.2 Instrasplenic I m m u n i z a t i o n 151 4.5 G e n e r a t i o n o f a n A n t i - N e w b o r n B r a i n Par t i cu la te F r a c t i o n M o n o c l o n a l A n t i b o d y 152 4.5.1 T h e G e n e r a t i o n of I g M C l a s s of M o n o c l o n a l A n t i b o d y to C N S Par t i cu la te F r a c t i o n 152 4.5.2 C e l l type Specif ic i ty Demons t r a t ed b y P f (b ) -C3A8 153 4.5.3 T h e E lec t rob lo t t i ng Charac ter i s t ics of the R e c o g n i z e d A n a l y t e .154 4.5.4 C o m p a r i s o n of P f ( b ) - C 3 A 8 to O t h e r S i m i l a r l y R a i s e d M o n o c l o n a l A n t i b o d i e s 154 4.6 G e n e r a t i o n of a n A n t i - B o v i n e O l i g o d e n d r o c y t e M o n o c l o n a l A n t i b o d y 158 4.6.1 Subclass D e t e r m i n a t i o n a n d I m m u n o l a b e l i n g Charac te r i s t i cs of A n t i - B O L A n t i b o d y 159 4.6.2 C o m p a r i s o n of A n t i - B O L A n t i b o d i e s to O t h e r S i m i l a r l y R a i s e d M o n o c l o n a l A n t i b o d i e s 159 4.7 S u m m a r y a n d c o n c l u s i o n 163 5 B I B L I O G R A P H Y 165 1 1 INTRODUCTION Our basic understanding of the nervous system has grown substantially during the past few years, due largely to the many recent advances made in the field of immunology and from adopting an immunological approach in innovating neurobiological studies. Most significant of these developments has been the implementation of somatic cell hybridization techniques for the generation of hybridoma cell lines capable of producing and secreting monoclonal antibody. The present study describes the novel application of intrasplenic injection in conjunction with the established techniques of neural cell culture towards the generation of monoclonal antibodies directed against surface analytes of specific mamallian neural cell classes and subclasses. The development and ready availability of neuronal, astroglial, and oligodendroglial-specific immunological probes represents a fundamentally unique approach towards circumventing the technical limitations imposed by the use of heterclonal antisera in immunocytochemical investigations of the nervous system. It does so by providing the opportunity to specifically identify, isolate, and thus characterize a cells individual properties and by allowing a viable means with which to observe the multitude of intercellular interactions as they occur under controlled experimental conditions The purpose of this introduction is to familiarize the reader with not only how monoclonal antibodies compare to their heteroclonal counterparts, but also to attempt to establish the versatility of these immunoreagents in the fields of immunodiagnostics, immunotherapeutics, and basic research. Emphasis is placed on immunospecific identification in neurobiology with examples given of available heteroclonal and monoclonal antibodies to neural cell types and neural antigen preparations of a more 2 complex nature. Lastly, we present some basic information on the more contemporary methods used in tissue culture as it pertains to both neurobiology and the generation of hybridomas producing desired monoclonal antibody. 1.1 Background Immunology and Early Itnmunocytochetnistry. The basic ingredient in all immunological reactions involve both antigens and antibodies. Deliberate or accidental exposure (by vaccination and infection) to a particular foreign element or pathogenic agent, wi l l elicit an immune response which wi l l normally lead to the processing and subsequent elimination of the substance from the body [for rev. see Tonegawa 1985; Haynes and Fauci 1987]. The fundamental aspect of such a response requires the generation and subsequent secretion of antibody by the B-lymphocyte. There are perhaps a million different lines of plasma-cell unmaturated-progenitor B-lymphocytes in the mammalian spleen, each having the potential for producing monospecific antibody [Seiler et al. 1985]. A n immunologically-determined maturated B-cell progenitor (and its successor; the plasma cell) produces one antibody type of predefined specificity. During the differentiation of the lymphocyte clone, there are rearrangements which may occur to the constant region of the immunoglobulin molecule, however, the singular specificity demonstrated by the variable region of the molecule is never lost. The mechanisms involved in achieving this degree of specificity are extremely intricate [Lane and Koprowski 1982], requiring both somatic mutation (associated with its clonal development) as well as extensive alterations to the genetic template which codes for the antibody [for rev. see Tonegawa 1983; Milstein 1986]. The hypothesis of "clonal-selection" [Burnet 1959] forms the basis for the theory of monoclonal antibody production [for rev. see Ada and Noussal 1987]. The organism, in such a response, undoubtedly generates a multitude of monospecific antibodies directed against the antigenic stimulus, with each individual immunoglobulin molecule expressing related specificity and patterns of reaction. Such a family or collection 3 of i m m u n o g l o b u l i n s are bet ter k n o w n as h e t e r o c l o n a l (or p o l y c l o n a l ) a n t i s e r u m . A l l B -l y m p h o c y t e s are d e r i v e d f r o m a c o m m o n s t em c e l l a n d have a n i n d e p e n d e n t capac i ty to p r o d u c e m o n o c l o n a l a n t i b o d i e s w h i c h r e c o g n i z e d i f f e r e n t o r i d e n t i c a l a n t i g e n i c de te rminan t s a n d w h i c h m a y o r m a y no t also differ i n thei r f o r m o r s t rength of b i n d i n g . T h e s ign i f icance of an t ibod ies , qui te apar t f r o m the i r n a t u r a l func t ion , have l o n g been r e c o g n i z e d as a n i m p o r t a n t r e sea rch t o o l , w h e r e a d v a n t a g e i s t a k e n o f t he i r a b i l i t y to spec i f i ca l ly l abe l o r iden t i fy a pa r t i cu la r c e l l epi tope. In 1941, A . H . C o o n s first desc r ibed the i m m u n o l o g i c a l p rope r t i e s of a n a n t i b o d y w h i c h c o u l d be u s e d to i d e n t i f y suspens ions of p n e u m o c o c c a l o r g a n i s m s b y means of a n a t t ached f l u o r o c h r o m e : thus , w a s i n t r o d u c e d the concep t o f i m m u n o h i s t o - a n d cy tochemis t ry . T h i s r e v o l u t i o n a r y m e t h o d has n o w become f i r m l y e s t a b l i s h e d i n a l l f i e lds of p a t h o l o g y a n d n e u r o b i o l o g y . W h e r e once the ent i re d i s c i p l i n e o f C N S e n z y m o l o g y a n d h i s t o c h e m i s t r y l a r g e l y d e p e n d e d o n a s s i g n i n g the l o c a l i z a t i o n of a p a r t i c u l a r e n z y m e , for example , b y i ts re la t ive b i o c h e m i c a l react ions in situ [Persi jn et al. 1970; P r o h a s k a et al. 1973; S i m s a n d C a r n e g i e 1976; M a t t h i e u et al. 1977; S p r i n k l e et al. 1978; O l a f s o n et al. 1979] o r p h y s i c a l {i.e. S D S - P A G E ) charac ter i s t ics [Sweadne r 1979; B i z z o z e r o et al. 1985; D e i b l e r et al. 1986], i m m u n o c y t o c h e m i c a l m e t h o d o l o g i e s h a v e l a r g e l y s u p e r s e d e d e v e n the use of e n z y m a t i c b i o c h e m i c a l reac t iv i t i es i n the l o c a l i z a t i o n of these pro te ins [Rousse l a n d N u s s b a u m 1982; H e y d o r n et al. 1985; D o b e r s e n et al. 1985a; M o n t z et al. 1985; B i e g o n a n d W o l f f 1986; C a m m e r et al. 1986; C a m m e r a n d T a n s e y 1986; O c h n i o et al. 1987]. A l t h o u g h i m m u n o f l u o r o c h e m i c a l t echn iques w e r e i n i t i a l l y c o n s i d e r e d to be v e r y c u m b e r s o m e a n d d i f f icu l t to e m p l o y , b y the 1960s the pract ice h a d become rout ine . O f specia l c o n c e r n , h o w e v e r , w a s spec i f i c i t y ; c o n s i d e r i n g the h e t e r o c l o n a l na tu re of the i m m u n e response to a c o m p l e x an t igen . T h i s r e q u i r e d subs tan t ia t ion of m u c h of the w o r k d o n e u s i n g the p o l y c l o n a l i m m u n o r e a g e n t s w i t h o ther i n d e p e n d e n t l y d e r i v e d me thods . 4 T h e m o s t s i g n i f i c a n t p r o b l e m e n c o u n t e r e d i n i m m u n o c y t o c h e m i c a l i nves t i ga t i ons has to d o w i t h the spec i f i c i ty o f the p r i m a r y an t i body . I m p r o v e m e n t s i n i m m u n o s t a i n i n g techniques s u c h as use of h ighe r p r i m a r y a n t i b o d y d i l u t i o n s , cons is tency i n s t a in ing pat terns, a n d c o m p a r i s o n o f s t a i n i n g in tens i t ies w i t h different a n t i s e r u m a n d an t ibod ies ( in va r i ous r e g i o n s o f t he s a m e s e c t i o n ) , h e l p to s u b s t a n t i a t e o r a l t e r n a t i v e l y n e g a t e the i m m u n o s p e c i f i c i t y o f the a n t i s e r u m b e i n g tested. T h e p r o b l e m s inheren t w i t h the use of c o n v e n t i o n a l a n t i s e r u m (such as f l u c t u a t i n g i m m u n o s p e c i f i c i t y a s soc i a t ed w i t h d i f ferent c o n v e n t i o n a l a n t i s e r u m prepara t ions , a n d the consequence of w h e t h e r or no t resul ts s h o u l d be u s e d w i t h o u t fur ther c o r r o b o r a t i o n ) , c o u l d be ad re s sed b y one of t w o h y p o t h e t i c a l s i tua t ions . T h e first w o u l d to be to s i m p l y isolate o n l y d e s i r e d monospec i f i c a n t i b o d y f r o m w h o l e he te roc lona l an t i s e rum. A l t e r n a t i v e l y , one c o u l d select o r c lone a specif ic p l a s m a ce l l , that syn thes izes a n d secretes a d e s i r e d i n d i v i d u a l (monoc lona l ) a n t i b o d y d i r e c t e d against a g i v e n an t igen ic de te rminan t . T h e re la t ive p h y s i c a l a n d c h e m i c a l s i m i l a r i t y of the a n t i b o d y m o l e c u l e s w h i c h c o m p r i s e c o n v e n t i o n a l an t i se ra , h o w e v e r , a l o n g w i t h the h e t e r o c l o n a l na tu re of the i m m u n e response m a k e s i t ex t r eme ly c u m b e r s o m e to i so la te a n a n t i b o d y of p r e d e t e r m i n e d spec i f i c i ty f r o m w h o l e s e r u m . S e c o n d l y , a n t i b o d y secre t ing p l a s m a cel ls d o no t possess the capac i ty to s u r v i v e i n t issue cu l tu re for the t i m e necessary to p r o d u c e usefu l a m o u n t s of a n t i b o d y [Benacerraf a n d U n a n u e 1979]. 12 Advent of Somatic Cell Hybridization Techniques. A s i g n i f i c a n t t e c h n i c a l p r o b l e m as soc i a t ed w i t h the g e n e r a t i o n of m o n o s p e c i f i c a n t i b o d y i s r e l a t ed to the t e r m i n a l l y d i f ferent ia ted status o f a n a n t i b o d y p r o d u c i n g p l a s m a ce l l , c o m p o u n d e d w i t h the fact that s u c h cel ls have a v e r y l i m i t e d l i fe s p a n a n d therefore can no t n o r m a l l y be g r o w n i n c u l t u r e for a n e x t e n d e d p e r i o d of t ime . H o w e v e r , a n t i b o d y p r o d u c i n g ce l l s w h i c h have u n d e r g o n e m a l i g n a n t t r a n s f o r m a t i o n d o no t o n l y pro l i fe ra te 5 r a p i d l y a n d i n d e f i n i t e l y i n cu l tu re , bu t c a n be a lso a c h i e v e d i n m o s t a n i m a l s w i t h repeated inject ions of m i n e r a l o i l . T h e " m o n o c l o n a l " a n t i b o d y ( m y e l o m a pro te in ) secreted, t h o u g h , is a lmos t ce r t a in ly o f a n u n d e s i r e d or u n k n o w n specif ic i ty . I n 1970, H o r i b a t a a n d H a r r i s were able to adapt M O P C - 2 1 (an IgG} k- l igh t c h a i n secret ing B a l b / C m y e l o m a cell) to t issue cul ture a n d r e n a m e d i t P 3 K . Subsequen t a t tempts to i n d u c e th is m y e l o m a c e l l l i n e to generate speci f ic a n t i b o d y to, for example , repea ted injections o f an t igen have a l l been unsuccess fu l . A t th i s t i m e h y b r i d i z a t i o n e x p e r i m e n t s b e t w e e n m o u s e a n d rat m y e l o m a c e l l l i n e s es tab l i shed that a l l e l i c e x c l u s i o n d i d not occur [Co t ton a n d M i l s t e i n , 1973]. In other w o r d s , genet ic i n f o r m a t i o n f r o m each i n d i v i d u a l parent c e l l l i n e w a s c o d o m i n a n t l y expressed b y the f u s i o n p r o d u c t . It w a s these ear ly essent ia l f i n d i n g s w h i c h l e d to the first s p e c u l a t i o n that s u c h a t u m o r c e l l ( i m m o r t a l c lones o f cel ls de scended f r o m a s ing le p rogen i to r ) m a y be fused to a l y m p h o c y t e w h i c h c o u l d generate a n t i b o d y of p r e d e t e r m i n e d spec i f ic i ty , a n d that the p r o g e n y o f s u c h a f u s i o n m a y possess the c a p a c i t y to secrete m o n o s p e c i f i c a n t i b o d y character is t ic o f the sp lenocy te c o m b i n e d w i t h the i m m o r t a l geno type inheren t of the s econd parent ce l l [ K o h l e r a n d M i l s t e i n 1975]. T h e successfu l a p p l i c a t i o n o f somat i c c e l l h y b r i d i z a t i o n techniques i n the genera t ion of m u r i n e m o n o c l o n a l a n t i b o d i e s b y K o h l e r a n d M i l s t e i n represents p r o b a b l y the m o s t s i g n i f i c a n t a d v a n c e m e n t to the b i o l o g i c a l sc iences i n the past 10 years . T h e a d v e n t o f h y b r i d o m a t e c h n o l o g y has c i r c u m v e n t e d m a n y of the ma jo r p r o b l e m s a s soc ia t ed w i t h reagent i m m u n o s p e c i f i c i t y a n d r e p r o d u c i b i l i t y . C o n c u r r e n t w i t h this d e v e l o p m e n t w a s the a b i l i t y to generate essent ia l ly u n l i m i t e d s tock of exquis i te ly-spec i f ic , h o m o g e n o u s a n t i b o d y a n d to d o th is w i t h a n i n i t i a l i m m u n i z i n g agent of ques t ionable p u r i t y . T h e p r o t o c o l u s e d b y K o h l e r a n d M i l s t e i n has f o r m e d the bas i s o f e s sen t i a l ly a l l subsequen t s tud ies i n v o l v i n g the p r o d u c t i o n of m o n o c l o n a l an t ibod ies . B a s i c a l l y , B a l b / C m i c e are s ens i t i z ed to a n an t igen (usua l ly b y repeated inocu la t ion ) after w h i c h the sp leen is 6 r e m o v e d a n d a l y m p h o c y t e s u s p e n s i o n p repa red . T h e sp lenocytes are then m i x e d together w i t h a c o m p a t i b l e m y e l o m a c e l l s u s p e n s i o n . A fusogen (i.e. p o l y e t h y l e n e g l y c o l [ P E G ] , i n a c t i v a t e d S e n d i a v i r u s [SV] , o r a m i x t u r e thereof) is then a d d e d to p r o m o t e i n i t i a l fus ion a n d subsequen t h y b r i d i z a t i o n . D e s p i t e the use o f P E G or S V , f u s i o n i s s t i l l c o n s i d e r e d a r e l a t i v e l y rare occur rence [ G o d i n g 1980]. F u r t h e r m o r e , the o v e r g r o w t h o f h y b r i d o m a s b y u n d e s i r e d non- fused cel ls w i l l occur r a p i d l y i n c e l l cu l tu re . A t echn ique first d e s c r i b e d b y L i t t l e f i e l d (1964), w a s s u b s e q u e n t l y a d o p t e d to c i r c u m v e n t t h i s p r o b l e m of m y e l o m a e x p a n s i o n b y se lec t ing f u s i o n p r o d u c t s w h i c h possess a res is tance to b o t h 8 - a z a g u a n i n e ( inco rpora t ed in to the c e l l u l a r D N A v i a the e n z y m e h y p o x a n t h i n e g u a n i n e p h o s p h o r i b o s y l transferase) a n d 5 - b r o m o d i o x y u r i d i n e ( ind ica t ive of a n absence of t h y m i d i n e k inase ac t iv i ty ) . T h e r a t i o n a l e b e h i n d the use o f th is m e t h o d l ies i n the m a i n p a t h w a y s o f p u r i n e a n d p y r i m i d i n e b iosyn thes i s . T h e techn ique p r o v e d to be i d e a l l y su i t ed for p r o v i d i n g se lec t iv i ty agains t non- fused cel ls i n the p r o d u c t i o n of h y b r i d o m a s . If a c e l l is def ic ient i n ei ther of the e n z y m e s t h y m i d i n e k i n a s e ( T K " ) , o r h y p o x a n t h i n e g u a n i n e p h o s p h o r i b o s y l t ransferase ( H G P R T " ) it c a n be p l a c e d i n cu l tu re m e d i u m c o n t a i n i n g a m i n o p t e r i n (a fo l ic a c i d reductase an tagonis t ) so that D N A syn thes i s (both de novo a n d the a l t e rna t ive s a lvage p a t h w a y s ) ceases a n d the c e l l subsequen t ly dies [Fig.1.1]. A specif ic m y e l o m a c e l l l i ne can , therefore, be fused to a sp l enocy t e ( w h i c h possesses b o t h T K a n d H G P R T , bu t canno t i t se l f s u r v i v e i n cu l tu re ) , so that cu l t u r e m e d i a s u p p l e m e n t e d w i t h exogenous h y p o x a n t h i n e a n d t h y m i d i n e (but a l so c o n t a i n i n g the se lec t ive agent a m i n o p t e r i n ) w o u l d s u p p o r t the g r o w t h of s u c h a f u s i o n p r o d u c t . T h e m y e l o m a c e l l l i ne u s e d i n the ea r ly fus ion exper imen t s (es tabl ished i n M i l s t e i n s lab) w a s a n a z a g u a n i n e resistant descendent o f the P 3 K B a l b / C m y e l o m a : P 3 X 6 3 -A g 8 (or X63) . T h e genera t ion of a T K " (azaguanine resistant) m y e l o m a c e l l l i n e w a s the first to be d e v e l o p e d , as o n l y a s ing le m u t a t i o n a l event to the e n c o d i n g gene ( w h i c h exists o n the X c h r o m o s o m e ) w a s r e q u i r e d . F u r t h e r m o r e , the X 6 3 m y e l o m a t u m o r c e l l ( i m m o r t a l a n d capab le of g r o w i n g i n t i ssue cu l tu re ) , does no t h a v e the e n z y m e H G P R T a n d therefore canno t b y i t se l f s u r v i v e a m i n o p t e r i n se lec t ion . H o w e v e r , no t a l l h y b r i d ce l l s are u se fu l Fig 1.1: The main biosynthetic pathways for purine and pyrimidine biosynthesis displaying the sites of aminopterin blockage Q . Alternative pathways, in the presence of this folic acid antagonist, depend on the salvage enzymes thymidine kinase (TK) and hypoxanthine guanine phosphoribosyl transferase (HGPRT) 8 p r o d u c e r s of monospec i f i c a n t i b o d y after fus ion . H y b r i d o m a s at a n ear ly stage o f g r o w t h con t a in the genetic i n f o r m a t i o n con ta ined b y b o t h pa ren ta l c e l l l i nes a n d consequen t ly are not capable of s y n t h e s i z i n g the t w o classes of b o t h h e a v y a n d l i gh t a n t i b o d y chains . T h e re la t ive i n s t ab i l i t y inheren t w i t h the p o l y p l o i d a l na ture of these ea r ly fus ion p roduc t s is e v i d e n c e d b y the r a p i d loss of 32 to 42 ch romosomes d u r i n g the first m i t o s i s , a n d as a consequence , the p ro te ins w h i c h w o u l d h a v e n o r m a l l y been c o d e d for b y these ch romosomes . The process of se lect ing a d e s i r e d h y b r i d c lone that secretes not o n l y specif ic a n t i b o d y bu t a lso consis ts of o n l y a s ing le p a i r of l i g h t a n d heavy cha ins c a n be s i m p l i f i e d b y the i n i t i a l use of a specif ic c e l l l i n e w h i c h syn thes izes o n l y the l i g h t c h a i n o r expresses no i m m u n o g l o b u l i n at a l l . A p l a s m a c e l l m a y devo te u p to ha l f of its to ta l p r o t e i n syn thes i s to i m m u n o g l o b u l i n p r o d u c t i o n [Eisenbar th 1981]. It is b e l i e v e d that a " n o n - p r o d u c i n g " h y b r i d o m a ( w h i c h does no t have this a d d i t i o n a l me tabo l i c bu rden) w i l l g r o w m u c h m o r e r a p i d l y t h a n a n an t ibody-p r o d u c i n g v a r i a n t a n d thus w i l l t e n d to p r e d o m i n a t e the c u l t u r e , t h o u g h there are n o w f i n d i n g s to ind ica te that this m a y not be the case [Wes te rwoud t et al. 1984]. Never the less , it is g e n e r a l l y c o n s i d e r e d i m p o r t a n t that p r i m a r y sc reen ing o f cu l t u r e superna tan ts for specif ic a n t i b o d y p r o d u c t i o n a n d the subsequen t c l o n i n g o f d e s i r e d h y b r i d o m a s be p e r f o r m e d q u i c k l y . It is also i m p o r t a n t to ensure the p rese rva t ion of other po ten t i a l l y use fu l h y b r i d s b y f reez ing a n d subsequen t ly s t o r i ng t h e m i n l i q u i d n i t r ogen [Patel a n d B r o w n 1984; H a r w e l l et al. 1984]. O n c e su i t ab l e c lones have been i d e n t i f i e d , cu l tu r e s are s l o w l y e x p a n d e d a n d s u b c l o n e d so that even tua l ly the r e q u i r e d a n t i b o d y m a y be p r o d u c e d i n large quanti t ies. . F i g . 1.2 s chema t i ca l ly ou t l ines the fus ion p rocedure . Fig. 1.2: The conventional procedures involved with the generation of monoclonal antibodies in the murine system. Note that the three different methods of cloning employed in the present study have also been represented. 10 13 Comparison Between Heteroclonal and Monoclonal Antibodies. In general, there are considerable overall advantages afforded by the use of monoclonal antibodies over their conventionally-raised counterparts. Many of the currently available immunoreagents are from heteroclonal sources in that they are prepared from the sera of immunized animals and are comprised of a mixture of antibody as synthesized by the various sensitized B-lymphocytes; each directed to different or overlapping epitopes in response to a particular complex antigen. The fact that not all of these immunoglobulins are directed against identical analytes helps to explain why the immunological properties of a given antiserum tend to differ markedly between different animals and even from the serum obtained from different bleeds of the same animal [Calabrese et al. 1981]. These differences are often reflected in the immunoassay that uses conventional antisera and will, as a consequence, require repeated standardization. The antibodies which make up an antiserum are more likely to bind and therefore cross-react with structurally similar components. In addition, the specificity of a polyclonal-based assay system is related to the antibodies cumulative ability to recognize multiple epitopes, which subjects the assay to more frequent false positives due to varying degrees of cross reactivity to antigenically similar compounds [Yasuda 1984a, b]. A productive hybridoma cell line, in contrast, will produce a continuous source and unlimited supply of identical (monoclonal) antibody and in this respect is far superior as a standard reagent to conventional antiserum. Furthermore, the specificity afforded with the use of a monoclonal antibody usually circumvents cross-reactivity even when the antigen contains very similar determinants. In its more refined application the use of monoclonal antibodies provides detection of the most subtle antigenic differences as in, for example, those expressed among various strains of bacteria and virus. There, may be some disadvantage, however, with this property of monoclonal antibodies. One such situation could exist if, for example, a prosthetic group 11 w a s c o m m o n to t w o e q u a l l y accessible bu t o the rwi se d i s s i m i l a r p ro te ins . C ros s - r eac t i v i t y w i t h the m o n o c l o n a l a n t i b o d y i n s u c h a s i t u a t i o n c o u l d r each 100% whereas c o n v e n t i o n a l s e r u m w o u l d c o n t a i n an t i bod i e s d i r e c t e d to o ther u n i q u e d e t e r m i n a n t s a n d thus d i s p l a y s ign i f i can t ly m i n i m i z e d cross-react iv i ty [ C a m p b e l l 1986]. A l t e r n a t i v e l y , it is also occas iona l ly poss ib le to generate a n a n t i b o d y w h i c h d i s p l a y s too h i g h a speci f ic i ty for its final app l i ca t ion . T h i s m a y be the case w h e r e no t a l l s t ra ins of a v i r u s o r bacter ia express a c o m m o n ana ly te necessary for a genera l d i agnos i s . I r on i ca l l y e n o u g h , i t m a y i n s o m e instances be necessary to c o m b i n e a v a r i e t y of i m m u n o s p e c i f i c m o n o c l o n a l an t ibod ies to decrease the chance of not de tec t ing a n epi tope pecu l i a r to a pa r t i cu la r c o m p l e x ant igen. There is a genera l p r e s u m p t i o n that the h i g h degree of a f f in i ty ava i l ab l e f r o m the use of he t e roc lona l an t i se ra m a y not be r e a l i z e d w i t h the use o f m o n o c l o n a l an t ibod ies . T h i s hypo thes i s is based o n the apparent coopera t ive effects that have been demons t r a t ed to exist a m o n g m u l t i p l e a n t i b o d y types [ E h r l i c h et al. 1982]. O n e can , h o w e v e r , select a m o n o c l o n a l a n t i b o d y of r e q u i r e d a s soc ia t ion constant i n sc reen ing . F o r example , a n eas i ly d e n a t u r e d p r o t e i n w h i c h r e q u i r e s p u r i f i c a t i o n w i t h a p r e p a r a t i v e a f f i n i t y c o l u m n w o u l d be less v u l n e r a b l e to the ex t reme c o n d i t i o n s of p H or salt concen t ra t ion n e e d e d i n e l u t i o n [ G o d i n g 1980; M a s o n a n d W i l l i a m s 1980] w h e n h a n d l e d w i t h a l o w a f f in i ty m o n o c l o n a l a n t i b o d y spec i f i ca l ly se lected for th is characterist ic . Therefore, the a b i l i t y to select a n i m m u n e reagent of specif ic af f in i ty (such as at the t ime o f screening) is of spec ia l s ignif icance. A fu r ther advan t age to u s i n g h y b r i d o m a m e t h o d o l o g i e s comes f r o m b e i n g able to select a spec i f i c i m m u n o g l o b u l i n sub type . M u l t i v a l e n c e , for example , is b e l i e v e d to affect assay s e n s i t i v i t y [ R o d w e l l et al. 1983], o r m a y be r e q u i r e d i n a c o m p l e m e n t f i x a t i o n assay [ G o d i n g 1986]. 12 Another important consideration when comparing monoclonal and polyclonal antisera is in the yields of antibody that each source provides. The yield of useful antibody in conventional serum may be as high as 1 mg/ml. However, contaminating immunoglobulin may comprise up to 100% of the immunoreagent. Therefore, the immunizing agent used to produce the desired heteroclonal antibody must be either pure or the serum must be absorbed to minimize undesirable cross-reactivity. Alternatively, hybridomas maintained in tissue culture will yield in the order of 100 u,g/ml of useful antibody. This antibody (unlike its polyclonal counterpart), will be essentially free of all contaminating immunoglobulin except for those found in fetal calf sources required in culture medium. Hybridomas may also be raised as ascities tumors in mice, where ascities fluid yields slightly more (10%) contaminating immunoglobulin while the amount of useful monospecific antibody recovered can be as high as 20x the maximum amount realized with conventional methods [Campbell 1986]. One final significant consideration in selecting between both methods of producing antibody is the time and costs involved. The production, testing, cloning, and methods for assaying hybridomas for the generation and use of antibody must be simple, reliable and capable of being performed quickly and in large numbers. Often conventional serology will be sufficient for one's needs. There are, however, many instances where there exists a paramount need for highly specific antibody. In such situations, the generation and use of a monoclonal antibody becomes unparalleled. 1.4 Applications of Monoclonal Antibodies. The substitution of heteroclonal antiserum by monoclonal antibody has led to improved immunoassay technology. Virtually unlimited quantities of standard reagent of 13 known and usually higher specificity can be obtained from a given hybridoma cell line. The applications of monoclonal antibody technology has been well reviewed in recent years [Kennet et al. 1980; Edwards 1981; Yelton and Sharff 1981; Eikens 1981; Hamerling et al. 1981; McMichael and Faber 1982; Larrick and Bourla 1986; Langone and Van Vunakis 1986;]. The immense and still growing numbers of publications dealing with hybridoma technology makes it increasingly difficult to keep abreast of the more recent innovations in this relatively young field. It is, however, possible to profile some of the more significant implementations of monoclonal antibody technology currently being undertaken, and in doing so summarize the scope and range of the more popular methods encountered. 2.4.2 Diagnostic Application of Monoclonal Antibodies. The potential use of monoclonal antibodies as specific immunodiagnostic reagents has dramatically increased. They offer many advantages over similar applications using conventional antibodies by avoiding many of the limitations associated with the use of heteroclonal antisera [see Sec. 1.3] [Kemshead et al. 1983; Keller et al. 1984; Bolhuis and Haaijman 1985; Wang et al. 1986]. The unlimited availability (at standard titer) of a monoclonal antibody perhaps exemplifies its most superior characteristic relative to its heteroclonal counterpart. Such features afford a relatively straight forward method with which direct comparisons of information generated by a variety of investigators may be made [Cone 1985; Sevier 1985]. The use of highly specific standardized immunoreagent as provided by monoclonal antibodies should also add greatly to both the speed and accuracy with which a diagnosis is obtained. Monoclonal antibodies have become an important tool for the differential diagnosis 14 of v i r a l , bac te r i a l a n d pa ras i t i c infec t ions b y b e i n g ab le to detect a n d i m m u n o s p e c i f y the pa r t i cu l a r pa thogen . F o r example , a v e r y large n u m b e r of m o n o c l o n a l an t ibod ies have been p r o d u c e d to a w i d e v a r i e t y of v i ru se s i n c l u d i n g : C y t o m e g a l o v i r u s [ R e d m o n d et al. 1986; W i r g a r t a n d G r i l l n e r 1986], E p s t e i n B a r v i r u s [ H o f f m a n et al. 1980], Hepa t i t i s v i r u s [Shih et al. 1980; W a n d s et al. 1984], In f luenza v i r u s [Ge rha rd et al. 1981], H e r p e s S i m p l e x v i r u s [ Z w e i g et al. 1979; Pe re i r a et al. 1980; D r u y t s - V o e t s 1986; S u t h e r l a n d et al. 1986], P o l i o v i r u s [Ferguson et al. 1982], Rab i e s v i r u s [ W i k t o r a n d K o p r o w i s k i 1978], R u b e l l a v i r u s [Chan t l e r , p e r s o n a l c o m m u n i c a t i o n 1987], a n d S e m l i k i Fores t v i r u s [ K h a l i l i - S h i r a z i et al. 1986]. There has even been a recent r epor t o n the genera t ion o f m o n o c l o n a l an t ibod ie s to the " p r i o n " p ro te ins of the causa t ive agent o f Scrapies [Bar ry a n d P r u s i n e r 1986]. M o n o c l o n a l an t ibod ies have also been u s e d to measure v i rus - spec i f i c I g M a n t i b o d y to ind ica te a recent v i r u s in fec t ion rather than d e p e n d i n g o n a m o r e d e l a y e d increase of s e r u m I g G a n t i b o d y ti ter [Krau leda t et al. 1984]. M o n o c l o n a l a n t i b o d i e s w h i c h h a v e been u s e d for d i a g n o s i n g bac t e r i a l pa thogens h a v e a l so b e e n r e p o r t e d . O n e i m p o r t a n t e x a m p l e of t h e i r use fu lness i n a d i a g n o s t i c a p p l i c a t i o n i s for the r a p i d a n d spec i f ic d e t e r m i n a t i o n o f s e x u a l l y t r a n s m i t t e d bac te r i a l disease (STD) . M o n o c l o n a l an t ibodies n o w exists w h i c h can be u s e d i n the d i agnos i s of S T D s u c h as Chlamydia trachomatis, Neisseria gonorrhoeae [ N o w i n s k i et al. 1983] a n d i n the de tec t ion of the causa t ive agent o f s y p h i l i s ; Treponema pallidum [ M o s k o p h i d i s a n d M u l l e r 1986]. A p r o b l e m m a y exist i n the i r use as i m m u n o d i a g n o s t i c reagents , h o w e v e r , i n that there m a y be a l a c k of cross-genera reac t iv i ty ; w h e r e m o n o c l o n a l spec i f ic i ty is too h i g h . F o r example , r eac t iv i ty to a pa thogenic bacter ia (especial ly w h e r e a large n u m b e r of s t rains exist) m a y not be apparen t i f the r e c o g n i z e d ana ly te is no t c o m m o n to a l l s t rains . O n e m e t h o d of a v o i d i n g th is p r o b l e m w o u l d be to screen a n d spec i f i ca l l y select a m o n o c l o n a l a n t i b o d y -p r o d u c i n g c lone w i t h cross-genera spec i f ic i ty [ D u n n et al. 1986]. A pa r t i a l a n d i n some cases, p r o v e n s o l u t i o n to "over -spec i f ic i ty" is to c o m b i n e w h o l e pane ls o f m o n o c l o n a l an t ibodies , each d i r e c t e d to a different ep i tope [ N o w i n s k y et al. 1983]. T h e use of specif ic m ix tu r e s of 15 monoclonal antibodies should provide the advantage of titre standardization (and its associated reproducibility) over that realized with the use of heteroclonal antisera [Tagliabue et al. 1986]. In addition, monoclonal antibodies have been produced and characterized to various bacterial toxins [Remmers et al. 1982; Sheppard et al. 1984]. Finally, the study of the more common parasitic diseases such as schistosomaiasis [Smith et al. 1982] and malaria [Rener et al. 1980; Yoshita et al. 1980] has benefitted a great deal from the development and application of hybridoma technology. Another area of immunodiagnostics where the use of monoclonal antibodies appears to be well-suited (by providing a reliable degree of specificity at high titer and substantial quantity) is in tissue typing. The necessary immunological reagents (which until recently where obtained from exhaustive preparative techniques on sera obtained from, for example, multiparous women, multiple blood transfusion recipients or volunteers) are now, through somatic cell hybridization methodologies, being generated by a variety of investigators [Howard et al. 1979; Trucco et al. 1979; Bohnhoff et al. 1986], Furthermore, the generation of monoclonal antibodies to, for example, the many H L A determinants will facilitate a greater understanding of the underlying intricacies involved in the mechanics of the H L A system [for rev. see Bodmer and Bodmer 1984]. Monoclonal antibodies are particularly well-suited for the diagnosis of tumors (where they can identify the presence of tumor-associated antigens [Del Villano 1985; Haaijman 1985]), and in investigations concerning the unambiguous identification of onco-developmental antigens [Feizi 1985]. The ability to quickly and accurately detect both tumors and tumor-associated antigens (TAA) is also a very necessary extension of the post-operative monitoring of various cancer patients [Royston and Sobol 1985]. The immunospecificity of the monoclonal antibody provides the means for recognizing and following the cellular 16 alterations (i.e. the development or loss of antigens, the expression of fetal antigens and/or structural/functional macromolecular alterations of a given cell) associated with malignant transformation. The fact that some tumors share antigenic determinants with analytes which can be found on a number of different normal tissues makes interpretation of investigations obtained with heteroclonal antisera somewhat suspect [Wellerson and Kaplan 1986]. It seems obvious that a definitive immunological test for tumors and TAAs will require the properties inherent of monoclonal antibodies [Haisma 1985]. Monoclonal antibodies have been developed which can discriminate between a wide variety of lymphocyte-specific antigens as expressed on the surface of the various classes and subclasses of this cell type [Trucco et al. 1978]. In addition, monoclonal antibodies have been developed against the sialic acid residue of glycoproteins on non-T leukemic cells as generated by immunizing mice with antigen preparations from a pre-B leukemic cell line [Fukukawa et al. 1986]. Numerous monoclonal antibodies have been produced against defined tumor markers including: carcinoembryonic antigen (CEA) [Accolla et al. 1980; Haism 1985; Halpern et al. 1985], the squamous cell lung carcinoma-associated antigen (KA-32) [Kasai et al. 1981; Hanai et al. 1986], human melanoma-associated antigen [Koprowski et al. 1979; Fukaya et al. 1986], large-cell lung carcinoma [Mulskine et al. 1983], small-cell lung carcinoma [Cuttitta et al. 1981; Stahel et al. 1983; Leij et al. 1985; Takahashi et al. 1986; Sobol et al. 1986], human breast carcinoma [Kufe et al. 1984; Tagliabue et al. 1986], human epithelial ovarian carcinomas [Leoni et al. 1986; Friedman et al. 1986], human bladder carcinomas [Starling et al. 1982; Fradet et al. 1986], colon cancer-associated antigens [Drewinko et al. 1986; Hadas et al. 1986], and human malignant lymphomas [Meijer et al. 1985]. To illustrate how much information has been amassed on a single T A A , for example, an entire issue of a recent publication was dedicated solely to the immunodiagnostic and therapeutical values of monoclonal antibody CA-17.1A which "specifically" recognizes gastrointestinal cancer [Hybridoma, April 2-3,1986]. 17 The search for new tumor-specific antigens has been greatly facilitated with the implementation of hybridoma technology. However, investigators have yet to produce a truly tumor-specific immunological probe capable of recognizing analytes restricted to only tumors and metastases [Marx 1982; Hadas et al. 1986]. Instead what seems to have been produced are monospecific immunoreagents which not only recognize those antigens preferentially expressed on certain types of tumor cells, but which are also apparent on some types of normal cells [Frantz et al. 1986]. In addition, monoclonal antibodies clearly demonstrate that tumor cells can express differentiation antigens restricted to cells from a common origin. Of particular interest are monoclonal antibodies directed against tumor antigens of the brain or to antigens of neuroectodermal origin [McLendon et al. 1986; Stavrou et al. 1987]. For instance, anti-melanoma monoclonal antibodies were found to cross-react with gliomas and neuroblastomas [Kennett and Gilbert 1978; Herlyn et al. 1980; Seeger et al. 1981; Carrel et al. 1982; Liao et al. 1981]. The opposite situation of having an anti-glioma monoclonal antibody reacting with melanomas as well as gliomas and neuroblastomas, has also been reported [Kennett and Gilbert 1979; Herlyn et al. 1980; Liao et al. 1981; Seeger et al 1981; Carrel et al. 1982; Caincross et al. 1982]. Such findings support the notion that these monoclonal antibodies may actually recognize a common class of neuroectodermal differentiation antigen [Wilkstrand and Bigner 1982]. In a recent report by De Muralt et al. 1985, a panel of three anti-glioma and five anti-melanoma monoclonal antibodies were used to phenotypically characterize glioma and neuroectodermal tumors which included a series of malignant and low grade astrocytomas, malignant ependymomas and neuroblastomas [De Muralt et al. 1985]. These results also substantiate previous findings that many analytes associated with a cell line of neuroectodermal origin can be recognized by a given monoclonal antibody, and that immunocytological identification of glioma cells are severely hampered by the tumors marked antigenic heterogeneity or pleomorphism. 18 2.4.2 Monoclonal Antibodies as Immunotherapeutic Agents. The prospects for the therapeutic implementation of monoclonal antibodies are just now being realized [Larrick and Bourla 1986]. There are only a few reports on the therapeutic effects of monoclonal antibodies in vivo. [Hanai et al. 1986; De Pinho et al. 1986; Dillman et al. 1986], which no doubt is in part due to the infancy of the technology, as well as in part to the persisting technical problems which will have to be overcome before clinical benefits are fully realized [De Pinho et al. 1986]. Despite the recent reports of successful delivery of chemotherapeutic or radioiodinated immunoconjugates across the blood brain barrier [Neuwelt et al. 1986], evidence that human malignancies regularly express unique or restricted antigens is lacking, thus severely limiting the feasibility of clinical trials in humans. Another obvious disadvantage is encountered when one considers that the majority of monoclonal antibodies which display a potential therapeutic significance are those raised in mice. It appears that there is to date no suitable human myeloma cell line available to serve as a fusion partner [Boyed et al. 1984; Cole et al. 1984; Seiler et al. 1985; Lee 1986], although there have been infrequent reports of human-human hybridomas secreting monoclonal antibody [Lowe et al. 1984; Gaffar et al. 1986]. There are from time to time, as well, descriptions of stable human-murine hybridomas secreting antigen-specific human monoclonal antibody [Maeda et al. 1986]. Whether or not mouse immunoglobulins will themselves elicit too vigorous an immunogenic response in humans (serum sickness) so as to prohibit their use in vivo has yet to be determined [De Pinho et al. 1986; Dillman et al. 1986]. The use and potential of murine monoclonal antibody as an immunotherapeutic agent for the treatment of gram-negative bacterial sepsis [Dunn et al. 1986] as well as for the generation of specific immunoreagents directed against bacterial toxins, have been described 19 [Remmers et al. 1982; Kozbor et al. 1982; Sheppard et al. 1984]. Problems of a more technical nature, when considering a therapeutic application for monoclonal antibodies, include generating the class or subclass of immunoglobulin which demonstrates a high level of affinity and specificity even to analytes of a particularly weak immunogenic nature [De Pinho et al. 1986]. A further concern is the stability of the therapeutic immunoconjugate (i.e. radio pharmaceutical [Halpern et al. 1985]). One must also keep in mind the multiple variables of an in vivo application which is beyond the technical aspect of the immunotherapeutic agent including: accessibility, which in turn is influenced by factors such as blood flow to the site, physical barriers (i.e. blood brain barrier or that incurred with a capillary cell wall), and the possible variability and modulation (for example by antigen-antibody internalization) of antigenic expression associated with the target cell [Halpern et al. 1985; Dillman et al. 1986]. There are several aspects of immunotherapy where the use of murine monoclonal antibodies have been shown to be of value. The high degree of specificity for immune cells, as provided by monoclonal antibodies [Pilkington et al. 1984] with the subsequent large scale immunoselection and removal of certain lymphocyte subsets (i.e. helper T-cells: L3T4+ and Lyt-2 +) have been shown to help promote both marrow tolerance as well as accentuate the abrogation of graft rejection in the murine system [Cobbold et al. 1986a, b; Benjamin and Waldmann 1986]. The advent and use of monoclonal antibodies in the practice of bone marrow transplant (in conditioning regimes and for prevention of post-operative infection [Dunn et al. 1986]) will make a substantial impact towards the effective prevention of graft-versus host disease [Brenner et al. 1986c]. There are similar, recent, examples in the literature describing the use of murine monoclonal antibodies to manipulate the immunologic responses as seen in Theiler's Virus-induced demyelination [Rodrigues et al. 1986] and in progressive multiple sclerosis [Hafler et al. 1986]. 20 2.4.3 Monoclonal Antibodies in Basic Research. There exists inherent difficulties associated with the study of the constituent protein and lipid elements of the cellular membrane, including their relative scarcity in pure form, and in some cases, the loss of any measurable biological activity upon solubilization for analysis [for rev. see Fleischer and Packer 1974]. The use of polyclonal (and more recently monoclonal antibodies) has provided a means to avoid the problems associated with the characterization and purification of membrane molecules. Such an approach has proven fruitful in the search and recognition of novel cell surface antigens, identification and characterization of specific antigens at different stages of cellular differentiation, and in determining an analyte's functional significance. An excellent example of this is in the use of commercially available monoclonal antibodies which define human lymphocytes and their particular subsets [for rev. see Seiler et al. 1985; Eisenbarth 1981]. The expression of a particular molecular ensemble and the quantitative immunoreactivity of a given lymphocytes' surface antigens (as defined by these monoclonal antibodies) correspond to a specific state of cellular differentiation. Conventional antisera which are to be used against complex surface and intracytoplasmic antigens, however, are highly heterogenous and do not recognize single molecules without the use of more elaborate preparative methodologies. 15 Imtnunospecific Identification in Neurobiology. There is no doubt that immunocytology is at the present time undergoing profound changes, and that these changes stem largely from monoclonal antibody technology. New insight has been gained into the elements and framework of many biological structures which, when using classical immunocytological and biochemical methods, could only be detected (if at all) with great difficulty. The need for cell type-specific markers which 21 recognize particular antigenic components are of paramount importance not only in rapidly establishing a cell's lineage but also in recognizing the existence of cellular subsets. 2-5.2 The Availability of Conventional Cell Type-Specific Antibodies to Neural Antigens. It has been 80 years since Harrison (1907) first described the successful in vitro culture of embryonic frog spinal cord. Today a variety of both central nervous system (CNS) and peripheral nervous system (PNS) tissues may be routinely prepared and maintained from a multitude of animal species [see Sec. 1.6]. Despite all that has been discovered about the conditions affecting the characteristics, growth, and development of neural cells in culture [for rev. see Varon 1977], there persists problems in the general inability to unequivocally discriminate the major classes of neuronal and glial cell types [Raff et al. 1979; Kim et al. 1985]. The identification of the various classes of neural cells in culture has up until recently depended largely on their morphological (phase-contrast) characteristics [Bunge et al. 1967; De Mey 1983]; a method which has been demonstrated to be in many instances unreliable [Cowden 1985]. For this reason, cell specific or related macromolecules have been sought which may be used in conjunction with immunocytochemical techniques in an effort to overcome these difficulties [see Fig. 1.3]. The essential prerequisite in the development and characterization of a cell type-specific surface or intracytoplasmic antigenic marker is to generate an antibody directed against a neural cell-restricted constituent. The expression of this specific component as recognized by the immunoreagent can, therefore, be used in the subsequent identification of the neuronal or glial cell type. The use of immunocytological methodologies in neurobiology have been well reviewed [Fields et al. 1978; Raff et al. 1979; Kennedy 1980, 1982; Mirsky 1980a; Roots 1981; Weiner et al. 1982; Shachner 1982a, b, 1983a; Beutner et al. 1983; Brockes 1984; Kim 1985; Landis 1985; Reichart 1985; Valentino et al. 1985] 22 OLIGODENDROCYTE MARKERS ASTROCYTE MARKERS NEURONAL MARKERS Galactocerebroside Myelin-Associated Protein Myelin Basic Protein Sulfatide G M 3 Glial Fibrillary Acidic Protein S-100 Protein Glutamine Synthetase SSEA-1 Neurofilament Triplet Proteins Neuron Specific Enolase Tetanus Toxin Fig. 1.3: Schematic representation of the major constituents of the mammalian nervous system. Note the major cell class markers (see text for more complete lists and further explanations). 2 3 Heteroclonal Antiserum to Oligodendroglia. The ability to isolate highly enriched cultures of glia is essential for delineating the pathology of myelination [Raine 1983] [see Sec. 1.6.2]. With the use of the recently available immunoreagents, neural cell types may be identified and enumerated even when only constituting a small proportion of the total cell population. Cell surface markers that can be used to distinguish live cells, their properties and interactions, are essential for producing purified cell constituents [Hussey et al. 1986] or alternatively, in obtaining these populations through selective sorting or killing procedures [Meier and Schachner 1982; Meier et al. 1982b; Assouline et al. 1983; Abney et al. 1983; Smyrnis et al. 1986]. Efforts to produce oligodendrocyte and/or myelin specific heteroclonal antibody to the relatively simple compliment of myelin proteins [Moore 1973; Norton 1981; Morell 1984] has proved relatively successful. Human CNS myelin consists essentially of 20% to 25% protein [Brady et al. 1981; Brostoff 1984; Stefansson et al. 1986]. There are two predominant types of protein which make up the major protein elements of the myelin membrane. The first, known as A | , encephalitogenic, or myelin basic protein comprises about 30% of the total protein present in the oligodendroglial membrane [Boggs and Moscarello 1978a] and appears to play an integral role in maintaining the highly ordered spiral arrangement of the natural multilamellar membrane system [Boggs and Moscarello 1978b; Stollery et al. 1980; Boggs et al. 1980a; 1981; Brady et al. 1981]. The second major protein constituent which makes up approximately 50% of the total protein found in myelin, and which constitutes most of the fraction [Folch and Lees 1951], is called the lipophilin or N2 protein [Gagnon et al. 1971]. 24 Heterogeneous antisera which have been raised successfully against many of the constituents of myelin include: myelin basic protein [Sternberger 1978; Hartman et al. 1982; Itoyama et al. 1980b; Roussel and Nussbaum 1982; Kies 1982; Meller and Waelsch 1984; Mikoshiba et al. 1985; Rome et al. 1986], Wolfgram protein (WP) [Nussbaum et al. 1977; Roussel and Nussbaum 1982; Delaunoy et al. 1982], myelin associated glycoprotein (MAG) [Sternberger et al. 1979; Quarles 1981; Webster et al. 1983], proteolipid protein (PLP) [Agrawal et al. 1977; Fischer and Sapirstein 1986], and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) [Sprinkle et al. 1980a; Sheedlo and Sprinkle 1985]. The expression of myelin-basic protein (MBP) on various non-myelinating [Mirsky et al. 1980b; Barbarese and Pfeiffer 1981; Bologa-Sandru et al. 1981], and myelin forming oligodendroglial cultures [Gonatas et al. 1982; Kim et al. 1983] has been amply demonstrated with the use of heteroclonal antisera. Data on the antigenicity [Ludwin 1981; Hockfield and McKay 1983] and chemical nature of MBP indicate that the amino acid sequence is highly conserved as isolated from several different mammalian species [Dunkley and Carnegie 1974; Day and Potter 1986]. Proteolipids on the other hand are operationally defined by their ability to dissolve in organic solvents [Folch and Lees 1951] and display a wide distribution in the membrane of many plant, animal, and bacteria species [for rev. see: Lees 1982; Lees and Brostoff 1984]. Regardless of their relative abundance (particularly in the CNS white matter), such an ubiquitous distribution of the proteolipid and its constituents would necessarily curtail its usefulness as an oligodendrocyte/myelin specific marker. However, many of the proteolipids' biochemical [Cockle et al. 1978; Boggs and Moscarello 1978a; Bizzozero et al. 1985] and physical characteristics [Moscarello et al. 1973; Vail et al. 1974; Wood et al. 1980; Aguilar et al. 1983] appear to be distinct from those possessed by proteolipids of a non-myelin 2 5 origin [Fischer and Sapirstein 1986; Dubois-Dalcq et al. 1986; Duncan et al. 1987]. The proteolipid protein (PLP) (also known as N2, P7, or lipophilin) in addition to its water soluble apoprotein, constitutes the major protein elements that make up the proteolipid fraction [Gagnon et al. 1971]. It is of special interest to note that like MBP and M A G , PLP appears to be abnormally expressed (albeit independently) in various mutant strains of dismyelinating mice [Dautingny et al. 1986; Sorg et al. 1986]. Furthermore, investigations using heteroclonal antisera have demonstrated an antigenic constituent common to PLP and MBP capable of eliciting an encephalitogenic response in various species [Yamamura et al. 1986; Trotter et al. 1987]. This finding suggests the existence of partial sequence homology shared between both of these myelin components [Hashim et al. 1980]. Of course any amount of peptide homology between PLP and MBP could increase the degree of non-specific reactivity seen in a comparative, heteroclonal antisera based immunoassay. The activities of as many as 20(+) enzymes have been observed in myelin purified from CNS tissue [for rev. see Schousboe 1982; Norton and Cammer 1984]. Of these enzymes, only about one half can be classified as myelin specific, the rest being myelin-localized or non-specific components. One example of a non-specific myelin-localized enzyme is carbonic anhydrase-C (CA). Significant amounts of C A activity have been immunocytochemically and biochemically demonstrable in other (non-oligodendroglial) subcellular fractions, as in for example, astrocytes. [Gandour et al. 1980b; Church et al. 1980; Cammer et al. 1985; Cammer and Tansey 1986]. Also considered a cell type-specific marker for oligodendrocytes is the NADH-linked enzyme glycerol phosphate dehydrogenase (GPDH) [Leveille et al. 1980; Montz et al. 1985] responsible for the generation of a-glycerol phosphate from dihydroxyacetone phosphate. However, it has also been reported to be present in very small amounts on astrocytes (a second major glial cell type) as well [McCarthy and de Vellis 1980; Leveille efaZ. 1980]. Another example of perhaps a more myelin specific (with respect to myelin localized) 26 enzyme is 5'-nucleotidase. This enzyme has recently been used to produce a heteroclonal antisera which demonstrates 5'-nucleotidase possessing selective activity in the brain and myelinated CNS fibers of adult mice [Cammer et al. 1986]. It has also shown a similarity to the immunostaining characteristics of carbonic anhydrase-C [Cammer and Tansey 1986]. We have shown [E.S. and Kim, independently generated, unpublished observations] that the frequency distribution of murine and porcine oligodendroglia immunolabeled with specific polyclonal antisera (directed against the intracytoplasmic markers CNPase or MBP) can be, to a large extent, associated with a positive immunolabeling (as determined with FACS analysis) of HNK-1 expression by these cells in culture; substantiating the notion for the existence of antigenic determinants common to both the nervous system, and immune system [Oger et al. 1982] and which may have immunological significance in the induction of an immune response involving these neural cells. The remaining 75% to 80% of human CNS myelin essentially consists of lipid [Brady et al. 1981]. Since the first demonstration of a positive immunofluorescence staining of anti-galactocerebroside antibody on the surface of cultured rat oligodendrocytes by Raff et al. [1978b], antisera directed against GalC (the major glycolipid of myelin [Norton and Autilio 1966]), has served as the most dependable cell type-specific indicator of these myelin-producing cells [Raff et al. 1979; Mirsky et al. 1980a; Kim 1985; Saneto and de Vellis 1985; Kohsaka et al. 1986]. We have recently described a method in which populations of oligodendrocytes from whole brain were enriched, labeled with heteroclonal antisera and subsequently immunoselected using the fluorescence activated cell sorter (FACS) [Smyrnis et al. 1986; Kim et al. 1987]. Through multiparameter gating on both a defined forward-angle light scatter peak (corresponding to cell size) and positive GalC immunolabeling, oligodendroglia exceeding 98% purity were obtained that were suitable for long term culture and further immunological and neurobiological investigations. 27 The generation and characterization of a polyclonal antiserum to sulfatide has been described by Hakomori et al. 1974, and more recently by Ranscht et al. 1982, as being useful in demonstrating a localized-specificity to oligodendrocytes in culture, and as also having potential use in investigations dealing with the process of myelination [Raff et al. 1979; Ranscht et al. 1982; Kim 1985]. Sialosylgalactosylceramide (GM4) a unique ganglioside which is abundant in the CNS tissues of humans and birds [Ledeen et al. 1973] is specifically localized in CNS myelin and reported to be specific for oligodendroglia [Ledeen et al. 1973; Yu and Iqbal 1979]. Recently, however, Kim et al. 1986 has demonstrated this ganglioside to also exist on the surface of a majority of astrocytes as well. Finally, it should be noted that there are indications that Fc receptors exist on the surface of oligodendrocytes which may bind non-specifically to a variety of different immunoglobulins [Traugott et al. 1979; Ma et al. 1981]. Heteroclonal Antisera to Astrocytes. Perhaps the most reliable and versatile specificity used for the unequivocal identification of astroglia is the intracytoplasmic marker glial fibrillary acidic protein (GFAP)[Bignami et al. 1972; Gilden et al. 1976]. The GFAP protein is associated with the glial-specific class of intermediate (10 nm) filament [Schachner et al. 1977], and is specifically expressed by only both protoplasmic and fibrous astrocytes. It has been demonstrated in immunohistological [Bignami et al. 1972; Pelc et al. 1986; McLendon et al. 1986], and immunocytological preparations [Raff et al. 1979; Kim et al. 1984a, b; Vernadakis et al. 1986; Newcombe et al. 1986; Malloch et al. 1987] of neural cell cultures from a variety of species, and is believed to be responsible for stabilizing and maintaining the general overall 28 m o r p h o l o g i c a l appearance o f the ma tu re astrocyte [ T r i m m e r et al. 1982] A n a l te rna t ive , ye t less p o p u l a r , i n t r a c y t o p l a s m i c m a r k e r e m p l o y e d to i m m u n o l a b e l a s t rog l i a i s the e n z y m e g l u t a m i n e synthetase (GS) . G S is a k e y e n z y m e i n v o l v e d i n the d e t o x i f i c a t i o n o f a m m o n i a i n the b r a i n , as w e l l as i n the m e t a b o l i s m of c e r t a i n neu ro t r ansmi t t e r s [ W e i l - M a l h e r b e 1962; M c G e e r a n d M c G e e r 1981; J u u r l i n k 1982]. G S has been d e m o n s t r a t e d i n f rozen sect ions of b r a i n a n d is expressed e x c l u s i v e l y b y p r o t o p l a s m i c a n d f ib rous astrocytes [ N o r e n b e r g a n d M a r t i n e z - H e r n a n d e z 1979]. H o w e v e r , i t appears that i n i m m u n o c y t o c h e m i c a l s t u d i e s o f h u m a n o l i g o d e n d r o c y t e s i n e a r l y c u l t u r e , o n l y a m a x i m u m of 20% G F A P + astrocytes express pos i t i ve G S i m m u n o l a b e l l i n g [ K i m 1985]. T h e S-100 p r o t e i n (a h i g h l y a c i d i c c a l c i u m - b i n d i n g p r o t e i n o f l o w m o l e c u l a r weight ) is also c o n s i d e r e d to be a c e l l type-specif ic m a r k e r for astrocytes [Hans son et al. 1980] a n d has a d i s t r i b u t i o n w h i c h ( t h o u g h no t en t i r e ly r a n d o m ) i s qu i t e w i d e l y v a r i e d [ K u m a n i s h i et al. 1984]. Its presence i n w h i t e mat te r a n d b u l k - i s o l a t e d adu l t g l i a (both o l i godend rocy t e s a n d as t rocy tes ) u s i n g b i o c h e m i c a l m e t h o d s [ H a g l i d et al. 1977] a n d i m m u n o c y t o c h e m i c a l techniques [ K i m 1985] have been w e l l d o c u m e n t e d . It has also been demons t r a t ed i n va r ious n o n - n e r v o u s t issue a n d s h o w s p r o m i s e as a u se fu l m a r k e r i n d i agnos t i c s u r g i c a l p a t h o l o g y [ K a h n et al. 1983]. T h e use of these as t rogl ia l -specif ic marke r s , h o w e v e r , suffers f r o m the fact that the ce l l m u s t be f i x e d p r i o r to l a b e l l i n g w i t h antisera. S ince these marke r s are expressed i n t r a c y t o p l a s m i c a l l y i t is no t poss ib l e to use these p re sen t ly a v a i l a b l e i m m u n o r e a g e n t s to separate v i a b l e as t rocytes f r o m other ce l l s s ince the a s t rog l i a d o not s u r v i v e the f i xa t i on process ; necessary for a n t i b o d y access. D e s p i t e the g r o w i n g n u m b e r s of c e l l type-speci f ic ma rke r s b e i n g d e v e l o p e d against the astrocyte, there is at the present t ime s t i l l no satisfactory sur face spec i f i c m a r k e r a v a i l a b l e w h i c h c o u l d be u s e d for i m m u n o s e l e c t i o n o f v i a b l e a s t r o g l i a f r o m m i x e d c u l t u r e [ K e n n e d y 1982]. O t h e r m e t h o d s w h i c h p r o v i d e acceptable l eve l s of c e l l v i a b i l i t y a n d w h i c h m a k e i n t r a c y t o p l a s m i c de t e rminan t s a v a i l a b l e to specif ic 29 antisera without killing the cell are just now being developed [Schroff 1984; Cowden 1985]. Heteroclonal Antisera to Neurons. It is somewhat more difficult to distinguish neurons from non-neuronal cells in whole brain culture using cell type-specific surface markers because there is comparatively little in the way of such neuron-specific immunolabels currently available [Stallcup et al. 1983]. Gangliosides which are found in abundance on neuronal tissues can be considered to be of potential value as cell type-specific surface markers for the identification of neurons (which contain many major and minor gangliosides) and their subclasses [Kim et al. 1986]. These sialic-acid containing glycolipids are considered by many to be responsible for a variety of cellular interactions [Adinolfi and Brown 1983]. They, for example, have been identified as serving as membrane receptors for specific bacterial neurotoxins [Van Heyningen 1963], in playing a role in memory generation and synaptic transmission [Hollman and Seifert;l986], and in promoting axonal regeneration [Kim et al. 1985] by stimulating neurite outgrowth in neural cell cultures [Rybak et al. 1983; Toffano et al. 1986]. They have also been shown to be able to alter the morphological appearance and growth characteristics of astrocytes in culture [Hefti et al. 1985]; possibly through their ability to increase the accumulation of tubulin mRNA [Rybak et al. 1983] and the activity of choline acetyltransferase [Heft et al. 1985]. An example of a neuronal marker that has been shown to bind principally (greater than 96% [Mirsky et al. 1978] ) to the surface of neurons in both CNS and PNS cultures, from a variety of vertebrates, is the neurotoxic product of Clostridium tetani: tetanus toxin [Mirsky et al. 1978; Raff et al. 1979; Kennedy 1982]. There is considerable evidence suggesting that the specific gangliosides Gj)ib arid Gfi bind specifically to this neurotoxin [Van Heyningen 1963; 30 Holmgren et al. 1980; Yarvin 1984; Critchley et al. 1986]. The use of tetanus toxin, however, has the obvious disadvantage of being extremely toxic. The primary complex of toxin and surface marker possess the additional disadvantage (should incubation temperatures rise above 0 ° C) of being rapidly internalized during the labeling procedure [Critichley et al. 1985], and that the toxin itself displays high non-specific binding to cellular debris [Mirsky et al. 1978]. Heteroclonal antisera raised against the major ganglioside, Gy[\, can positively immunolabel approximately 80% of neurons from cultured human fetal spinal ganglia [Kim et al. 1986]. This specific class of ganglioside has been shown to help facilitate recovery from partial mechanical lesioning of specific CNS areas [Agnati et al. 1983], to influence neuronal plasticity [Toffano et al. 1986], as well as functioning as a receptor molecule for another bacterial neurotoxin; cholera toxin [King and Van Heyningen 1973; Raff et al. 1979]. Unfortunately, all major glial cell types predominantly express this epitope and in doing so, anti-G]yii antibodies can not serve to discriminately label specific neural cell types [Kennedy 1982]. This monosialosyl-ganglioside, however, may still prove useful as a general marker for cells of neuroectodermal origin [Kim et al. 1986]. Antisera raised against a neuron-specific glycolytic enzyme, enolase, has been shown to discriminately label neurons [Heydron et al. 1985] in dissociated cultures of embryonic mouse dorsal root ganglia (DRG) [Pelc et al. 1986], spinal cord [Hooghe-Peters et al. 1979] and preparations of rat CNS [Schmechel 1978]. The recent implementation of new biochemical and immunofluorescence methodologies have distinguished intermediate filaments (IF) as being a fibrous network comprised primarily of chemically heterogenous subunits which lend themselves to further subclassification; each being immunogenically and biochemically distinct [for rev. see 31 Lazarides 1980]. Of the five major classes of IF, neurofilaments (the neuron specific class of intermediate filament) and glial filaments (expressed only in astrocytes) are the major cytoskeletal components of the neural cells which posses them [Chiu et al. 1981]. Immunolabeling of the higher molecular weight microtubule associated proteins (MAPI and MAP2) [Matus et al. 1981; Couchie et al. 1986]; brain-specific spectrin (Fodrin) [Lazaridis and Nelson 1983; Hesketh et al. 1986], and even the various neurofilament subunits [Hirokawa et al. 1984] have demonstrated a highly differential distribution with the use of heteroclonal antiserum. Antibodies specifically directed against each subunit of the neurofilament triplet protein, for example, have demonstrated that this triplet is expressed only in neurons [Shaw et al. 1981, 1984; Lee et al. 1982a] . However, there exist a class of neuron (i.e. the cerebellar granule cell) which lack neurofilaments and consequently do not immunolabel positively [Shaw et al. 1981]. Ultrastructural examination demonstrates that each antibody pocesses the ability to recognize the same filament [Sharp et al. 1982] in both a periodic (200 KDa) or a continuous pattern of distribution [Valentino et al. 1985]. The specific role of cytoskeletal filaments is poorly understood, yet, are believed to perform a central function in the mechanical integration of their respective organelles. Conventional antibodies have been used to examine the distribution of these and other cytoskeletal elements in cultured neurons [Yen and Fields 1981; Gambetti et al. 1983]. These neurofilament components, being distinct individual elements, however, may be used to immunologically identify neuronal cell types from non-neuronal cells in mixed culture [Lazarides 1980; Bigbee and Eng 1982; Jones et al. 1986]. The first intermediate class of IF to be purified [Saneto and de Vellis 1985] was initially prepared from central white matter [Bignami et al. 1972]. These are clearly demonstrable in glia and predominantly comprised of GFAP [see Sec.]. Alternatively, the oligomeric neurofilament proteins purified from mammalian PNS (a glial-free source of material) are comprised of three major polypeptide subunits of approximate molecular weight: 200 KDa, 32 150 KDa, and 70 KDa [Lee et al. 1984, 1986a; Carden et al. 1985]. The differences which exist between these classes of intermediate filament develop from variations in the peripheral N H 2 - and - C O O H terminal sequences whereas the core sequences display structural preservation. Polyclonal immunocytochemical studies have affirmed the hypothesis that these three neurofilament polypeptides are immunologically distinct in any one species and that expression of corresponding neurofilament subunit-specific antigens are conserved across various mammalian species [Lee et al. 1982a; Shaw et al. 1984]. A problem exists, however, with the high degree of cross-reactivity observed with the use of conventionally raised heterologous antisera directed against these protein subunits [Liem et al. 1978; Lee et al. 1982b]. The unequivocal identification of both common and unique determinants expressed by the neurofilament proteins could only be achieved with monospecific antibodies. However, production of monospecific heteroantisera necessitates the availability of highly purified antigen. Heteroclonal Antisera to Complex Neural Antigens. The preparation of antigens from fluid and solid material has been reviewed [Davies 1971]. The need for pure antigen presents a serious drawback in neuroimmunological investigations considering the strict limitations in the availability of purified nervous system antigens. Antigens which may be comprised essentially of protein, carbohydrates, lipid, nucleic acid, or a complex thereof (i.e. glycoprotein, lipoprotein, glycolipids, and so on) are frequently prepared or used in impure form. It may, however, not matter in the context of a particular use (such as in vaccination), that substances of a non-antigenic nature be also present. Nevertheless, the implementation of pure antigen preparations would more often provide clearer results [Calabrese et al. 1981]. 3 3 Several investigators have generated antibodies using a highly complex immunogen. For example, heteroclonal antisera has been raised against whole nervous system homogenate [Schachner 1982b], particulate fraction of nervous system [Schachner et al. 1975; Zimmermann et al. 1976; Calabrese et al. 1984; Wikstrand et al. 1982], and even nervous system derived tumors [Doyle et al. 1977; Hedley-White et al. 1978]; including their cloned cell lines [Schachner 1974]. However, in studying the many reports it becomes evident that the majority of the cell-type "specific" heteroantisera raised to a given neural antigen could only be obtained through techniques involving exhaustive absorption against epitopes common amongst all other neural antigens; since many such surface and intracytoplasmic constituents exist. As new methods for purifying neural antigens become available, researchers may apply them to investigations concerning a particular cell or culture system. Immunologically distinguishable substances must possess structural and chemical differences. It is unlikely, therefore, that the methods employed in the purification of any one antigen will lead to the purification of an other. The development of hybridoma technology has revolutionized the prospect for developing novel cell type-specific markers in neurobiology by making it feasible to immunize an animal with a complex mixture of antigens and subsequently select a hybridoma cell line producing a given antibody of desired reactivity, without the concomitant interference of antibodies that express activity toward unwanted specificities as seen with heteroclonal perparations. In other words, the use of these methods negates the need for highly purified immunogen. The frequency of desired or expected immunoreactive hybridomas, however, may decrease with the "crudeness" of the immunizing agent, but it produces at the same time an opportunity to discover new, more subtilely expressed (and possibly even more specifically associated) immunogenic cellular constituents. 34 The composition of brain tissues is primarily of membranous elements containing many different proteins and lipids which may function as enzymes, receptors, or structural components. Attempts to characterize the proteins of defined tissue fractions have been hampered largely by the presence of significant quantities of exogenous protein and proteolytic enzymes used in the isolation procedure [De Vries et al. 1981]. As a consequence, the molecular features of the CNS supporting cell and the neuronal (excitable) membrane remains virtually uncharacterized [Stanley et al. 1979; De Vries and Zmachinski 1980]. To a great extent, the more readily accessible nerves (with their component axonal and glial elements) have been dissected out and partially investigated [Fischer et al. 1970]. There now exists methods for the relatively routine purification of CNS axons from mammalian white matter which has been reviewed by De Vries et al. 1972. Use of these methods provides the means for preparing external membranes of CNS axons by fractionation of subcellular particles (following initial homogenization), and the additional opportunity to generate against these fractions, both heteroclonal and monoclonal antibodies. Various physical methods have since been used extensively in the concomitant preparation of both axolemma-enriched [Stanley et al. 1979; Harford et al. 1979; Zetusky et al. 1979; De Vries 1981; Cullen et al. 1981; De Vries et al. 1983; Yoshino et al. 1983; Sobue et al. 1983], and myelin enriched [Norton and Poduslo 1973; Matthieu et al. 1977; Haley et al 1979; Cullen et al. 1981] fractions. Implementation of these techniques have led to the biochemical characterization of both: the constituent proteins [De Vries et al 1976a; Micko and Schlaepfer 1978; Sweadner 1979] and lipids [De Vries et al 1976b, 1981; Schook and Norton 1976; Henderson et al. 1984] of an axolemma-enriched fraction, as well as to the enrichment of the proteins found in myelin [Zimmerman et al. 1975; Autilio-Gambetti et al. 1975; Shapira et al. 1978; Matthieu et al 1979; Kolehmainen et al 1982] and the proteins' lipid subfraction [Sweadner 1979]. Further studies have uncovered a significant ability to induce proliferation of cultured Schwann cells when exposed to these subfractions [Raff et al 1978a; Salzer et al 35 1980a, b; Yoshino et al. 1984; Sobue et al. 1984; Meador-Woodruff et al. 1985; De Vries et al. 1982], and the potential mediators of such a mitogenic response [ Meador-Woodruff et al. 1984; Sobue and Pleasure 1985]. 15.2 Monoclonal Antibodies in Neurobiology. Until recently, the in situ origin of a CNS subcellular fraction largely depended on indirect lines of evidence. For example, enrichment of a CNS axolemma subfraction may be judged on the basis of the relative increase in activity of those components (i.e. enzymes) most closely associated with the axonal plasma membrane. These include the enzymes 5'-nucleotidase [Cammer and Tansey 1986; Cammer et al. 1986], acetylcholinesterase [Koelle 1954; Cauna and Naik 1963; Karnovski and Roots 1964;] and sodium-potassium activated ATPase [Jorgensen 1974; Mitchell et al. 1982]. There is also an increase in the binding of tetrodotoxin [Baker and Van Der Togt 1986] and scorpion toxin [De Vries and Lazdunski 1982] to these fractions; reflecting a relative enrichment of the functional sodium channels in these preparations [Berwald-Netter and Koulakoff 1982]. It is also interesting to note that the expression of scorpion-toxin binding is believed to be a useful quantitative indicator of both neural maturation and differentiation. An alternative, more direct approach with which to define the origin of such a membrane fraction is to raise an antiserum specific to the axolemma, and subsequently use the immunoreagent to demonstrate an increased relative immunoreactivity with the majority of the membrane vesicles of various isolated membrane fractions [Bigbee et al. 1985]. Use of an indirect enzyme-linked immunosorbant assay (ELISA) has demonstrated its usefulness in the quantitation of even microgram amounts of both analyte and anti-axolemma enriched antibody [Calabrese et al. 1981]. A subsequent preliminary report of a monoclonal antibody generated to an enriched subfraction of bovine splenic-nerve axolemma [Bigbee et al. 1986] will be useful in characterizing the specific antigenic determinants associated with the axonal plasma 3 6 membrane, and may be ultimately employed for the purification of the recognized specificities. An alternative though somewhat similar strategy of using crude fraction or subfractioned homogenate in the production of a previously undefined antibody comes from using nervous system-derived or related tumors (for example: meduloblastomas, neuroblastomas, and gliomas) as the primary immunogen. Such methods have been used to provide in some instances, large amounts of starting material which may express a high degree of cellular and genetic homogeneity [Liepkalns et al. 1982]. These methods offer a relatively available starting material, while also maintaining the possibility that these cells have preserved (or can be induced to express) some of the characteristic properties displayed by the original neural cell type [Akeson et al. 1981; Lee et al. 1982b; Koppel et al. 1986]. This has been substantiated by, for example, their cerebroside, sulphatide [Jungalwala et al. 1985], and ganglioside [Miller-Podraza and Fishman 1983] metabolism; in their biochemical expression of glial differentiation [Westermark et al. 1982; Volpe and Obert 1983; Raff et al. 1983a, b]; their elaboration of nerve growth factor (NGF) receptors [Schulze and Perez-Polo 1982; Ross et al. 1984]; and in their expression of the various cell type-specific markers: CNPase [McMorris et al. 1984] neurofilament (NF) triplet proteins [Lee and Andrews 1986], neural cell adhesion molecules (N-CAM) [McMorris et al. 1984], GFAP [Giotta and Cohn 1981; Bigbee et al. 1983a, b; Nishiguchi et al. 1985; Stanton et al. 1987], S100 protein [Lomneth et al. 1985], and vimentin [Pilkington et al. 1985]. Monospecific antisera which has been raised against a specific tumor cell line may establish a previously unrecognized demonstration of antigenic constituents shared with normal cells in culture [Lawson et al. 1985]. For example, specific surface epitopes of the embryonal carcinoma cell line PC-13 (clone 5) was shown to exist on a subset of rat DRG neurons when preparations of this tumor cell line were used for the immunization and 37 subsequent development of the monoclonal antibody 2C5 [Lawson et al. 1985]. Whole cells or crude membrane fractions have both been used successfully in the production of a variety of antibodies to intracellular and surface antigens. Perhaps the best recognized example of such a strategy comes from the use of a human neuroblastoma cell line in the development of human natural killer cell (HNK-1) monoclonal antibody. HNK-1 was originally prepared for the specific identification of a human lymphocyte subset with natural killer activity [for rev. see Porwit-Ksiazek et al. 1983a, b]. The HNK-1 antibody has also recently established itself as a cell type-specific marker for glia of the CNS [ McGarry et al. 1983, 1985a; Schuller-Petrovic etal. 1983]. The use of tumor cell cultures in the immunization and subsequent generation of antibody producing hybridomas has provided other fascinating immunolabeling characteristics. Anti F9-teratocarcinoma (SSEA-1 [Solter and Knowles 1978]), and anti-neuroblastoma ( M l / N l [Keamshed et al. 1981]) monoclonal antibodies have the capacity to recognize not only the original immunogen (as derived from their respective tumor cell line), but can also immunologically discriminate developmentally restricted expression of normal fetal antigens on cultures of whole newborn cerebellum [Kennet and Gilbert 1979] and on a subpopulation of GFAP-immunolabeled astroglia [Dickson et al. 1983], respectively. Alternatively, the antigenic relationship between tumors of neuroectodermal origin and fetal brain antigens has been corroborated in studies which show that monoclonal antibodies raised against fetal brain homogenate can also distinguish glioblastoma (medulloblastoma), and neuroblastoma (melanoma) associated antigens [Wilkstrand and Bigner 1982; Wilkstrand et al. 1982]. Thus far, the evidence appears to suggest that many if not all oncodevelopmental determinants recognized by these various monoclonal antibodies (including those recognized by SSEA-1) are a carbohydrate entity carried on the glycoproteins and glycolipids of the cell surface membrane. Most do not as yet have a well defined physiological function other than that which may be biochemically inferred [for rev. see 38 Feizi 1985]. These antibodies seem to specifically recognize gene products which have developed through the process of neoplastic transformation but which are as well normally expressed by embryonic cells as fetal antigens [Robbins, et al. 1986]. Such antibodies may in fact be able to recognize a shared neuroectodermal differentiation antigen [Wikstrand and Bigner 1982] as suggested by the high degree of cross-immunolabeling observed between cells which have embryologically originated from the neuroectoderm [De Muralt et al. 1985]. Monoclonal antibodies that discriminate many of the specificities recognized by heteroantisera in neurobiology have been produced and will probably augment or replace their corresponding heteroclonal antibody in most applications. The availability of monoclonal antibodies in neurobiology has been reviewed [McKay et al. 1982; Valentino et al. 1985; Kim 1985]. Monoclonal antibodies which can accentuate specificity, relative antibody yield, and maintain a constant standard of reactivity in many cases justifies the higher cost and labour involved in their production. In addition, the many novel antigenic determinants which stand to be uncovered by initially immunizing with crude preparations of whole brain or tumor homogenates (or a subfraction thereof), may be preferred over predefined antigen preparations. Monoclonal Antibodies to Astrocytes. Despite the availability of heteroantisera for the relatively accurate immunolabeling of the main constituents of intermediate filaments (IF) in astroglia, a closer investigation of the antigenic "anatomy" of GFAP and its distribution in the nervous system required the development of anti-GFAP monoclonal antibodies [De Bus et al. 1983]. These monospecific immunoreagents were demonstrated to recognize even the most subtle differences between the various epitopes of the GFAP molecule [Lee et al. 1984; Newcombe et al. 1986; Courel et al. 1986]. They have therefore, allowed an immunological "dissection" and subdivision of this 39 particular IF class [De Bus et al. 1983; Albrechtsen et al. 1984; Ashtstatter et al. 1986]. Monoclonal antibodies which specifically recognize GFAP, of course, also serve to differentially immunolabel astrocytes in general, from other neural cells in culture [Kim et al. 1984b]. They may also provide a method for estimating the degree of glial differentiation in specific types of neoplasia [Velasko et al. 1980, Tascos et al. 1982; Collins 1984; Ashtstratter et al. 1986]. Hybridoma technology has provided a means with which to identify the unique and common properties associated with each of the various subclasses of these intermediate cytoskeletal components [for rev. see Lazarides 1980], by discriminating between specific properties of both glial and neuronal filaments [De Bus et al. 1983; Lee et al. 1984]. The SI 00 protein apart from being a well-established component of astrocytes also shows a differential, species-dependent expressivity on oligodendrocytes as well as (though less frequently) neurons. The subunit composition of brain specific S100 protein in various neural cell types and in different animal species has not been done in detail as most investigations involve the use of heteroclonal anti-SlOO-a and anti-S100-p antisera. The recent development and use of ASA-1 (a monoclonal anti-SlOO-a antibody), to substantiate previous findings has determined that employing conventionally raised anti-SlOO antiserum may not be well advised for investigations where certain animal species are involved [Kumaniski et al. 1984]. A monoclonal antibody, P - l l , which discriminately labels the NG2 glycoprotein molecule on a subpopulation of adult neural cells (displaying both glial and neuronal properties) has been described [Stallcup 1981; Stallcup et al. 1981]. Dual immunolabeling of NG2 and GFAP in corpus callosum suggests that these cells are astrocytes. However, GFAP-positive radial glial fibers in the cerebellum do not express NG2. Moreover, a subpopulation (approximately 10%) of tetanus toxin binding neurons have also been demonstrated to express the NG2 antigen. 40 Investigations concerning the distribution of the various ganglioside classes (among both neurons and glia) encounter a significant problem in the unavailability of truly immunospecific heteroantisera. Contributing most conspicuously to these difficulties in the high degree of structural similarity which exists between the different types or classes of a given ganglioside. Most significantly, also, is a ganglioside's inability to elicit a strong immune response. A panel of 11 different hybridomas secreting individual monospecific antibody (directed against the major ganglioside G M I ) were used to illustrate the problem of varying cross-specific immunogenicity between Gjyjl a n d five other classes of ganglioside; especially Grj)iD [Mahadik et al. 1986]. This feature of cross reactivity to elements common to both G M I and Grjib can be further illustrated by the concomitant immunostaining of these gangliosides with a serum IgM M-protein obtained from patients displaying monoclonal gammopathy [Freddo et al. 1986]. The availability and use of these monosialoganglioside class-specific monoclonal antibodies should compliment biochemical and biophysical evidence suggesting quantitative differences in expression of G j ^ l and GM.4 m Quaking mice [Iwamori et al. 1985]. Recent findings suggest that the various epitopes expressed by the ganglioside Gjyjl ( ° r more specifically determinants recognized by this panel of murine monoclonal antibodies) are in most cases different from the specificities recognized by conventionally raised rabbit heteroantisera. This peculiarity of cross-reactivity with GDib no doubt contributes to the immunolabeling seen on some neurons by heteroclonal, and in even some cases, monoclonal antibodies. Cross reactivity could therefore be avoided with the use of a carefully selected monoclonal antibody. Monoclonal antibody, AbR24, is directed specifically against the ganglioside G7J3 [Pukel et al. 1982], and is useful in identifying glia in culture [Kim 1985]. It is interesting to note that previous studies have reported increased levels of this ganglioside in reactive astrocytes [Seyfried et al. 1982], as well as in tumors of neural origin [Eto and Shindo 1982]. However, only a fibroblast-like subpopulation of astrocytes (5% to 10%) immunolabel with 41 this anti-Gj_)3 monoclonal antibody [Kim et al. 1986]. In addition, all oligodendrocytes, but no neurons, demonstrate this epitope on their surface [Kim et al. 1986]. Modulation in the expression of cell type-specific gangliosides by astroglial subclasses is not peculiar to immunolabeling by AbR24 alone but can be also illustrated with the anti-C - Q l C " c l a s s monoclonal antibody F.12A2B5 [Eisenbarth et al. 1981]; more commonly known as A2B5. A2B5 was originally raised and used to specifically identify retinal neurons [Eisenbarth et al. 1979, 1981; Miller and Raff 1984]. There is a special significance, however, in using A2B5 to immunolabel these astroglial subtypes in that it has been suggested that the expression of A2B5 (in conjunction with GFAP and GalC immunolabeling) on cultures of developing [Raff et al. 1983a, b; Abney et al. 1981] and adult rat CNS cultures [Ffrench-Constant and Raff 1986] may provide a suitable model for studying glial development by helping to identify undifferentiated glial progenitor cells [Raff 1984; Raff and Miller 1984; Temple and Raff 1986]. These progenitor (0-2A) cells label with A2B5 and (depending on culture conditions) will rapidly acquire GFAP positivity to become type-2 astrocytes, or alternatively, will begin to express GalC (with a more retarded loss of A2B5 expression) to immunologically represent oligodendroglia. Differentiation of these progenitor cells does not require cell division or even the presence of other cell types [Temple and Raff 1985]. There are, however, indications that the morphological expression of these antigenic phenotypes (as demonstrated by rodent brain cells) may not pertain to the developmental patterns observed in cultures of human fetal neuroglia [Kim et al. 1986]. Monoclonal Antibodies to Neurons. A2B5, as previously mentioned, was generated against and used initially to identify retinal neurons [for rev. see Eisenbarth et al. 1981]. A2B5 is also believed to weakly react with the ganglioside Gi\\y [Kundu et al. 1983] which may (as with Grjjb) contribute to its ability to 42 immunolabel neurons in culture (see Section on tetanus toxin binding: However, its cross-reacting specificity to particular subclasses of astrocytes in white matter cultures (i.e. astroglia expressing the neuron-like phenotypic compliment of the gangliosides G Q I c and those representing the tetanus toxin receptor Gj-[\, and Gj)ib) restricts its usefulness as a neuron-specific marker per se. A2B5 expression and its use as a method for distinguishing neurons in culture, however, also seems to suffer significantly from species interdependencies [Schachner 1982a; Kim et al. 1986]. The discrepancies encountered when using A2B5 to specifically identify neurons in whole brain cultures may be largely circumvented with an antibody directed against the better defined neuron-specific class of intermediate filament. The neurofilament (NF) is also of clinical significance as changes in their subunits may account for eliciting the presence of auto-antibodies in the serum of some neuropathological conditions [Bahmauyar et al. 1983]. Unique shifts in antigenic expression may occur from the incorporation of new proteins or from post translational modifications, which in turn could account for the immunochemically defined ultrastructural development of neurofibrillary tangles [Miller et al. 1986]; the hallmark of alzheimer's disease [Gambetti et al. 1983; Hussey et al. 1986; Ksiezak-Reding and Yen 1987]. In a report by Miller et al. 1986, paired helical filaments inherent in neurofibrillary tangles were electronically purified using a cell sorter in conjunction with the discriminate immunolabelling afforded by 8D8: a monoclonal antibody which has been shown to specifically recognize these by bifilar helices. More recently, it has been reported that the A2B5 monoclonal antibody of Eisenbarth et al. 1979 [see Sec.] also recognizes neurofibrillary tangles, and as such, suggests the existance of shared specificities common to these structural elements and components expressed by human fetal neurons [Emory et al. 1987]. 43 Considering the fact that the three protein subunits which comprise normal neurofilaments are separate gene products [Czosnek et al. 1980], the only practical method for unambiguously resolving these individual constituents would be with the monospecificity afforded by monoclonal antibodies [Wood and Anderton 1981; Brown et al. 1981; De Bus et al. 1983; Lee et al 1982a, 1984; Trojanowski et al. 1986; Ksiezak-Reding and Yen 1987]. The preparation and characterization of a variety of monoclonals have unequivocally demonstrated the presence of unique and common antigenic determinants associated with each of the three distinct proteins [Carden et al. 1985; Wood et al. 1985; Lee et al. 1986a; Pelc et al. 1986], as well as with the NF proteins expressed between a variety of different species [Lee et al. 1986a; Dahl et al. 1986]. The use of monoclonal antibodies has even suggested the existence of a particular antigenic determinant shared among all intermediate filaments in general [Pruss et al. 1981]. Such naturally-occurring expression of epitopes common to the NF classes makes the validity of studies using heteroclonal antisera somewhat questionable. Newly developed panels of monoclonal antibodies have also substantiated previous findings using conventional heteroantisera for the high degree of differential distribution seen with the higher molecular weight microtubule-associated proteins [Bloom et al. 1984] and in mapping (on a molecular basis) the erythroid-spectrin related protein: fodrin [Glenney et al. 1983]. It is unfortunate that antibodies raised against the neurofilament proteins when implemented as cell type-specific markers possess the inherent disadvantage of being expressed intracytoplasmically, making their use with live cell cultures impractical. The elucidation of neuronal markers which can specifically recognize neurons and their individual subclasses appears to be a major goal in characterizing the diverse expression of antigens as elaborated by these cell types in culture. Conventional antiserum preparations, however, have yet to indicate such a sophisticated level of sensitivity. The application of 44 monoclonal antibody methodologies, on the other hand, should provide great potential for attaining this much needed degree of discrimination in cell type-specific immunolabeling [Schachner 1982b]. In a recent report by Guentert-Lauber et al. 1985, the monoclonal antibody ASCS-4 was used to immunocytochemically distinguish a normal surface protein in aggregate cultures of fetal rat telencephalon. The specificity of ACSA-4, however, awaits further characterization . The monoclonal antibodies A4 and 38/D7 are reported to distinguish between classes of neurons from CNS cultures including cerebral, cerebellar, and retinal (but not from DRG or superior cervical ganglial) neurons [Cohen and Selvendran 1981; Cohen et al. 1981], and to epitopes expressed specifically on the surface of PNS (including DRG and superior cervical ganglia, but not to any A4 positive) neurons [Vullimay et al. 1981], respectively. It is interesting to note that A4 was produced by immunizing mice from dissociated cultures of rat cerebellum, whereas the monoclonal antibody 38/D7 was generated with dissociated cultures of neonatal DRG. Moreover, while A4 seems to also be able to immunolabel a specific cell population of astrocytes in optic nerve cultures, the antigen is not expressed by mouse or guinea pig CNS cultures. Conventional [Wilson et al. 1981; Stallcup 1981; Stallcup et al. 1983] and increasing reports of monoclonal antibodies [Adinolfi and Brown 1983; Brockes 1984; Valentino et al. 1985] have been used in a wide variety of experiments to detect, characterize and monitor the development of antigens that distinguish one type of neuron from another. The hybridoma antibody used is commonly raised against a random antigen which is subsequently immunocytochemicially defined. These studies are based on the logical assumption that differences among specific functional classes of neuronal cells may be expressed in part by differences in cell surface molecules. Examples of monoclonal antibodies that distinguish between subclasses of neurons 45 include 2C5 which immunolabels some but not all small-class neurons in cultures of DRG [Lawson et al. 1985]; H9 to H12 which immunolabel neurons and/or neuronal cell structures in young rat cerebella [Ghandour et al. 1984]; 8-6A2 which can specifically label purkinje cell bodies and main dendrites (also in cerebella) [De Bias 1984]; RT97 a monoclonal anti-NF antibody [Wood and Anderton 1981] which was used in conjunction with a heteroclonal antisera to tyrosine hydroxylase, substance P, somatostatin, and histochemical determinations of acid phosphatase to provide further evidence of heterogeneity amongst the "large light" phase-contrast classification of adult rat DRG [Price 1985]; and the preliminary report of a monoclonal antibody directed to the axolemma of cultured DRG neurites of the PNS [Bigbee et al. 1986]. Other monoclonal antibodies which discriminately recognize antigens preferentially expressed on only a specific subclass of both CNS and/or PNS neurons have also been described. These include N l [Schnitzer et al. 1984], a monospecific antibody that can discriminate between subclasses of murine cerebellar and cerebral neuronal cells, yet which does nc-t label the surface of DRG, retinal, or spinal cord neurons. Expression of the reactive specificities as recognized by N l were shown to be dependent on both starting material and culture conditions [Schnitzer et al. 1984]. Using a similar strategy (of immunizing with dissociated trigeminal ganglia), Semenenko et al. 1987, in an attempt to raise a monospecific antibody to specific sensory neuron subsets, have generated the monoclonal antibody GTES2, which recognizes a novel intracellular antigen unevenly distributed among various cell types of the adult CNS including oligodendrocytes, some (but not all) GFAP 4" astrocytes, as well as certain subclasses of sensory neurons. Immunogens prepared from formalin-fixed human spinal cord and human DRG have generated the monoclonal antibodies MA-229 which immunolabels intracytoplasmically expressed determinants of human spinal cord and DRG, but not neurons of the cerebral cortex, cerebellar hemispheres, or brain stem nuclei [Antel et al. 1985]. Similar techniques have generated the monoclonal antibody 4D7 46 which has demonstrated a specificity for the developmentally regulated markers present on the surface plasma membrane of a neurite cell population [Yamamoto et. al 1986]. The monoclonal antibody, YOL/34, directed against the alpha-subunit of tubulin has also proven to be useful in ultrastructurally subclassifying neurons in the cerebellum [Cumming et al. 1983]. One final example of a monoclonal antibody (CAT-301 [Hockfield and McKay 1985]) that immunolabels many but not all pyramidal and non-pyramidal neurons in discrete areas of monkey striated cortex has been demonstrated [Hendry et al. 1984]. This previously undefined epitope has been shown to be differentially expressed even among neurons with what was considered identical morphological characteristics [Hendry et al. 1984]. There is another class of large, functionally similar sialoglycoproteins which can be found primarily (though not exclusively) on the surface of neuronal cells, but which are nevertheless considered by many investigators to be useful in the immunodiscrimination of neurons from mixed culture. This family of surface epitopes is known as cell adhesion molecules (CAM) and have been reviewed by Edelman (1983, 1985). The functional implication of these molecules, as their name suggests, appears to deal predominantly with the stabilization of intercellular contacts, and as such perform an integral part in the cellular interactions which occur during the early and subsequent development of the nervous system. Therefore, the production and use of monoclonal antibodies to these cell adhesion molecules should provide a way of immunologically characterizing the mechanisms associated with recognition between cells, and those interactions pertinent to the morphogenesis and ontogeny of the nervous system [Goridis et al. 1983; Frelinger and Rutishauser 1986; Sunshine et al. 1987; Kintner and Melton 1987]. For example, a specific subclass of cell adhesion molecule, LI [Schachner et al. 1983a; Faissner et al. 1984], may have functional significance in the migration of granule-cell neurons of mouse cerebellar cortex [Linder et al. 1983], has demonstrated a specific association to neurons, and has been shown to mediate only neuron-neuron interaction [Keilhauer et al. 1985]. Another functionally 47 similar yet immunogenically distinct glycoprotein is the N g - C A M molecule [Grumet et al. 1983, 1984]. L2 [Schachner et al. 1983b; Kruse et al. 1984] and N g - C A M surface antigens provide a useful target with which to immunocytochemically discriminate neurons in mixed culture [Him et al. 1981]. Ng-CAM, prepared from neuronal membranes, purified by 10F6 monoclonal antibody [Grumet et al. 1984], and shown to be specifically expressed on the surface of neurons, has been determined (through adhesion neutralization assay) to mediate neuronal-glial cell adhesion [Grumet et al. 1983]. Another glycoprotein possessing similar functional significance: cytotactin [Grumet et al. 1985], has been identified, isolated, and reported to resemble N g - C A M in mediating glial-neuron interactions. Unlike Ng-CAM, however, cytotactin (comprised of 220 KD, 200 KD, and 190 KD polypeptides) is not immuno-cytochemically expressed on neurons. Cytotactin, moreover, is not even exclusive to the CNS [Grumet et al. 1985]. L l has been shown to act synergistically with N - C A M [Hirn et al. 1981] in promoting cell-cell adhesion [Keilhauer et al. 1985]. This phenomenon is thought to be differentially associated to the 180 K Da component of the molecule which is believed to show greater stability through a possible spectrin-linked association with the cytoskeleton [Pollerberg et al. 1986] but not with the smaller (140 KDa and 120 KDa) and possibly even less differentiated molecular forms [Pollerberg et al. 1986] of the glycoprotein constituents [Thor et al. 1986]. The following three independently reported putative cell adhesion molecules: D2 (originally isolated from the rat) [Jorgensen and Bock 1984]; BSP-2 (from mouse brain glycolipid [Hirn et al. 1981]; and N - C A M (from the chick neural retina) [Thiery et al. 1977], have been judged to be equivalent surface antigens [Noble et al. 1985; Langley and Aunis 1986]. This is substantiated by the seemingly identical peptide maps provided by the molecular forms of their protein triplets: 180 KDa, 140 KDa, and 120 KDa [Sadoul et al. 1986]. Alternatively, the peptide maps of L l and N g - C A M display a marked dissimilarity [Noble et al. 1985]. These molecules (including MAG), however, have been shown with the use of the monoclonal 48 antibody, L2/HNK-1, to share a common carbohydrate moiety [Kruse et al. 1984; martini and Schachner 1986]. Recent ultrastructural analysis employing monoclonal antibodies (which recognize defined epitopes of the N C A M polypeptide near both its - C O O H and -NH2 terminus) suggests that binding/aggregation is represented by the occurrence of molecular associations near the -NH2 terminus [Hall and Rutishauser 1987]. Some degree of heterogeneity between the N g - C A M and the N - C A M molecules has also been suggested in studies using the monoclonal antibodies anti-N-CAM 4 and 5. These two independently derived monoclonal antibodies are said to recognize at least one common epitope expressed by the otherwise distinct molecules [Grumet et al. 1984]. On an immunocytochemical basis LI, N g - C A M and N-CAMigg can be considered as being unique neuronal-specific surface antigens [Hirn et al. 1981; Meier et al. 1982a; Valentino et al. 1985]. Expression of the N - C A M / D 2 adhesion molecule has been used to characterize bovine paraneurones [Keith and Aunis 1986] but is not specifically restricted to neurons alone; it is also demonstrable on a wide variety of the major glial cell types including astrocytes and oligodendrocytes. A possible explanation for the apparent discrepancy and surrounding controversy over the specificity expressed in immunolabeling of post-embryonic forms of N-C A M / D 2 [see Hirn et al. 1981, 1982; Noble et al. 1985] may be attributed to the use of heteroantisera that can not distinguish the subtle differences expressed by each of the three comprising elements. Other operationally similar glycoprotein molecules isolated from brain homogenates which have been demonstrated as having a role in neural cell-cell adhesion include the Jl [Kruse et al. 1985] and L2 antigens [Kruse et al. 1984]. Again, many of these molecules do not appear to be exclusively distributed on either neuronal or glial cells; thus limiting their usefulness for distinguishing between neural cell types in whole brain cultures. The Jl glycoprotein (isolated from murine membrane fractions) can be discriminated on the surface 49 of both astrocytes and oligodendrocytes, and has been shown to selectively disrupt only neuron-astrocyte adhesion [Kruse et al. 1985]. Furthermore, Jl may be recognized by the L2 monoclonal antibody but not by monoclonal antibodies which recognize specificities expressed by the myelin-associated glycoprotein (MAG); including HNK-1 [Kruse et al. 1985]. Alternatively, the L2 analyte (similarly responsible for neural cell adhesion) has been found to share a carbohydrate moiety with the glycoconjugates M A G [Kruse et al. 1984] and BSP-2 antigen [Hirn et al. 1981]. L2 expression can be immunocytochemically localized using the respective monoclonal antibody to M A G and HSB-2 as being on the surface of oligodendroglia [Kruse et al. 1984; McGarry et al. 1985b]. Monoclonal Antibodies to Oligodendroglia. The significance of unique and shared carbohydrate side-chain moieties (as recognized by these various independently generated immunoreagents) lies in what seems to be their ability to differentially modulate (between adult and embryonic forms) the adhesive affinities expressed by C A M molecules; either by acting directly as ligands for lectin-like receptors, or alternatively by stabilizing the conformational properties of the glycoprotein in question [Feizi 1985]. There has been ample demonstration of the usefulness of monoclonal antibodies in, for example, the localization of even the quantitatively minor glycoproteins of myelin (such as M A G [Quarles et al. 1983]) in immunohistological preparations of CNS tissues [Steck et al. 1983; Dobersen et al. 1985a; Eyas et al. 1985]. The use of these monoclonal reagents have proved invaluable in the discrimination of myelin specific components, and in investigations relating to the pathology of myelin [Hays et al. 1987; Rudnicki et al 1987]. There now exists several reports on the development and partial characterization of monoclonal antibodies (for example D7E10, G7C8 to C6E11, and F7F7) which recognize 50 specific analytes expressed by M A G [Miller et al. 1984; Dobersen et al. 1985a; Nishizawa et al. 1986; Ilyas et al. 1986]. Generation of monospecific anti-MAG antibody (C6B5) has uncovered expression of an as yet uncharacterized antigenic component shared between M A G and the principal protein constituent of myelin: myelin basic protein (MBP) [Miller et al. 1984]. Other monoclonal antibodies which recognize M A G have also served to support earlier evidence [Hirn et al. 1981, 1982] of a similar immunoreactivity between specific carbohydrate epitopes associated with particular glycosphingolipids of the CNS and PNS [Ernerudh et al. 1986; Ilyas et al. 1986; Jonsson et al. 1987]. Although M A G is not itself encephalitogenic (as is MBP), it is highly immunogenic and is believed to play an important role in the normal processes and pathology of myelin formation [Quarles 1983; Gendelman et al. 1985; Dal Canto and Barbano 1986]. Abnormal expression of M A G polypeptides have been reported in the dismyelinating nervous system of quaking [Trapp et al. 1984], jimpy [Frail and Braun 1985], and trembler [Inuzuka et al. 1985] mice. Other evidence implicating M A G as a probable primary target molecule comes from investigations of a certain pathological condition known as "benign" paraproteinemia [Hays et al. 1987; Jonsson et al. 1987; Rudnicki et al. 1987; Kusunoki et al. 1987]. These patients present a disproportionately high incidence of peripheral neuropathy; a complication which is now largely attributed to specifically generated anti-MAG IgM "monoclonal" autoantibodies [Kahn et al. 1985; Ernerudh et al. 1986]. These autoantibodies are also known as paraproteins, myeloma or M-proteins, and cryoglobulins. Myelin is an integral part of nervous tissue which has unique function, composition, and structure [Golds and Braun 1976]. These characteristics make it most suitable as a subject of neuroimmunological investigations. Monoclonal antibodies which have been raised against oligodendroglial-specific surface and intracytoplasmic antigens are regularly directed against the oligodendroglial plasma membrane; namely myelin and its comprising elements. Of the components which make up the CNS lipid fraction, approximately 20% to 25% are cholesterols, 40% to 45% are phospholipids, and the final 27% to 30% are 51 galactosphingolipids [Norton 1981]. In spite of the fact that no lipid is singularly specific to myelin, immunohisto- and cytochemical investigations indicate that antibody directed against galactocerebroside (galactosylceramide), considered to be the most myelin-typical lipid, specifically labels the oligodendrocyte-myelin unit in mixed CNS cultures [Raff et al. 1978b; Kim et al. 1984a, 1985], as well as whole brain sections [Dubois et al. 1979]. However, general inconsistencies in titer, questionable specificity, and limited supply of the polyclonal antibody has prompted several investigators to independently generate monoclonal antibody against this surface glycolipid [Sommer and Schachner 1981; Ranscht et al. 1982; Rostami et al. 1984]. Although GalC is a comparatively minor glycolipid, it seems to possess a high degree of immunogenicity. For example, Sommer and Schachner (1981) produced a panel (Ol to 04) of monoclonal antibodies (and subsequently 05 to O i l [Kettenmann et al. 1985]) to white matter obtained from bovine corpus callosum. Three of these (Ol, 02 and 07) reacted with glycolipids corresponding to GalC on a thin layer chromatogram. In a similar study, Ranscht et al. 1982 developed an IgG3 class monoclonal antibody to GalC, yet the immunizing agent was a preparation of synaptic plasma membranes originating from bovine hippocampus. The explanation offered for how a monoclonal antibody that was essentially generated against the axolemma (but which instead specifically recognizes galactocerebroside) was that the true immunogen was most likely a glial contaminant and not necessarily an analyte common to both the synaptic plasma membrane preparation and a myelin membrane component [Ranscht et al. 1982]. Finally, the generation and biological properties of a hybridoma cell line reportedly producing monospecific antibody to GalC (using purified bovine lower spot cerebroside) has been reported [Rostami et al. 1984], but unfortunately has since stopped producing antibody [Kim, personal communication]. These monospecific antibodies to GalC have already provided useful information concerning the developmental expression [Schachner et al. 1981; Ranscht et al. 1982], and functional relevance [Kettenmann 5 2 et al. 1985] o f th is i m p o r t a n t m y e l i n const i tuent . T h e s p e c i f i c i t y o f a s e c o n d , l e s s c o m m o n g a l a c t o l i p i d : s u l p h a t i d e , o r su l faga lac tosy lce ramide (a genera l class o f cerebroside su l fur ic esters) to the o l i g o d e n d r o g l i a l -m y e l i n u n i t i s s o m e w h a t less ce r t a in . N e v e r t h e l e s s , m o n o c l o n a l a n t i b o d i e s 0 3 to 0 6 [ S o m m e r a n d Schachner 1981; K e t t e n m a n n et al. 1985] w h i c h have been s h o w n to spec i f ica l ly c o r r e s p o n d to g l y c o l i p i d s c o - m i g r a t i n g w i t h s u l p h a t i d e s o n a t h i n - l a y e r c h r o m a t o g r a m [ S c h a c h n e r 1982a] , h a v e p r o v e d u s e f u l i n d e m o n s t r a t i n g the r e l a t i v e d e v e l o p m e n t a l e x p r e s s i o n o f su lpha t i de s w i t h respect to G a l C i n ea r ly (1 to 5 D I V ) cu l tu res of e m b r y o n i c m o u s e b r a i n [Schachner et al. 1981; S o m m e r a n d Schachner 1982]. O f less quan t i t a t i ve s ign i f i cance is the g l y c o s y l c e r a m i d e - d e r i v e d s i a l i c - a c i d b e a r i n g g l y c o s p h i n g o l i p i d s (gangl ios ides) w h i c h d i s p l a y a u n i q u e d i s t r i b u t i o n i n m y e l i n as c o m p a r e d to that o f w h o l e b r a i n g a n g l i o s i d e s . T h e g a n g l i o s i d e w h i c h seems m o s t r e l i a b l e for i m m u n o c y t o c h e m i c a l l y d i s c r i m i n a t i n g b e t w e e n h u m a n o l i g o d e n d r o c y t e s (adu l t o r fetal), fe tal neu rons , a n d to a large extent a d u l t o r fetal astrocytes i n cu l tu re o r t h i n sec t ion , i s Grj)3 [ K i m et al. 1986]. T w o other classes of gang l io s ide , Gpyj i / a n d the o n l y ga lac tocerebros ide-d e r i v e d g a n g l i o s i d e , G M 4 ( s i a l o sy l g a l a c t o s y l c e r a m i d e ; a l so k n o w n as Gy), h a v e been d e m o n s t r a t e d o n the surface of fetal a n d a d u l t h u m a n o l i g o d e n d r o c y t e s [ K i m et al. 1986]. H o w e v e r , an t i se ra w h i c h i m m u n o l a b e l s G j y j l is not l i m i t e d to o n l y th is n e u r a l c e l l type a n d its m y e l i n p l a s m a m e m b r a n e [ A s o u et al. 1985]. M o r e o v e r , recent d e s c r i p t i o n s of G]yj4 e x p r e s s i o n b y a m a j o r i t y o f b o t h c u l t u r e d fe ta l a n d a d u l t h u m a n as t rocy tes seems to con t rad ic t ear l ie r ev idence w h i c h sugges ted that Gjyi4 express ion w a s res t r ic ted o n l y to the p l a s m a m e m b r a n e s of o l i g o d e n d r o c y t e s [ Y u a n d I q b a l 1979]. T h e re la t ive concent ra t ions of g a n g l i o s i d e i n m y e l i n o n l y a m o u n t s to 0.3% to 0.7% o f the t o t a l l i p i d i n th i s f r ac t ion [ C o c h r a n et al. 1982]. T h e subsequent p u r i f i c a t i o n o f these gang l io s ide s , therefore, for the p r o d u c t i o n o f h e t e r o c l o n a l a n t i s e r u m is v e r y t i m e c o n s u m i n g . A n anti-Grj>3 p r o d u c i n g 53 clone of the hybridoma AbR24 [Pukel et al. 1982] has circumvented many of the problems associated with the use of heteroclonal antisera, and thus should better facilitate investigations concerning the underlying mechanisms associated with reactive gliosis [Yu et al. 1982] and neuroectodermal differentiation [Goldman et al. 1984]. A panel of monoclonal antibodies which recognizes both unique [Deibler et al. 1986] and overlapping [Carnegie et al. 1983, 1985] structural sequences of myelin basic protein (MBP) [Hruby et al. 1985; Groome et al. 1985; Carnegie et al. 1983; Carnegie and Dowse 1983; Fritz and Chou, 1983; Sires et al 1981; Alvord et al. 1986; Chou et al. 1986], have made it possible to "immunodissect" and subsequently investigate cross species conservation and distribution of not only the encephalitogenic peptide but also of other sequences expressed in normal as well as in pathological CNS tissue samples [Sires et al. 1981; Carnegie et al. 1983; Miller et al. 1984; Brenner et al. 1986b]. The number of individual antigenic specificities associated with the basic protein as determined by conventional and monospecific antibodies may be as high as 27(+), and appear to define at least eleven separate regions of the molecule [for rev. see Day and Potter 1986]. To better understand the processes involved in the immunopathology of myelin and even for the potential diagnosis of these diseases, high purity MBP is required [Tigyl et al 1984]. Despite the many different purification procedures employing conventionally raised heteroantisera for the isolation of MBP, most suffer from variability and incompleteness [Kolehmainen et al. 1984]. Quite often the purified encephalitogenic protein is contaminated with other proteins making standardization almost impossible [Day and Potter 1986]. The invariability and high degree of specificity realized with the use of anti-MBP monoclonal antibodies on the other hand provides the opportunity to prepare extremely pure myelin [Dowse et al. 1983; Tigyl et al. 1984]. Pure MBP should in turn contribute to a better understanding of the events involved with the normal processes of myelination as well as in helping to subsequently determine how deficiencies in MBP expression can lead to a dismyelinating condition [Zimmerman and Cohen 1979; Kim 54 and Pleasure 1980; Kimura et al 1985]. The exquisite specificity of binding provided by monoclonal antibody to PLP can also prove superior in establishing the functional relationship between myelin membrane proteins and their lipids [Anthony and Moscarello 1971; Papahadjopoulos et al 1975; Boggs and Moscarello 1978b; Boggs et al 1980a, b], as well as in affording a better understanding of how these and other membrane constituents can influence the underlying mechanisms associated with normal myelination [Trotter et al 1984a; Bizzozero and Pasquini 1984] and its pathology [Boggs et al 1978b; Boggs and Moscarello 1980; Trotter et al. 1984b]. Dautigny et al. 1986 have recently generated a complimentary-DNA probe (P-23) which corresponds to a PLP gene transcript and can therefore provide essential information about the activities involved with PLP-synthesis [Dautigny et al 1986]. Development of a monoclonal antibody to this intracellular marker, however, has yet to be reported. A largely heterogenous group of high molecular weight proteins (which are insoluble in both aqueous and organic solvents) provide the final 15% to 20% of the total protein fraction in myelin [Norton 1981]. Within this classification are included several of the glycoproteins (including MAG) [see Sec.], as well as the so-called Wolfgram proteins [Wolfgram 1966]. There are two predominant forms of Wolfgram proteins, designated W l and W2. However, as many as 15 to 20 individual bands have been resolved using polyacrylamide gel electrophoresis [Fisher et al 1974; Morell et al. 1972] thus contributing to much of the confusion in the literature concerning the nomenclature of these proteins [Batteiger et al 1982]. It has been suggested that W l and W2 differ only in their conformational states and that the two proteins possess a closely related amino acid composition. Subsequent studies, however, have failed to demonstrate any immunological similarity between W l and W2 [Sprinkle et al. 1980b]. The high molecular weight component of this family of protein, W2, was demonstrated to co-migrate specifically with 55 tubulin [Gozes and Richter-Landsberg 1978], whereas the lower band of the Wolfram protein doublet, W l , was shown to possess corresponding electrophoretic mobility, amino acid composition, and immunogenic characteristics to that of the myelin-specific enzyme 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) [Sprinkle et al. 1980b]. Further investigations have shown that polyclonal antiserum raised to CNPase immunolabels both CNS and PNS myelin [Sprinkle et al. 1983], suggesting common antigenic determinants expressed by both peripheral and central myelin. The recent implementation of independently developed monoclonal antibodies in studying the distribution of CNPase in the CNS [Fujishiro et al. 1986; Brenner et al. 1986a] substantiates the hypothesis that W l and W2 are immunologically distinct myelin proteins and that the protein components of W l (Wla and Wlb) are for the most part [Fujishiro et al. 1986] immunogenically indistinguishable [Sprinkle et al. 1983]. In addition, no expression of central CNPase was immunohistochemically, or immunocytochemically demonstrable in either prefixed bovine siatic nerve sections [Fujishiro et al. 1986] or in cultures of neonatal rat DRG [Brenner et al. 1986a], respectively. These findings appear to support the belief that this myelin specific enzyme exists as two immunologically distinct molecular forms in the CNS and PNS [Matthieu 1986]. 1.6 Tissue Culture in Neurobiology. I have already stressed that the ability to accurately identify and subsequently purify whole populations of neural cells will help to circumvent the obstacles in recognizing and investigating many of the physical constituents and specific biochemical activities associated with each neural cell type [see Sec. 1.5.1]. The most fundamental method with which to examine the overwhelming number of intra- and intercellular interactions as they occur in situ [Hendelman et al. 1985], or in mixed whole brain cultures [Kim 1986], would be to simplify the system at the level of initial or primary culture [Varon 1977]. Studies on 56 nervous tissue cell markers in vitro are generally performed on dissociated cultures which can now be easily prepared using standard technique. Cell counting and visualization of individual cells is much easier in single cell suspensions and monolayer cultures than in tissue explants [Kim 1986]. Tissue culture technology has been adopted into many scientific disciplines and has become an integral part of neurobiology. A major advantage of this technology resides in the ability to easily manipulate the physical, chemical (pH, temperature, osmotic pressure, CO2 tension), and the physiological conditions of a given culture. There is no easier method with which to investigate intracellular activity (protein synthesis and energy metabolism), intracellular flux (modulation of receptor complexes and in metabolism), cell-cell interactions (adhesion, recognition, population kinetics), and "cellular ecology" (nutrition, pharmacology, infection, transformation) than in a dissociated culture-system [Blank et al. 1974; Allt and Ghabriel 1982; Henderson et al. 1984]. Many recently implemented modifications in tissue culture methodologies have been developed where the selective pressure of a culture condition in conjunction with specific methods of preparation differentially favour the survival of one particular neural cell type over that of another. These selective tissue culture methods, in addition to using cell type-specific immunolabeling techniques, have helped to circumvent the problems associated with studying neural culture samples that are invariably heterogenous; where even replicates from a single source display variability in the cells that constitute it [Varon 1977]. The use of specific culture conditions and methods of culture preparation can help to enrich and subsequently characterize a given neural cell type. There is a final, special significance also realized when using these improved techniques of providing enriched glial and neuronal preparations, in that such cultures are well-suited for use in the immunocytochemical assay of hybridoma culture supernatants [Furst and Mahowald 1984]. The preparation and cellular composition of cerebral hemisphere primary cultures 57 have been reviewed [Varon 1977; Raff et al. 1979; Hansson 1984; Bologa 1985]. Primary cultures of dissociated whole neonatal or newborn brain readily provide viable neurons, astrocytes, and oligodendrocytes [Hansson 1984; Saneto and de Vellis 1985; Bologa 1985]. These primary mixed cultures have therefore been used extensively in (for example) developmental investigations concerning neuronal and glial proliferation, as well as for aiding in their morphological and functional differentiation [Bhat et al 1981; Bologa et al. 1982; Hirayama et al. 1984; Meller and Waelsch 1984; Hirano et al. 1985; Eccleson and Silberberg 1985; Bologa 1985; Rome et al. 1986; Wernicke and Volpe 1986; Bhat and Pfeiffer 1986]. Identification of the temporal and spacial expression of the neural cell type-specific markers including the myelin associated components: GalC [Hirayama et al. 1984; Bhat and Pfeiffer 1986], sulfatide [Abney et al. 1981; Bologa-Sandru et al. 1981], MBP [Barbarese and Pfeiffer 1981; Sternberger et al. 1978; Meller and Waelsch 1984; Rome et al. 1986], M A G [Sternberger et al. 1979; Quarles 1983], N2 [Hartman et al. 1982], carbonic anhydrase C [Ghandour et al. 1980; Cammer et al. 1985], glycerol phosphate dehydrogenase (GPDH) [Vernadakis et al. 1984; Montz et al. 1985] and CNPase [McMorris 1983; Hirano et al. 1985; Bansal and Pfeiffer 1985]; studies of GFAP expression in astrocytes [Vernadakis et al. 1984; Meller et al. 1984]; as well as neurofilament protein [Lee et al. 1986a] and neuron-specific enolase (NSE) [Bologa 1985] for identifying neurons in developing cultures, will lend greater insight into the physical and biochemical milieu which optimize neural cell growth and function [Pruss et al. 1982; Eccleston and Silberberg 1985; Merril et al. 1984]. In other words, culturing dissociated CNS tissue not only serves to verify and quantitate a proliferative response, it also provides a means with which to morphologically identify, classify [Meller and Waelsch 1984; Vernadakis et al. 1984; Hirayama et al. 1984; Bansal and Pfeiffer, 1985], and subsequently separate the various neural cell classes present in a mixed primary culture [Poduslo et al. 1985; Bhat and Pfeiffer 1986; Rome et al. 1986]. A simple manipulation of culture conditions as described by McCarthy and de Vellis (1980), for example, is based on a process of neural cell stratification which occurs naturally in mixed cultures of astrocytes and 58 oligodendrocytes, and the selective detachment and harvest of the overlying oligodendroglia in response to a given shear force or shaking. This technique has been successfully used by many investigators for the preparation of separate oligodendroglial and astroglial cultures from whole mixed primary cultures of mammalian brain [Pettmann et al. 1982; Merril et al. 1984; Saneto and de Vellis 1985; Poduslo et al. 1985; Bhat and Brunngraber 1986; Bhat and Pfeiffer 1986]. 1.6.1 Preparation and Maintenance of Astrocytes. It is desirable to isolate and culture the astrocyte (both its fibrous and protoplasmic subtype) as there is increasing evidence suggesting that these glia modify the neuronal microenvironment. Astrocytes are known, for example, to play an active role in regulating the levels of adenosine [Lewin and Black 1979], as well as in preventing excess build-up of potassium in the brain [Hertz and Chaban 1982]. It is also well established that astrocytes possess binding sites for a variety of neurotransmitters (nor adrenaline [NA], dopamine [DOPA] and serotonin [5HT]), that they participate in specific uptake, turnover and metabolism of the neurotransmitter y-aminobutyric acid (GABA) [Schubert 1975] and glutamate (GA) [McGeer and McGeer 1981; Juurlink 1982; Katz and Kimelberg 1985; Pearce et al. 1986], and that the enzyme responsible for this, glutamine synthetase (an astrocyte specific marker), is also responsible for ammonia detoxification in the brain [Yudkoff et al. 1983]. Most exciting, however, is the recent evidence which strongly infers that astroglia are potent immune regulating cells [Frank et al. 1986; Takiguchi and Frelinger 1986; Fontana et al. 1986; Sun and Wekerle 1986; Schnyder et al. 1986; Pulver et al. 1987]. For example, astrocytes seem to functionally resemble accessory cells [Traugott and Raine 1985; Stefansson et al. 1986; Fontana et al. 1986] in their ability to induce proliferation of natural killer (NK)/cytotoxic T-cells and T-helper cells through the release of interleukin (IL)-l [Muller et 59 al. 1985; Yates et al. 1985; Fontana et al. 1986; Schnyder et al. 1986]. Also, human and murine astrocyte cultures, which have been previously reported as normally expressing only very low levels of the major histocompatibility (MHC) class antigens (I and II [or Ia]) [Vitetta et al. 1978; Williams et al. 1980; K i m et al 1985; Schnyder et al 1986], (or to not express them at all [Lisak et al. 1983]), have been recently shown to elaborate the immune-response gene product (Ia) on their surface after exposure to the specific T-cell lymphokine: y-interferon (IFN-y) [Vitetta et al. 1978 Hirsch et al. 1983; Wong et al 1984; D u Bois et al 1985; Tedeschi et al 1986; Takiguchi and Frelinger 1986]. Induction of Ia expression in these neural cells has been reported as well in response to virus particles [Suzumura et al. 1986; Massa et al 1986; Massa and Meulen 1987] and/or bacterial adjuvants [Massa and Meulen 1987]. Furthermore, class II M H C antigens have even been demonstrated in significantly higher frequencies on astrocytes obtained from the various lesion areas of multiple sclerosis (MS) brain [Traugott et al. 1985] and within the gliosis surrounding metastases and abscesses [Frank et al. 1986]. Investigation of the neuronal-glial and oligo-astroglial interactions, as they occur in mixed primary culture, first requires a better understanding of individual properties associated with each of the neuronal, oligodendroglial, and astroglial populations. The system could, therefore, be simplified by studying the various neural cell constituents in separate culture systems. Ironically enough the methods used to establish astroglial-enriched cultures are an extension of the techniques used for selectively isolating oligodendrocytes from enzymatically digested whole white matter [McCarthy and de Vellis 1980; K i m et al. 1985]. In some culture systems, astrocyte proliferation (even if only a few percent of growing astrocytes exist at the time of initial isolation and plating) will with time eventually overwhelm the oligodendroglia. It may be possible to use these demonstrated differences in g r o w t h characteristics to selectively kill proliferating astroglial cells thereby enriching the relative 6 0 numbers of oligodendroglia in culture. 1.6.2 Preparation and Maintenance of Oligodendrocytes. The myelin sheath, produced and maintained by the oligodendrocyte, is in fact a modification or extension of its plasma membrane [Raine 1981; Norton 1983]. The ability to isolating pure or enriched populations of oligodendrocytes, therefore, is an essential pre-requisite in achieving a better understanding of the various stages and events involved in the myelinating [Ludwin 1981; Poduslo et a/.1982b] and demyelinating [Billings-Gagliardi et al. 1984; Silberberg et al. 1984] processes. Studies have shown that newborn and adult mammalian oligodendrocytes can be isolated [Farooq et al. 1981; Poduslo et al. 1982a; Brammer 1984; Kohsaka et al. 1986], and even maintained in culture for extended periods of time [Gebicke-Harter et al. 1981; Szuchet et al. 1980, 1983; Bhat et al. 1981; Hnrayama et al. 1983; Kim et al. 1983; Norton et al. 1983; Suzumura et al. 1984; Smyrnis et al. 1986]. Current techniques for the preparation of enriched cultures of oligodendrocytes (apart from the shaking method of McCarthy and de Vellis), involves the use of density gradients prepared from a variety of material including: Percoll (polyvinylpirrolidone coated silica gel) [Lisak et al. 1981; Gonatas et al. 1982; Hirayama et al. 1983; Lubetzki-Korn et al. 1983; Kim et al. 1983; Wood and Bunge 1986a], sucrose [Farooq et al. 1981; Poduslo et al. 1982a, b], and more recently, density gradients produced from metrizamide [Doering and Fedoroff 1984]. These preparative techniques for specific cell isolation and enrichment will yield almost pure cultures of oligodendrocytes. It has been suggested, however, that a tendency exists for residual contaminating astrocytes (a by-product of these enrichment processes), with time to totally overgrow the non-dividing oligodendrocytes in certain long term culture systems, keeping in mind the paucity of mitosis by these myelin producing cells in culture [Latov et al. 1979; Lisak et al. 1981]. It may therefore be necessary to supplement the culture media with antimitotic agents. Cytosine arabinoside, for example, is a commonly used selective agent for 61 the differential destruction of replication-competent cells in tissue culture. This latter method of course, has the notable disadvantage of the loss, rather than the segregation, of the vulnerable cell population. The use of anti-mitotic agents, on the other hand, may not even be required when dealing with certain mammalian neural culture systems. In human mixed glial cultures, for example, astrocytes have been described as being unable to undergo division [Kim et al. 1983]. Differential destruction of specific neural cell populations may also be achieved by using cell surface-specific antibodies in eliciting a complement-mediated cell lysis [Varon 1977]. However, another more straight forward method for the preferential destruction of a particular cell type may accomplished by simply withholding an essential nutrient or media supplement requisite for the survival of a given population of cell (yet not necessarily needed by a second cell type) even though both cells may have originated from a common precursor. 1.6.3 Preparation and Maintenance of Neurons from Dorsal Root Ganglia (DRG). Disaggregated cultures of dorsal root ganglia provide to the investigator a mixed source of Schwann cells (the myelinating cell of the PNS, and sister cell to the oligodendrocyte of the CNS), neurons, as well as some fibroblasts [Fields et al. 1978; Shahar 1983]. These mixed cultures may also be further separated into highly enriched populations of individual cell-types by simply manipulating culture conditions [Salzer and Bunge 1980; Kreider et al. 1982]. For example, withholding nerve growth factor (NGF) from DRG cultures will lead to the obtention of a purified population of Schwann cells [Varon 1977], as sensory root neurons will not survive for any extended period of time in culture without it [for rev. see Okun 1972]. Alternatively, differential adhesion may also be used to isolate Schwann cells from sciatic nerve [Kreider et al. 1982]. On the other hand, supplementing the culture media with NGF as well as with the antimitotic agent fluorodeoxyuridine (Fudr) [Sobol et al. 1986], eliminates most non-neuronal cells while permitting the non-dividing neurons to 62 survive. A good recent example of the use of carefully selected starting material, primary isolation techniques, subsequent culture conditions, and differential cell destruction come from a study by Wood and Bunge [1986b]. Here, Percoll isolated adult oligodendrocytes were added to enriched cultures of D R G neurons. Oligodendrocytes (in some cases) were then eliminated using an antibody-dependent complement-mediated cell lysis. From their fundings the investigators were able to propose that there exists a requirement for GalC 4 " cells (oligodendrocytes) in order for neuronal myelination to occur. The considerable increase in the number of publications dealing with the techniques of neural culture and the methods by which each individual cell type can be identified, indicates a growing recognition of the value that these methodological approaches offer neurobiological investigations. The ability to isolate pure or enriched populations of neural cells substantially contributes to the simplification of the complexities associated with investigations concerning the nervous system. The development of cell type-specific immunoreagents further accentuates this simplification by providing a means with which to enrich or isolate specific neural cell subpopulations and thus allow greater insight into a cell's individual morphological, functional, and developmental characteristics. It also becomes evident, as in the present study, that the advantages offered by these recent advances in tissue culture methodologies wi l l serve to increase the generation and characterization of novel cell type-specific monoclonal antibodies by supplying specific sources of immunogen as well as providing the means with which to screen hybridoma culture supernatants. These markers will prove to be instrumental in analyzing cell lineage relationships where morphological criteria alone was previously insufficient . 63 2 MATERIALS A N D METHODS 2.1 Selection of a Suitable Myeloma Cell Line. In the present study the mouse myeloma cell line: P3-NSl-Ag4-l (NS-1), was purchased from the American Type Culture Collection (Rockville, MA). NS-1, a non-secreting clone of P3X63-Ag8, as derived from the P3K cell line established from the mineral oil induced plasmacytoma MOPC-21 [Horibata and Harris, 1970] (see Sec. 1.1). NS-1 were initiated from stock culture stored frozen in 80% fetal calf serum (FCS) 10% dimethyl sulfoxide (DMSO) in Dulbeco's modified Eagles medium (DMEM) at -220° C. After washing twice in Hanks Balance Salt Solution (HBSS, Grand Islands Biologicals Company, GIBCO) to remove residual DMSO, NS-1 cells were resuspended to a final concentration of approximately 1x10^ cells/ml and thereafter routinely maintained in D M E M supplemented with 5 mg/ml glucose, 1.0 mM sodium pyruvate, 40.3 m M sodium bicarbonate, 2 m M L-glutamine, 100 [ig/m\ streptomycin, and 100 IU/ml penicillin: DMEM(+) -10% FCS. Cultures were incubated in a humid atmosphere at 37° C with 10% CO2. Requirements for feeding and subculture was based on changes in pH, cell concentration, and cellular morphology. Once an exponential stage of growth was attained by the myeloma cell line, feeding and subculture was usually necessary every two days. Five days preceding the actual fusion, 5 x 10^  NS-1 cells were transferred to a sterile 250 ml double-side arm tissue culture flask equipped with a suspended stir bar (VWR Sci., CA) containing 100 ml of "gassed" DMEM(+) and 10% FCS. Gassing of media was performed by either directly blowing a 10% CO2 mixture over the DMEM(+) for 2 to 3 min., or by placing the flask containing the culture media into a 10% CO2 atmosphere (as contained in our culture incubator) for at least 6 hours. Once transferred, flasks were sealed with Parafilm, 64 placed on a magnetic stirrer (at a setting of 60 to 100 rpm) and allowed to incubate at 37° C in a warm room or alternatively in a shaking waterbath. Cultures were intermittently monitored to ensure that there would be adequate numbers of exponentially growing NS-1 cells available for fusion. Cultures propagated in suspension offered various advantages (for example: in providing media and in sampling of the cells) not realized with growing these cells in a "monolayer" system. 2.1.1 Myeloma Selectivity to HAT. It is essential that the parent myeloma cell line express a specific drug marker sensitivity to allow for the selective elimination of any myeloma cells which have not successfully fused to a given splenocyte. One of the most important characteristics of the myeloma cell line used in the present study was its resistance to azaguanine (or thioguanine) and bromodeoxyuridine, representing a deficiency in the enzymes HGPRT and TK, respectively. These NS-1 cells do not possess the means for de novo biosynthesis of purines and pyrimidines, and as well, do not have the ability to utilize artificially supplemented exogenous nucleosides when a dihydrofolate reductase antagonist (aminopterin) has been added to the growth medium. In order to ensure the successful elimination of any non-fused parent myeloma cells from our original hybridoma cultures, the efficacy of selectivity to HAT(+) medium (containing 0.1 mM hypoxanthine, 4.0 x 10"2 mM aminopterin, and 1.6 mM thymidine) was periodically tested on small aliquots sampled from our batch cultures. If cytotoxicity in HAT(+) (as determined by trypan blue exclusion) was not apparent within 48 h after plating, the culture was suspected of having reverted (i.e. HGPRT"1") and of again possessing functional nucleotide salvage pathways. 65 2.2.2 Selectivity to Azaguanine. On indication that the parent myeloma cell line displayed signs of mutational reversion (i.e. H A T resistance), sensitivity to a cytotoxic purine nucleotide analogue was determined. 8-azaguanine (Sigma, MO) was added to DMEM(+)-10% FCS, and fed to cultures at a final concentration of 2.5 u,g/ml. Cells were then allowed to grow with periodic feeding and subculturing. Myeloma cultures surviving 8-azaguanine treatment must have necessarily been deficient in the enzyme HGPRT and again could be selected against using H A T containing meduim. If cell death after azaguanine treatment was overwhelming, surviving cells were enriched over non-viable cells by using ficoll density gradient centrifugation. Culture suspensions diluted 1:1 in HBSS were carefully layerd onto a half-volume of ficoll (Pharmacia, Sweden) and the gradients centrifuged at 400 g for 10 min at 4 ° C. The resulting viable cell fraction was then carefully pipetted off and washed twice with HBSS (to remove residual ficoll) resuspended, and subsequently maintained in DMEM(+)-10% FCS. 22 Preparation and Characterization of the Immunogen. 2.2.1 Preparation of the Human Axolemma-Enriched Fraction. In the present study, isolation of an axolemma-enriched fraction was based on the method of De Vries 1981, and was used with only minor modification [Fig. 2.1]. Whole sections of temporal lobe from a human 73-year-old female was obtained at autopsy, 3 h post mortem and frozen in 30 gm portions at -70° C until needed. A preliminary dissection to remove the larger blood vessels and meninges was performed prior to freezing. Samples of H I G H S P E E D C E N T R I F U G A T I O N Fig. 2.1: Preparation of a Human axolemma-enriched (HAx) fraction m p from post-mortem material. Samples, once applied to the sucrose gradients, were collected from the 8.0/1.0 and 1.0/1.2 surface interface. These samples were then pooled and purified using a second density gradient centrifugation. Purified HAx was then analyzed for protein content and saved for subsequent SDS-PAGE characterization. 67 frozen white matter were then aseptically dissected from the gray material while on a chilled sterile petri dish. 2 gm (wet weight) portions of this myelinated material was then finely minced using sterile scalpels and quickly transferred to one of two 40 ml Dounce homogenizers (Kontes glass, NJ) each containing approximately 40 ml of freshly prepared flotation media. Flotation media consisted of ice-cold 0.85 M sucrose, 0.15 M NaCI, and 10 m M TES (N-tris[hydroxymethyl](methyl-2-aminoethanesulfonic acid) [Sigma, MO] at pH 7.5. Minced white matter was then homogenized using 10 complete up and down strokes of the loose (B) pestle of the homogenizer while on ice. Care was taken to dislodge any particles which tended to adhere to the sides of the preparation vessel. Approximately 20 ml of homogenate was then aliquoted equally amongst 2 Oakridge polyallomer tubes (Falcon, Becton Dickinson, CA) and centrifuged for 30 min at 15,000 rpm in a Beckman high-speed refrigerated centrifuge using a fixed-angle JA-17 rotor. Following centrifugation the floating layer of myelinated axons was carefully removed using a pre-wetted spatula and added to a second 40 ml aliquot of flotation media. Homogenization and centrifugation was then repeated. Following the second centrifugation a small aliquot of purified myelinated axons was taken for phase microscopy and checked for the absence of capillaries and cell nuclei. If these components were still apparent, a third flotation step was performed. Once purification of the floating fraction was complete, myelin was osmotically stripped away from its axolemma by homogenizing the material in 37 ml of ice-cold hypotonic (10 mM) TES buffer (pH 7.5) using 10 complete up and down strokes of the tight (A) pestle. Osmotically shocked myelinated axons were again centrifuged at 15,000 rpm for 30 min at 4 ° C. Combined pellets were subsequently resuspended in 20 ml of 0.75 M sucrose containing 1 mM TES and 1 mM EGTA (ethylene glycol bis [b-aminoethyl ether]N, N'-tetraacetic acid) [Sigma, MO] at p H 7.5. 68 4 ml aliquots of the shocked myelinated axons from 2 gm of white matter in 20 ml of the 0.75 M sucrose media was then carefully applied to five discontinuous sucrose density gradients pre-constructed in 13 ml nitrocellulose ultracentrifuge tubes (Beckman, CA). Gradients consisted of 2 ml of 1.2 M sucrose overlaid with 3 ml of 1.0 M sucrose and finally superlayered with 4 ml of 0.8 M sucrose. All sucrose solutions contained 1 m M EGTA and 1 mM TES at p H of 7.5. Gradients were kept on ice until their transfer to a swing-bucket SW-27 rotor and centrifuged at 25,000 rpm for 60 min. at 4 ° C using a Beckman model LA55 ultracentrifuge. The myelin was found to be uniformally distributed throughout the 0.75M sucrose fraction and easily removed with careful aspiration. After centrifugation, pellets constituting the myelin-enriched fraction were resuspended in a given volume of 0.25M sucrose-10 mM TES (pH 7.5) and frozen at -70° C for future study. The axolemma-enriched fractions have been reported to collect at both the 0.8 /1.0 M and 1.0 /1.2 M interface [De Vries 1981]. Each fraction was collected and similar fractions pooled, diluted with two volumes of 10 m M TES (pH 7.5), and centrifuged in the SW-27 (25,000 rpm for 30 min at 4 ° C). Similar pellets, reported by De Vries to be cross-contaminated with one or the other axolemma-enriched fraction, required additional purification using further discontinuous sucrose density gradient centrifugation. Each fraction was therefore resuspended in 8 ml of 1.0 M sucrose and 4 ml superlayered onto 4 ml of 1.42 M sucrose solution. This in turn was overlaid with 4 ml 0.8 M sucrose. Again, all sucrose solutions contained 1 m M TES and EGTA (pH 7.5). The second discontinuous sucrose density gradient was centrifuged for 30 min at 25,000 rpm and 4° C. Similar fractions were pooled and added to two volumes of 10 m M TES. Preparations were then centrifuged (as above) and resuspended in 2 ml of 0.25 M sucrose/0.01 M TES (pH 7.5). Small samples were subsequently taken to determine the protein concentrations of the individual fractions. 69 Aliquots of 200 u.1 each of axolemma fraction were distributed to microeppendorf tubes and frozen at -20° C. 2.2.2 Preparation of Human Newborn Brain Particulate Fraction The particulate fraction used for intrasplenic injection was produced by the method of Ghandour et al. 1984, and is illustrated in Fig. 2.2. Whole human newborn brain was obtained approximately 12 h post mortem. The larger blood vessels and meninges were first removed from the tissue with sterile forceps before 4 gm (wet-weight) were transferred to an ice-cooled glass petri dish. Brain material was finely minced, transferred to a chilled 40 ml Dounce homogenizer containing 10 m M (1:10 w/v) Tris([hydroxymethyl]aminomethane)-HC1 buffer (pH 7.4) and subsequently homogenized using 10 full strokes of the loose (B) pestle. One volume of freshly prepared 0.64 M sucrose was then added to the homogenate and the mixture centrifuged at 12,000 g for 20 min at 4 ° C. The resulting supernatant now largely devoid of crude nuclear fraction, capillaries, and debris, was carefully collected and re-centrifuged at 32,000 rpm for 60 min at 4 ° C using a TY-60 fixed angle rotor. Resulting pellets of human newborn cerebellar particulate fraction were resuspended, pooled, and washed twice in a similar buffer. The protein concentration of our particulate fraction were determined using the method of Lowry et al. 1951, and adjusted to give 1 mg protein/ml. Material was then dispensed as 200 u,l aliquots, transferred to microeppendorf tubes and frozen (-20° C) until use with our enzyme linked immunosorbent screening assay (ELISA), or alternatively for sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis fractionation and characterization. R c CO c > B^ C c — — * 7&- :*•*•• -ft: •Vi"-: •: • Fig. 2.2: Preparation of human newborn brain particulate fraction. Once tissue is minced and homogenized, differential centrifugation is used to obtain a particulate fraction. SDS-PAGE subfractionation, excission of selected protein bands, and concentration of dialysed preparations are then used for subsequent intrasplenic immunization and biochemisrty. 71 On the basis of our results [see Sec. 3.1.1] and those previously published [De Vries 1981], the majority of the axolemma-enriched protein fraction was determined to have a relative molecular weight of between 45,000 Da to 60,000 Da. To increase the probability of producing a monoclonal antibody specific for these molecular weight proteins it was decided to subfractionate and subsequently isolate various newborn brain particulate fractions using SDS-PAGE. 2.2.3 Preparation of Bovine Oligodendrocytes (BOL). The methods used for the bulk preparation and subsequent enrichment of adult bovine oligodendrocytes (BOL) have been discussed in detail in the sections concerning the preparation of disaggregated neural cell cultures [see Sec. 2.11.1]. After re-seeding primary BOL cultures, aliquots of oligodendrocyte-enriched suspensions (approximately 6 x 10^  cells) were gently removed by scrapping two to three plates with a rubber policeman and washed in two changes of HBSS (1,200 rpm for 7 min at 4 ° C). Cells were then resuspended in phosphate buffered saline (PBS), pH 7.5, for a final volume of 100 jxl and immediately used for intrasplenic injection. The remainder of this material was saved and used for subsequent SDS-PAGE characterization. 2.2.4 SDS-Polyacrylamide Electrophoresis Subfractionation and Characterization. Discontinuous 10% polyacrylamide gels were prepared according to the methods of Laemmli 1970, with only minor modification. Briefly, stock solutions of 30% (w/v) acrylamide (Biorad, CA) and 0.8% (w/v) of Bis (N, N'-methylenebisacrylamide) [Fisher,NJ] were combined in 3.75 M Tris-HCl (pH 8.8) and 0.1% SDS to produce a 10% discontinuous running (separating) gel. Chemical polymerization of this mixture was catalyzed with the 72 with 50 |il to 100 u,l of a 10% ammonium persulphate solution (Biorad, CA). A Biorad model-220 slab-gel electrophoresis apparatus-clone was used to prepare the completed polyacrylamide (180 mm x 120 mm x 2 mm) gel. Contamination of gels by exogenous protein was largely circumvented through the exhaustive cleaning of the glass casting plates. Seams were carefully sealed with both 2% agar in DH2O (w/v) and Parafilm to prevent both leaks and air bubbles. The gel once poured was carefully overlaid with 5 ml of DH2O to prevent exposure to air and allowed to polymerize overnight. The following day, the water layer was removed with syringe and filter paper, overlaid with a 5% acrylamide stacking gel and the comb inserted. The stacking gel was also allowed at least 2 h for complete polymerization to take place. Newborn brain particulate fraction was combined with an equal volume of sample buffer containing 2% SDS, 5% 2-mercaptoethanol (Eastman Kodak, NY), 62.5 mM Tris-HCL (pH 6.8), as well as 10% glycerol and 0.001% bromphenol blue tracking dye to visualize the migrating front of the sample mixture. The proteins of both the particulate fraction and of the molecular weight standards (prepared for electrophoresis in a similar manner) were disassociated by first immersing the sample in boiling water for 3 min followed by sonication using a Bransonic-12 ultrasonic cleaner (Branson, CT) for 15 to 30 seconds. Al l residual aggregates where then removed by centrifugation in a microfuge (Eppendorf, UK) for 3 minutes. Samples were then carefully transferred with a 1 ml syringe and a 27 g needle, through the running buffer (containing 25. mM Tris, 192 m M glycine, and 0.1% SDS at p H 8.8) and applied to a pre-formed 10 mm x 120 mm x 2 mm sample trough as evenly as possible. The molecular weight of the proteins comprising the particulate fraction were determined with the concomitant separation of molecular weight standards (Pharmacia, Sweden). The electrophoresis calibration kit (for the determination of proteins of low molecular weight) contained phosphorylase-B (94,000 Da), bovine serum albumin (BSA) (67,000), ovalbumin (43,000), carbonic-anhydrase (30,000), soybean trypsin inhibitor (20,100) 73 and a-lactalbumin (14,400). Each vial containing a total of 578 mg total protein was prepared for electrophoresis (as described for particulate fraction) in 1 ml of sample buffer. 15 ul were then measured and applied to either side of the stacking gel with a Hamilton syringe. Once loaded, the electrophoresis apparatus was transferred to a cold room (4° C.) where it was coupled to an electrophoresis constant power supply ECPS-3000/150 (Pharmacia, Sweden). Electrophoresis was carried out initially at a current of 5 mA for a time interval necessary for the tracking dye to migrate to the stacking /separating gel interface (usually about 1 h). At this point the current was increased to 10 mA and the sample run at this amperage until the tracking marker came within approximately 10 mm from the bottom of the gel (approximately 8 h). Once electrophoresis was complete, the gel slab was removed and the borders containing the molecular weight standards (and at least 10 mm into the region containing the electrophoresed particulate fraction) were excised and transferred to a staining chamber containing a 0.5125% Coomasie brilliant blue solution mixed 2:3 (v/v) in a methanol/acetic acid (5:1) fixative for 12 h at 37° C with constant shaking. Gel sections were subsequently destained in a model-556 gel destainer (Biorad, CA) with 3 to 4 changes of a 1:1 (v/v) 50% methanol 14% acetic acid solution. Once adequately destained (usually 6 h) the gel sections were placed back onto one of the glass casting plates and the edges of the gel aligned to that of the original unstained running gel. The glass plate was then placed onto a piece of graph paper and positioned over a light box. Recovery of Protein From Gel Excised Subfractions. Regions of the gel-containing electrophoresed particulate fraction best corresponding to where the major protein constituents of an axolemma-enriched fraction would exist (as determined by the molecular weight standards, the banding pattern of the particulate 74 fraction itself, and previous results of SDS-PAGE of axolemma-enriched fractions) were then carefully cut out for the entire width of the separating gel and transferred (as small pieces) to four 10 mm x 100 mm test tubes each containing 5 ml cold C a + + Mg + + - free phosphate buffered saline (CMF-PBS). Square sections of (approximately 46 mm x 46 mm) spectrapor membrane tubing (Spectrum Medical Ind., LA) of 6,000 to 8,000 molecular weight cut-off were secured over the ends of the test tubes. Tubes were then inverted and the protein containing gel dialyzed against CMF-PBS overnight in a cold room (4° C) with stirring and at least two changes of dialysis buffer. Dialysis membrane tubing was boiled for 20 min and rinsed in several changes of deionized water before use to denature contaminating proteolytic enzymes sometimes associated with dialysis tubing. Once dialysis was complete, concentration of the protein fractions leeched from the gel excised areas was determined [see Sec. 2.2.5]. Dialysed protein fractions were subsequently concentrated lOOx, down to 50u.l, in approximately two hours. Once concentration was complete, 5 u,g to 10 fig of these proteins were again electrophoresed (along with an equivalent amount of axolemma-enriched fraction), and the sample best approximating the major proteins of this fraction subsequently used as immunogen. The excised protein subfractions were re-electrophoresed on a 10% polyacrylamide gel. However, a smaller format (80 mm x 60 mm x 1 mm) PAGE microsystem (Marysol Ind., Japan) was used as only 5 |ig protein aliquots and a shorter running time of 2 h were required. The protein-binding pattern was visualized using Coomasie brilliant blue as previously described. Gels needed for future reference were dried onto filter paper using a model-334 slab gel drier (Biorad, CA) at the 80° C setting for 4 h under vacuum. 75 2.2.5 Determination of Protein Concentration. The protein concentration of our newborn brain particulate fraction and of our bovine oligodendroglial preparations were extrapolated from a calibration curve produced with serially diluted bovine serum albumin (BSA) protein standard and subsequent colourimetric development by either a modified Lowry, or protein dye-binding assay. Use of the Lowry Method. The first method used for determining the protein concentration of our samples was based on Petersons' modification [Peterson 1977] of the micro-Lowry technique [Lowry et al. 1951] which utilizes SDS to facilitate the dissolution of relatively insoluble lipoproteins from both of our sample preparations. The reagents used in the micro-Lowry assay were all supplied and purchased in kit form (Sigma, MO). A problem existed with the procedure, however, in that substances that were routinely used in the preparation of our antigens (for example Tris, EGTA, sucrose, and ammonium sulphate) interfered with the direct Lowry procedure [Bradford 1976], necessitating the precipitation of the protein with sodium deoxycholate (DOC) and trichloroacetic acid (TCA) prior to testing. Briefly, a standard curve using BSA at 50 M-g/ml to 40 Ug/ml was prepared in an appropriate number of plastic microcentrifuge tubes. To each tube (including our unknowns) was then added 15 ng of an aqueous DOC solution, after which samples were thoroughly mixed and allowed to stand at room temperature for 10 minutes. Incubation was followed with the addition of a l/10th volume of 72% stock T C A solution. Protein precipitates were subsequently collected by centrifugation at 12,000 rpm for 10 min to 15 min and the supernatants discarded. Each pellet was in turn dissolved into 1 ml of modified Lowry reagent solution, then mixed with an equal volume of water and allowed to stand at room temperature for at least 20 minutes. 76 Using a 2 N working solution of Folin-Ciocalteu's phenol reagent, 0.5 ml was then quickly added to each sample and thoroughly mixed. The subsequent development of colour (usually after 30 min) was assessed by absorbance (A750) as measured by a Beckman model-34 spectrophotometer. Use of the Protein Dye Binding Method. Alternatively, total protein quantitation was determined using the principles of a protein-dye binding method as described by Bradford, 1976. A given volume of protein assay dye-reagent concentrate (Biorad, CA) was carefully added to four volumes of both a BSA protein standard (Sigma, MO) of predetermined concentration (usually between 5 (i.g to 20 u,g quantities) as well as to a given concentration of our protein solution (undiluted, diluted 1:10 and diluted 1:100). The amounts of protein standard plotted against the corresponding absorbance (A595) was used to generate a standard curve and quantitate the protein in our unknown samples. Because the binding process is very rapid and the complexes dispersal in solution relatively stable, samples were usually read within five minutes (and up to an hour) after mixing. 23 Immunization Protocol. 23.1 Concentration of Immunogen Prior to Characterization and Immunization. Large volumes (up to 5 ml) of protein samples needing concentration (such as after dialysis) were first prefiltered through Whatman No 1 filter paper (London, England) and the macrosolute content of the sample determined [see previous section]. Dialyzed protein subfractions were then concentrated lOOx and volumes reduced to 50 |4.1 using a B15 Minicon clinical sample concentrator (Amicon, MA) rated at 15,000 molecular weight rejection/cutoff. 77 The concentration itself would take from 200 to 480 minutes. After the samples had reached the desired volume , they were removed from the concentration cell with a pasteur pipette and subsequently used for further SDS-PAGE characterization and intrasplenic immunization. 23.2 Intrasplenic Injection. The method used in the present study for inducing sensitization of murine splenocytes through intrasplenic immunization was based on the methods of Spitz et al. 1984. Gel-excised newborn brain particulate subfractions and bovine oligodendroglial cell culture in PBS were adjusted to give volumes of between 50 yl and 100 ul containing 20 |Ag of protein and 2 to 2.5 x 10^  live cells, respectively. Prior to injection Balb/C mice were weighed and anesthetized with an intraperitoneal injection of 6 ul to 7 (xl of a 5 m g / m l nembutal-saline solution per gram of body weight. In some cases intermittent use of anesthetic ether was also necessary. The animal was placed on its left side and shaved over the general area surrounding the lower edge of the facing rib-cage. This was then swabbed with 70% alcohol and a 1.5 cm to 2 cm long incision made over the area of the spleen (seen through the skin as a darkened strip 3 mm x 6 mm), with a pair of sterile straight-edge scissors. Forceps were then used to gently pull at the peritoneum and (taking care not to cut the larger blood vessels) another 1.5 cm to 2 cm long incision made to expose the dark-red coloured organ. The spleen was then exteriorized by gently lifting and pulling its lower pole with a second pair of sterile forceps. A 27 g needle fitted to a 1 cc turberculin syringe was then used to inoculate the antigen preparation by inserting the needle deeply into the spleen (at an acute angle) and delivering the immunogen as the needle was withdrawn. At least two separate sites were injected to ensure adequate introduction and distribution of the immunogen throughout most of the organ. The inoculum consisted of a 50 u,l bolus of 2 to 2.5 x 106 whole living BOL, or 50 u.1 to 100 pi of 20 u.g of SDS-PAGE subfractionated human 78 newborn brain particulate fraction. After injection the spleen was carefully returned into the peritoneal cavity and the peritoneal and muscular walls stitched back together. The incised borders of the skin were then approximated and sewn together, again, with 3 to 4 stitches using No 4-0 prolene (Ethicon, ONT) non-absorbable surgical suture. Cell fusions were performed on the fourth day after intrasplenic immunization. 2.4 Fusion Protocol. The fusion procedure used for generating hybridomas was based largely on the methods described by Galfre et al. 1977, with only minor modification. Basically, one day prior to fusion, cultures of P3-NS-l-Ag4-l (NS-1) plasmacytoma cells were adjusted to provide at least 1x10^ cells (at least 95% viability) necessary for fusion. 2x 100 ml ahquots of DMEM(+) (one containing 15% fetal calf serum) and 4x 10 ml aliquots DMEM(+) (2 containing 15% fetal calf serum) were allowed, along with a freshly prepared polyethylene glycol (PEG) solution [see sec. 2.4.3] to equilibriate with the atmospheric conditions contained by a 10% C02/37 0 C humidified culture incubator (for at least 6 h) prior to cell fusion. 2.4.1 Preparation of Sensitized Murine Splenocytes. Dissection of the Spleen. Immunized mice were sacrificed by etherization four days after intrasplenic injection. Animals were then sterilized by dipping in 70% ethanol and the spleen dissected out using sterile instruments in an area used only for the preparation of primary cultures. Once removed, the spleen was briefly washed by placing it into a sterile petri dish containing a few ml of DMEM(+) (without FCS) and transferred to a sterile hood for further processing. 79 The spleen was cleaned of any attached membranes and placed into a fresh 75 mm dish containing 10 ml gassed DMEM(+)-7.5% FCS (i.e. 5 ml each of DMEM(+)-15% FCS and DMEM(+)). The petri dish was then inclined by resting an edge on its lid, and the spleen brought to the highest level of the medium. While holding the spleen using blunt sterile forceps, the organ was then nicked with a pair of curved scissors at its opposite pole and its contents gently teased out by rubbing the outer capsule. This was continued until essentially all material contained within the fibrous capsule was released into the suspension media. The capsule was then discarded and any existing clumps dissociated into single cells by alternatively drawing and expelling the suspension through the opening of a 10 cc syringe (not fitted with a needle) held loosely against the bottom surface of the sterile dish. This procedure was repeated 9 to 10 times [Fig. 2.3]. Separation of a Plastic-Adherent Cell Population. The separation and removal of a plastic adherent cell population is based on the observations of Van Mourik et al. 1984. Basically, plastic adherent cells were separated from murine lymphocytes by transferring the splenocyte suspension into a 25 cc tissue culture flask (Corning, VWR, CA) and incubating it for at least 1 h at 37° C. Once differential adherence was complete, non-adherent cells were harvested by gently agitating the flask and then transferring the subsequent cell suspension to a graduated conical 15 ml centrifuge tube. In addition, a small aliquot was also taken and mixed 1:1 with 0.1% trypan blue to determine cell concentration and viability. Both splenocyte and myeloma cell preparations were subsequently washed at least 2x in gassed, serum-free DMEM(+) with centrifugation for 10 min at 1,200 rpm using a table top centrifuge. At this time a special aliquot of 2.4 x 10^  cells was put into a 15 ml tissue culture tube and the volume brought up to 48 ml for subsequent use as a feeder layer. Fig. 2.3: The methods used for intrasplenic injection and the subsequent isolation of sensitized splenocytes. Splenocytes were sensitized by directly injecting the spleen with antigen. Hybridomas were then prepared using an enriched suspension of a non-plastic adherent cell population. An aliquot of the splenocyte suspension was also saved for use as a feeder layer and subsequently added to the newly formed hybrids 81 2.4.2 Lymphocyte to Myeloma Ratio. Lymphocyte and NS-1 cell pellets were resuspended in serum-free DMEM(+) at a concentration of 2 x 10? and 4 to 5 x 10.6 cells/ml, respectively. 10 ml of each cell suspension was then added together in a 15 ml conical centrifuge tube (at a ratio of approximately 1 NS-1 cell to 10 splenocytes), inverted several times to mix, and centrifuged at 1,200 rpm for 7 min at room temperature 2.4.3 The Preparation and Use of Polyethylene glycol (PEG). Polyethylene glycol 6,000 (Fisher, NJ) was prepared by first autoclaving the fusogen for 20 min (115 lbs per sq in), and then allowing it to cool to approximately 75 to 80° C, at which time D M E M was mixed with the now liquid PEG to provide a 50% working solution (w/v). The refinement of adding DMEM(+) at double concentration to autoclaved PEG was not considered necessary [Campbell 1986]. The supernatant of the NS-1/splenocyte mixture was carefully removed by aspiration and 1.0 ml of freshly prepared 50% PEG 6,000 in serum-free DMEM(+) (w/v) added over a period of one minute with thorough mixing. To this suspension, a series of 1 ml, 4 ml, and 5 ml aliquots of serum-free DMEM(+) were added in drop-wise fashion over a 1 min, 2 min and again 2 min interval, respectively. PEG was removed from our cell suspensions by centrifuging samples at 400 g for 7 min at room temperature and discarding the supernatants. Cell pellets were then resuspended in 3 to 4 ml of DMEM(+)-15% FCS, slowly, so as to not disrupt the newly formed hybrids. The suspension volume was then increased and the cells carefully transferred to 24 well Limbro tissue culture plates (Flow, VA). Cultures were observed under the low power of a phase microscope to approximate the efficacy of the newly prepared fusion. The preparation of a splenocyte suspension for use in the technique of hybridization are outlined in Fig. 2.3. 82 2.4.4 Fusion Frequencies and Plating Densities. Hybridization frequencies will vary with the nature and purity of the lymphocyte preparation used, as well as with the ratio at which the cells are partnered to the NS-1 cell suspension [Goding 1986]. However, frequencies which fell in the range of 1 to 100 clones per 1x10? splenocytes were achieved. Hybrids were plated in 2 cc (3.5 ml capacity) 24-flat-bottom well Limbo tissue culture plates, regardless of the success of a fusion. Initial plating densities were maintained for all fusions at approximately 2.25 x 10^ total cells per well. Therefore, a typical fusion would require 2x 24-well plates. 2.5 Cell Culture Requirements for Hybridomas. 2.5.1 Basic Culture Requirements. Dulbeco's modification of Eagles' medium (DMEM) when purchased from Flow was supplemented with (as in our NS-1 cultures) 0.45 u.m filter-sterilized (Gellman, MI) 40.3 mM sodium bicarbonate, 1.0 m M sodium pyruvate, 2 mM L-glutamine, 5 mg/ml sucrose. 100 IU/ml penicillin and 100 M-g/ml streptomycin was also used to inhibit the growth of most gram-positive and negative bacteria, respectively. Amphotericin B (fungizone) (GIBCO) at 2.5 Ug/ml was used but only where contamination by fungus was suspected. Fetal Calf Serum. Fetal calf serum (FCS [GIBCO]) was used at an initial concentration of 15% and was heat inactivated at 56° C for 30 min prior to use. Concentrations of FCS were subsequently decreased step-wise to 10% in DMEM(+) as hybridomas displayed stability in growth. The 83 need for testing the suitability of FCS and of having an adequate frozen stock at hand is an absolute necessity. The Use of H A T Media. After fusion, hybridomas were generally allowed 2 to 3 days to recover before undergoing selection against non-fused cells. Selection was performed with DMEM(+)-15% FCS supplemented with 4 x 10"2 m M aminopterin (Flow, VA) (the minimal effective dose), 1.6 m M thymidine and 0.1 m M hypoxanthine. Once H A T was introduced into the culture medium, cells were inspected daily to confirm massive cell death expected of all cell types not successfully fused. Cultures were not fed again until there was signs of cell growth. If cell death was not apparent within four days after fusion, aminopterin (which is itself very unstable), was prepared and used fresh. Aminopterin, in addition to having properties which block folic acid synthesis (and so in turn prevent de novo nucleotide biosynthesis) can also inhibit other distinct metabolic pathways necessary for the synthesis of some amino acids. It was therefore necessary to ensure that the media used had incorporated into it whichever amino acids (for example glycine) which may have been affected. Use of HT Media. Aminopterin, for the reasons mentioned in the previous section, was removed from the culture media relatively soon (several weeks) after fusion. However, 0.1 mM hypoxanthine and 1.6 mM thymidine were still added to the culture medium for at least 2 weeks after aminopterin removal to allow cells to re-adapt to using their main purine and pyrimidine biosynthetic pathways. Cell cultures were subsequently maintained in DMEM(+)-15% FCS. 84 Feeding of hybridoma cell cultures was based on the colour of the indicator dye present in the feeding media and judged necessary every second to third day. Once microscopically visible colonies appeared in the culture wells (usually 10 to 15 days post-fusion) culture supernatants were assayed for desired antibody activity. 2.6 Initial Screening of Hybridoma Culture Supernatants. In terms of clonal growth, the time of assay was soon after clones could be seen under the microscope and again a few days later when clones became visible to the unaided eye. 2.6.1 Immunochemical determination of antibody activity. In all cases where hybridoma supernatants were assayed for antibody activity (using indirect immunofluorescence on various disaggregated cell cultures) saturated ammonium sulphate was used to precipitate and subsequently concentrate antibody contained in culture supernatants (from 5x to lOx) prior to their application. Concentration of Culture Supernatant by Ammonium Sulphate Precipitation. The method of immunoglobulin concentration using ammonium sulfate precipita-tion has been reviewed by Heide and Schwick 1978, and was used with little modification. Briefly, saturated ammonium sulphate solution was prepared by adding approximately 500 gm (NH4)S04 (Fisher, NJ) to 500 ml D H 2 O at 50° C with stirring until most is dissolved. The 45% saturated solution (percent by weight at room temperature) was then allowed to sit overnight and the p H adjusted (if desired) using ammonia or alternatively sulphuric acid. 85 Supernatants to be concentrated were transferred in 800 |xl aliquots to eppendorf tubes and combined with 700 ul of the saturated ammonium sulphate solution. Because the protein solution was very dilute, ammonium sulphate solution was added all at once. After admixture, proteins were allowed to salt out of solution for at least 1 h at room temperature or overnight at 4 ° C. Temperature proved not to be of critical influence. Once precipitation was complete, samples were centrifuged in a microfuge (12,000 rpm for 15 to 20 min at 4° C) where-upon supernatants were removed by careful aspiration, discarded, and the pellet resuspended in 80 |il to 160 ul of PBS. The tops were removed from the eppendorf tubes and dialysis of the sample performed essentially as described for our gel excised particulate fractions [see Sec.]. With such small volumes, however, it was necessary to "flick" the sample to overcome surface tension to draw the fluid down to the membrane after inverting the tube, and then placing it in CMF-PBS (4° C) to be dialyzed. Once dialysis was complete, the samples were capped and subsequently stored at 4 ° C until used for immunocytochemistry. Indirect Immunocytochemistry. Indirect immunofluorescence, based on Raff's modification of Coons' classical methodology [Coons 1941], was used for both suspended and attached cells using the cell-type specific markers previously described [Raff et al. 1979]. Reagents were diluted in HBSS with 0.1% (w/v) sodium azide: HBSS(+). Pre-incubating with 10% (v/v) normal goat serum in HBSS(+) was also employed to block the possible existence of Fc receptors [Traugott et al. 1979; Ma et al. 1981; Oger et al. 1982]. Cells were treated in rabbit anti-GalC serum diluted in HBSS(+) (1:40) and/or the concentrated (5x to lOx) monoclonal samples for 30 min on ice. Cultures were then washed twice in HBSS(+), followed by incubating in 50 u.1 of fluorescein or rhodamine-coupled goat anti-rabbit, rat, or goat anti-mouse immunoglobulin-F(ab')2 (1:40) for 30 min at 4° C. In double-labeling experiments, equal volumes of both primary and 86 subsequently secondary fluorochrome-coupled antibodies were added simultaneously at double (1:20) the normal concentration. Incubations involving the use of fluorochrome-coupled secondary antibodies was done in the dark to prevent photobleaching [Giloh et al. 1982]. After three changes of washing, the cells were fixed with 5% acetic acid/95% ethanol (v/v) at -20° C for 10 min, washed, and mounted in polyvinyl alcohol (PVA) media [Valnes and Brandtzaeg 1985]. PVA (Sigma type II) was mixed 1:4 (w/v) with a 1:8 (v/v) solution of glycerol in 25 m M Tris buffer (pH 9.0). In experiments involving the intracytoplasmic marker GFAP (used to unambiguously immunolabel astrocytes [Bignami et al. 1972 ]), cultures were pre-fixed in acid-alcohol before incubation in anti-GFAP (1:40) antiserum. The respective fluorophore-immunoconjugate was subsequently introduced as described above. Enzyme-linked Immunosorbant Assay (ELISA). Briefly, affinity purified antigen-specific goat anti-mouse IgG and IgM (whole molecule) alkaline phosphatase conjugate (Sigma, MO) at 1:1,000 in PBS-Tween and phosphatase substrate (Sigma 104 tablets: disodium p-nitrophenol phosphate dissolved in diethanolamine buffer) was used in the ELISA assay. ELISA was performed in rigid "U" bottom-based immunolon-I 96-well microtitre plates (Dynatech Lab, Sussex) in which all assay reagents were used at a volume of 100 ul/well. Frozen antigen stock was thawed and diluted in 0.05M carbonate buffer (pH 9.6) for a final concentration of 1 ug /ml, after which it was distributed to an appropriate number of microtiter plates and allowed to incubate covered and sealed overnight at 4° C. Plates were stored like this for up to 5 days prior to use. PBS-tween was used to wash off unbound antigen before a blocking agent (FCS) was added (usually at 1:100 to saturate all remaining non-specific binding sites) for 1 h at room temperature. The plates were again rinsed three times in PBS-tween and the hybridoma supernatants to be tested (added neat, or diluted 1:10 to 1:100) were applied. Test-samples were subsequently washed off after an incubation of at least 1 h at room temperature (or 87 overnight at 4 ° C) before the enzyme-immunoconjugate was introduced using an 8-channel multipipettor (Titertech, Flow Labs, VA). Once added, plates were stored in the dark for 1 h at room temperature, after which they were washed with PBS-tween to remove non-specifically bound enzyme. Bound phosphatase-conjugate was detected by adding enzyme substrate dissolved in 0.1 M diethanolamine buffer (pH 9.8) and allowed to undergo hydrolysis in the dark for 10 to 15 minutes. Absorbance (A405) was determined using a Dynatech model-MR600 microplate reader with initial readings being adjusted to zero using unreacted substrate preparations. Absorbances were recorded and stored by microcomputer. Antisera. Rabbit antibodies were raised against purified bovine GalC following the procedure as described by Fry et al. 1976. Rat anti-GFAP antisera was also prepared in our lab. Culture medium from our hybridoma cell lines was harvested and used as monoclonal antibody. NS-1 myeloma culture medium was used as a negative control for our various monoclonal antibodies. Rhodamine and fluorescein-coupled goat anti-rabbit or anti-rat immuno-globulin F(ab')2 fragments were purchased [Cappel Labs, PA]. All antiserum was centrifuged for 2 to 3 min at 12,000 rpm before use to ensure removal of any existing aggregates. 2.7 Handling of Productive fusion Products,. 2.7.1 In Vitro Expansion of Hybridoma Colonies. Hybridomas, initially seeded into 24-well tissue culture plates, were maintained at 37° C in a humidified 10%-CO2 atmosphere. Subculturing of "primary" hybridoma colonies into a second set of 24 well culture plates was necessary to ensure against the loss of culture due to accidental contamination. 88 2.7.2 Plating, Cell-Freezing and Recovery of Hybridomas. In instances where fusion produced an overwhelming number of hybridomas, fusion products were frozen so as to distribute labour and permit a more thorough assessment of antibody production. Freezing (-70° C) and thawing of hybrids was based on the methods as described by Patel and Brown 1984, and Harwell et al. 1984, and used with only minor modification. Briefly, once the fusate had reach a density of approximately 1 x 10*> cells/ml, cells were gently resuspended by pipette and the bulk of the cell suspension transferred to a sterile 75 mm x 100 mm tissue culture tube. Cells were then centrifuged at low speed (usually 1,000 rpm) for five min at 4 ° C. Supernatants were then set aside for further characterization and the cells resuspended in 0.5 ml cold DMEM(+)-80% FCS, and transferred to pre-cooled freezing vials (Nunc, Denmark). 0.5 ml of a 20% solution of sterile dimethyl sulfoxide (DMSO) in serum-free DMEM(+) (v/v) was combined with the cell suspension and served as cryoprotectant. Cells were then frozen using an intermittent rapid cooling where cells are first taken to a holding temperature of -20° C (for overnight) before being transferred to -70° C (again for incubation overnight) and subsequently to storage in liquid nitrogen (-220° C). Cells were recovered when needed by rapid thawing and subsequent distribution into 10 volumes of HBSS. Suspension were centrifuged (1,000 rpm for 5 min at 5 ° C), supernatants discarded, and the cell pellet resuspended for culture in 1ml to 2 ml of DMEM(+)-15% FCS. H A T or HT was added (depending at which stage hybridomas were taken for freezing) 2 to 3 days after thawing by changing half the culture volume and renewing it as it became necessary. 2.7.3 Cloning of Antibody Producing Hybridomas. During the various stages in the cloning procedure, hybridomas were frozen down. There are several hypotheses which advocate the need for cloning as early as possible once 89 hybridomas begin to demonstrate stability in culture [see Discussion]. This need is usually associated with the potential of non-secreting hybrids to overgrow those, actively producing and secreting antibodies. These cells have a tetraploid complement of chromosomes and as such must invest more energy into the replication of their DNA. In the present study, three different approaches were used .for the isolation of various individual antibody producing clones, including: cloning by micro-manipulation, limiting dilution, methyl-cellulose, or a combination thereof. The continued use of a feeder layer was not considered necessary once hybridomas indicated signs of tolerance to subculturing and expansion in vitro. Manual Microscopic Isolation of Hybridomas. The use of micro-manipulative techniques to clone hybridomas has been described [Zaguri et al. 1981; Sijens et al. 1983] and forms the basis of our adaptation of this particular method. "Early" immunopositive hybridoma culture wells were observed under the low power objective of an inverted microscope and individually monitored on a daily basis. The microscope was scrupulously cleaned with alcohol and placed into a sterile hood prior to use. Selection of visible colonies was done under the 20x objective using a pulled (Orifice at an approximate diameter of 100 |xm) sterile cotton-plugged pasteur pipette with rubber bulb. The pipette was angled near its tapered end to allow for easy access into the culture well, pre-wetted in sterile media to lessen cell adherence, and washed by pipetting in several changes of sterile media to prevent cross contamination of potentially different hybridomas. Clones were slowly expanded over a period of days to weeks (depending on a clone's individual growth characteristics), frozen, and was the method used to handle early fusion products when a fusion appeared to be "too successful". The use of microscopic selection would also constitute an integral part of the procedure employed for cloning in methylcellulose [see following section]. 90 Cloning in Methylcellulose. A more labour intensive method for the manual selection of visible (or even microscopic) colonies involved plating of a hybridoma cell suspension appropriately diluted in methylcellulose-containing medium. Briefly, 2.2 gm of methylcellulose (Fisher, NJ) was mixed with ct-medium (Terry Fox Labs, Vancouver) containing 7.5% soduim bicarbonate and 10% BSA to give a final volume of 50 ml. This methycellulose-containing stock solution (MCM) was kept refrigerated until required. Two to three days prior to cloning, M C M was supplemented with 2 mM freshly prepared L-glutamine (GIBCO), 1 x 10~2 mM 2-mercapto-ethanol (Eastman Kodak, NY), antibiotics, 15% FCS and the volume made up to 100 ml to give a final concentration of 1.1% methylcellulose (w/v); MCM(+). 3 ml aliquots of MCM(+) were dispensed into an appropriate number of 12 x 75 mm sterile tissue culture tubes covered with loose fitting lids and transferred into an incubator to equilibriate with the 10%-CO2 atmosphere. Productive, stable suspensions of hybridomas were tested on the day of cloning for viability and to determine cell concentration. An appropriate number of cells were then removed from each sample and added with thorough mixing to one of two tubes containing MCM(+) to give a final concentration of 1 x 10^ and 1 x 10 4 cells/tube. Samples were subsequently dispensed into one of two 30 mm sterile plastic petri dishes for each hybridoma cloned. Both dishes were then placed, along with a third dish (containing 3 ml of sterile water), into a 75 mm sterile plastic petri dish. The water-containing petri dish was used to prevent the evaporation of MCM(+). The lid of the larger plate was then used to cover the smaller dishes within, and the final assembly transferred to and maintained in a 10%-CO2 incubator . Cultures in MCM(+) were monitored periodically for growth. Visible single colonies of cells (clones) were aspirated out of the semi-solid media with a pulled sterile pasture pipette and transferred to individual wells of a 24-well Limbro plates containing 1 ml of DMEM(+) per well. Antibody production was evaluated after approximately 7 to 10 days post-cloning [Fig. 2.4]. 92 Fig. 2.4: The use of methyl cellulose for cloning. Hybridomas displaying desired immunolabeling activity are mixed at various dilutions (1:100 to 1:1000) in methyl cellulose to allow for easy selection once growth becomes apparent. The cell suspensions are then dispensed into small petri dishes which are in turn placed inside a larger dish. The larger dish contains an additional plate which itself contains sterile water necessary to prevent evaporation of the cell suspensions. The assembly is then placed into a culture chamber and the hybrids allowed to grow. Individual colonies are then selected manually with a "pulled" pasture pipete under the low power objective of an inverted microscope. These clones are subcultured and subsequently re-evaluated for antibody activity. 93 Cloning by Limiting Dilution. The method of cloning hybridomas by limiting dilution has been described [Michele et al. 1980] and used here with little modification. Two to three days prior to cloning and recloning, DMEM(+)-15% FCS was dispensed into an appropriate number of 96 well tissue culture plates (Limbro, Flow Labs, VA) using a 8-channel multipipettor. 100 ul volumes per well were used (except in the first and last columns which contained 200 ul and nothing, respectively. The media contained by the culture plates was then allowed to equilibriate inside a 10%-CC»2 incubator. Cells within the positive wells were gently resuspended using a micropipettor. From this suspension, approximately 2.0 x 10^ cells were removed and transferred to the bottom well in the first column of the 96-well plate. 100 ul was then removed from this well and transferred to the next well up the column. In this manner, the number of hybridomas in the first column was serially reduced as the top of the plate was approached. In a similar fashion, 100 ul volumes of the cell suspension was removed from the first column and systematically transferred (a column at a time) to each adjacent well, and so on, again reducing the numbers of cells per well until theoretically only one cell per well exists at some point before the last column. Usually one 96 well plate per column was used. Once dispensed, each well of every plate was examined under a microscope and wells containing single cells were marked. Culture supernatants were periodically sampled and tested for antibody productivity. Early cultures required ammonium sulphate concentration of the antibody [see Sec.]. 2.7.4 Propagation of Positive Clones. Once growth of clones had stabilized and numbers had sufficiently increased, the desired clones were, as before, slowly expanded. Expansion of positive clones was first to 24-well plates and subsequently to 25 cc tissue culture flasks. Once cell densities of between 10^  94 to 10^  cells/ml were achieved, samples were taken for freezing. 2.8 Handling of Monoclonal Antibody-Containing Culture Supernatants. 2.8.1 Large Scale Production of Antibody. Purification of our monoclonal antibodies was, as with many other examples in the literature, not considered necessary [for rev. see Campbell 1986]. Samples of culture media were harvested, centrifuged, and used as antibody. When larger amounts of antibody were required, hybridomas were seeded into 25 cc flasks (or larger), grown in bulk to exhaustion, and the cultures supernatants collected and used as antibody. 2.8.2 Storage of Monoclonal Antibody. In most cases, culture supernatants once harvested were kept at 4 ° C until required. In cases where the antibody was not to be used in conjunction with an immunoperoxidase reaction, sodium azide (Sigma, MO) was added for a final concentration of 0.1%. If a delay of greater than 3 to 4 days prior to the use of the antibody was anticipated aliquots were frozen at -20° C. IgM class antibodies, however, were not frozen to circumvent the potential lose of their full antigen-binding capacity [Campbell, 1986]. 2.9 Characterization of Monoclonal Antibody. Characterization of the presently generated antibodies involved determining their specific class and subclass, their ability to recognize electroblotted protein analytes, and the cellular localization of their antigen. 95 2.9.1 Class Determination of Monoclonal Antibody. Use of ELISA. Class determination of our anti-newborn brain particulate subfraction monoclonal antibody was established with an ELISA-based mouse typer sub-isotyping system (Biorad, CA). The procedure for ELISA has been described in section and was similarly used here except that a panel of rabbit anti-mouse immunoreagents (IgA, IgM, IgG}, IgG23/ IgG2b> and IgG3) (Biorad, CA) were employed as the secondary antibody, and that 10 culture supernatants were run at one time. Immunoglobulin class and subclass were then determined using horse radish peroxidase (HRP)-conjugated IgG fraction goat anti-rabbit IgG (heavy and light chain) antibody (Cappel Labs, PA) at a concentration of 1:1,000 (or 1:2,000 if background seemed to be excessive), incubating for 1 h at room temperature, washing excess enzyme conjugate off, reacting the conjugate with HRP substrate solution, and allowing 10 to 30 min for colour development. HRP reagent substrate solution contained 0.5 mg/ml 3,3'-diaminobenzidine tetrahydrochloride (DAB) dissolved in 0.1 ml of 1 m M imidazole and 0.02% hydrogen peroxide (Fisher, NJ) mixed with 40 ml of 10 m M tris buffer (pH 7.2). This was prepared fresh from stock DAB and 30% H202- Imidazole was stored at 4 ° C until needed. Because the assay was meant to be qualitative, reading the absorbance at 405 nm was not necessary. Use of a DOT Immunobinding Assay. A dot immunobinding assay used for the determination of immunoglobulin class and subclass was based on the method of Hawkes et al. 1982b, as modified by Beyer 1984, and Sternberg and Jeppesen 1984. Nitrocellulose membranes were always handled with forceps and gloves. 0.045 (xm pore size, No EA85 nitrocellulose membrane (Schleicher and Schrell, 96 NH) was cut into six (70 mm x 25 mm) rectangular pieces and marked with waterproof ink for identification. Membranes were then transferred into a 20 m M Tris-0.5 M saline buffer solution (pH 7.5 with HC1) for 10 min after which they were allowed to air dry for 5 min on filter paper. 5 ul to 10 ul of the mouse monoclonal antibody culture supernatants to be tested were applied (neat) as small spots to each of the membranes, and were again allowed to air-dry at room temperature. Once dried, membranes were incubated in a solution of 25% normal goat serum (NGS)-2.5% BSA, in Tris-HCl containing 0.1% tritonx-100 (Sigma, MO) for 30 min at room temperature to saturate and block any non-specific protein binding sites. Membranes were then rinsed together for 5 min in DH2O and individually soaked in a given ultra-pure lOx concentration (Amicon, MA) of rabbit anti-mouse subclass specific antiserum as supplied in the mouse typer sub-isotyping kit. Membrane preparations were then transferred to a humidified air-tight chamber and incubated overnight at 4° C. The subclass antibody generated was colourimetrically determined with a Vector stain ABC pre-mixed avidin-biotinylated peroxidase system (Vector Labs, CA). Blocked membranes were first washed twice in PBS buffer (pH 7.5) for 10 min each before introducing a biotinylated sheep anti-rabbit IgG (1:100). Nitrocellulose membranes were then incubated for 30 min at room temperature before being once again washed in PBS. ABC complex was prepared by adding 1 drop of avidin D H (reagent A) to 5 ml of Tris buffered saline (pH 7.5), adding, and then mixing in one drop of biotinylated peroxidase (reagent B). The reagent was then allowed to sit for 15 min at room temperature before use. Finally, membrane preparations were washed in PBS, transferred into substrate solution, and incubated for 10 to 30 min. Peroxidase substrate solution was prepared fresh by dissolving 50 mg of DAB in 10 ml of 10 m M Tris buffer (pH 7.4) containing hydrogen peroxide at a final concentration of 0.02% (w/v). The enzymatic reaction was terminated by rinsing in DH2O. Samples were then dried and photographed using Tri-X film. 97 2.10 Characterization of the Analyte by Western Blot. The preparation of whole BOL cultures and newborn brain particulate fraction has been previously described [see Sec.2.11.1 and 2.2.2, respectively]. BOL to be characterized by SDS-PAGE were first collected by scraping or pipetting cells free from approximately three 5 x 50 mm tissue culture dishes and diluting them in 10 ml PBS (pH 7.4). Harvested cells were then centrifuged for 5 min at 1,200 rpm, the supernatant discarded and the BOL pellet resuspended in 1 ml PBS. BOL was then homogenized using 10 to 15 completed up and down strokes of a 2 ml tissue grinder (Wheaton, NJ). The homogenized sample was electrophoresed in an identical manner as that described for our newborn brain particulate fractions [see Sec. 2.2.4]. Once electrophoresed, proteins were transferred onto a 0.45 u.m pore-size 140 mm x 120 mm sheet of nitrocellulose paper, using the methods described by Towbin et al. 1979. Prior to electroblotting the acrylamide gel was allowed to equilibriate in transfer buffer (25 m M Tris, [pH 8.3], 192 mM glycine, 20% [v/v] methanol), for 30 min at room temperature. Immunoblotting itself was performed using a Transblot cell and cassette assembly (Biorad, CA). The fiber-pads, filter paper and nitrocellulose membranes were also allowed to soak in transfer buffer for at least 20 min prior to assembling the transfer sandwich. Transfer assemblies were constructed by first carefully placing the slab gel onto thick filter paper, and then overlaying the gel with the nitrocellulose membrane. The nitrocellulose was eventually covered with a second piece of filter paper and any air bubbles removed using a clean testube by "roller pin" exclusion. The cassette assembly was then inserted into the center slot of the transfer chamber so that the gel was positioned facing the cathode side of the apparatus (relative to the nitrocellulose membrane). Low field electroblotting was carried out with 150 milliamp output (6 h to 12 h) from a model ECPS-3000/150 electrophoresis constant power supply (Pharmacia, Sweden). Heat generation was controlled by running the electroblot at 4 ° C. Heat dissipation was further improved by using a magnetic stir plate at a moderate setting. Once transfer was complete, the acrylamide 98 gel was placed in a filtered 25% methanol/10% acetic acid (v/v) fixative, covered, and left overnight on a Junior Orbital Shaker (Lab Line Instruments, IL) at a setting of 50 rpm. Acrylamide cells were subsequently silver stained to determine the efficacy of protein transfer. Alternatively, nitrocellulose membranes were treated so as to visualize the transferred proteins from our various sample preparations, including the molecular weight standards. The minimum time required for satisfactory protein transfer was determined with a preliminary electroblot where 20 mm wide nitrocellulose strip samples were intermittently removed from the main sandwich every hour and stained with amido black solution [see following Section]. 2.20.2 Detection of Electroblotted Protein Using Non-Immune Methods. Non-immune methods for detecting the transfer of molecular weight standards to nitrocelullose membranes was performed by staining sections with amido black (0.1% amido black [w/v] in 45% methanol/10% acetic acid [v/v]) for 10 to 20 min, rinsing for 30 sec in two changes of DH2O, and subsequently de-staining with 90% methanol/2% acetic acid (v/v) for 2 to 3 min and drying samples between pressed sections of filter paper. 2.20.2 Detection of Protein Transfer in Immunoblots Using Immunoreagents. The remainder of the electrophoretic blot (not stained with amido black) was transferred to a solution containing 25% NGS-2.5% BSA in 20 m M Tris-0.5 M buffered saline (pH 7.5 with HC1) containing 0.1% tritonx-100 for 30 min at room temperature to block additional binding sites. Once adequately blocked, western blots were thoroughly rinsed in DH2O and then transferred onto a Parafilm covered plastic grid. Membranes were then cut to give 35 to 40, 3 mm-wide strips. Each strip was then numbered in water-proof ink and carefully overlaid with approximately 500 \il of individual test supernatant from hybridoma 99 cultures. Test strips were then allowed to sit in a humidified chamber overnight at 4 ° C before colourimetric development using an immunoperoxidase system. In endeavoring to maximize the sensitivity of the HRP-based immunoassay (for both ELISA and immunoblot) one of three different horse-radish peroxidase (HRP) chromatogen solutions were used to visualize the binding of our monoclonal antibodies to the nitrocellulose. Two in which the precipitate was purplish-blue (reportedly more colour-dense [Mesulam and Rosene 1977] and, therefore, easier to detect), and a third which developed the standard brown (and reportedly less-photosensitive) reaction product [Mesulam and Rosen 1977; Bourne 1983]. Colour Development Using Diaminobenzadine (DAB). The methods for developing the HRP brown-colourimetric reaction and the preparation of the peroxidase substrate (DAB) solutions were as previously described when used with the ELISA based immunoglobulin class and subclass determinations [see Sec. Use of DAB-Nickel Disulphate Modified Solution. The "blue" reaction procedure for HRP immunochemistry largely resembles that which has already been described for developing the brown DAB precipitate. HRP colour development substrate when supplemented with nickel ions produces a purplish blue precipitate of DAB. The method for staining is similar to that described in Section Only minor modification of the peroxidase substrate solution, as described by Hsu and Soban (1982), and the conditions in which the reactions were allowed to occur was required. Briefly, 60 mg of nickel sulphate (Fisher, NJ), 50 mg of DAB, and 50 |il of a 30% hydrogen peroxide stock solution were mixed together in 100 ml of 10 m M Tris buffer (pH 7.2). The reagent was then added to immunoblots and allowed to develop in the dark for 2 to 15 100 minutes at room temperature. Use of 4-Chloro 1-Napthol. An HRP substrate working solution was produced by dissolving 4-chloro 1-napthol (Sigma, MO) in methanol to give a concentration of 3 mg/ml and subsequently mixing 20 ml of this solution with 5 volumes of 50 mM Tris buffer-200 mM saline (pH 7.4) containing 60 ul of a 30% aqueous stock solution of hydrogen peroxide. The immunoenzyme was then reacted with substrate for 2 to 15 min and membrane strips (once washed and dried) stored in the dark to prevent photobleaching [Hawkes et al. 1982a]. 2.10.3 Determination of Transfer Efficacy Using Silver Staining Techniques. Once the electrophoretic transfer of proteins onto nitrocellulose paper was complete, the efficiency of transfer was assessed by silver staining the "spent" polyacrylamide gel. The use of silver reagents for the colour development of spent gels has been reported to be 5 times more sensitive over staining with Coomasie blue [Filbin and Poduslo 1986]. Silver staining was based on the method of Merril et al. 1979, and used with little modification. In short, gels having been allowed to incubate overnight in an acid/alcohol fixative were rinsed in three changes of D H 2 O (20 min each at room temperature) prior to undergoing a second fixation step which consisted of a 30 min incubation with shaking in a 10% gluteraldehyde solution. Once complete, the gel was washed exhaustively in 6 to 8 changes of D H 2 O (10 min each at room temperature) before being placed into a diamine solution. Diamine solution was prepared by adding to 165 ml of 20% ethanol, 46 ml of 0.36% (w/v) sodium hydroxide, 3.1 ml of concentrated ammonium hydroxide, and 9 ml (added drop-wise) of a 20% solution (w/v) of silver nitrate dissolved in DH2O. Gels were allowed to incubate in the diamine for 101 15 min before washing in 3 changes of DH^O, 15 min each at room temperature. They were subsequently transferred into a developer consisting of 178 ml of 10% ethanol containing 0.5 gm citric acid and 1 ml of 3.7% formaldehyde. The colour Development of protein bands was best demonstrated by having the gel in a glass receptacle positioned over a light box. The colour reaction (if any) was allowed to proceed for 15 to 30 min, and quenched with several changes of water if necessary. 2.21 Dissociated Cell Cultures Techniques. 2.11.1 Preparation and Maintenance of Glial Cultures. Whole bovine brains were obtained from freshly killed animals and placed into a sterilized air tight receptacle containing 200 ml of ice cold CMF-HBSS supplemented with 2,000 U / m l penicilUn, 2 u.g/ml streptomycin and 100 M-g/ml bactrim. Higher concentrations of antibiotics were used to eliminate microbial contamination of starting material. Containers were kept on ice during transport to our laboratory. Upon arrival the brains were washed with 3 to 5 changes of refrigerated CMF-HBSS and antibiotics, followed with rudimentary dissection, and transferred to sterile glass petri dishes for further processing. The methods used for the bulk isolation of bovine, and murine oligodendrocytes has been previously described [Kim et al. 1983] and is a modification of the technique used by Farooq et al. 1981. The procedure is outlined in Fig. 2.5. Central nervous system (CNS) material, once washed and distributed to glass petri dishes, was handled aseptically. Material was removed of its meninges and larger blood vessels and cut into small pieces. Minced CNS material was then transferred using a large bore 10 ml plastic pipette (Falcon, Becton Dickinson, CA) to a 500 ml bottle containing 300 ml of CMF-HBSS with 0.25% trypsin (v/v) 102 (GIBCO) and 20 ug/ml DNAse (Sigma, MO). The mixture was incubated for 60 min at 37° C on a magnetic stirrer at a moderate setting. Trypsinization of the brain tissue was terminated with the addition of 3 ml horse serum. The enzymatic digest was then dissociated into single cells with gentle trituration (using a narrow bore pipette) followed by passage through a pre-wetted 100 um nylon mesh contained by a Buchner-funnel assembly. Passage through the nylon filter was facilitated with gentle stirring and mild vacuum. The filtrate was then transferred to 50 ml conical centrifuge tubes. Loose pellets were formed using a table top centrifuge with low speed centrifugation (1,500 rpm for 10 min at room temperature), and were subsequently diluted in 3 volumes of HBSS. 20 ml of this suspension was mixed with 9 ml Percoll (polyvinylpirroladone-coated silica gel, Pharmacia, Sweden) and 1 ml of concentrated HBSS (lOx) to form a 30% (v/v) solution. Continuous density gradients were spontaneously formed in 35 ml Oakridge-type centrifuge tubes (Falcon, Becton Dickinson, CA) upon centrifugation (at 15,000 rpm for 30 min) using a Beckman high speed refrigerated centrifuge with a JA-17 fixed angle rotor. After centrifugation, an upper myelin layer and lower erythrocyte (RBC) containing fraction were clearly identifiable [Fig. 2.5]. The myelin "cap" was removed with careful aspiration and the cloudy intermediate oligodendroglial fraction (specific gravity of 1.033-1.48 [Kim et al. 1984b] pipetted off and transferred to fresh 50 ml conical centrifuge tubes. Harvested cells were then diluted three-fold in HBSS and centrifuged at 1,800 RPM for 10 min again using a table-top centrifuge operating at room temperature. The resulting cell pellets were washed 2x with HBSS (1,200 rpm for 10 min) before being resuspended in an appropriate amount of feeding medium. 4 ml of cell suspension (approximately 1 x 10^  cells/ml) were subsequently transferred to 60 mm plastic petri dishes and maintained at 37° C in a humidified 5%-C02 atmosphere. Feeding medium consisted of Eagles minimal essential medium with Earl's salts (MEM), 5% FCS, 5 mg/ml glucose, 20 ug/ml gentamicin, and 2.5 ug/ml fungizone: MEM(+). Fig. 2.5: Bulk-Isolation of mammalian oligodendroglia using Percoll density gradient centrifugation. Cells, harvested from an intermediate layer, are subsequently used for immunizations, biochemistry, and tissue culture. 104 The methods for separating glia in mixed primary culture have been previously described [McCarthy and de Vellis 1980] and is based on an inherent property of oligodendrocytes and astrocytes to differentially adhere to plastic surfaces. Percoll-enriched BOL were maintained in culture for at least 24 hours before non-adherent oligodendroglia were selectively detached with shear-forces generated by a gentle swirling of the culture plates and the repeated aspiration of supernatant fluid, then the harvested non-adherent cells were placed into new petri dishes for subsequent use in immunization and biochemical characterization or alternatively seeded directly onto custom made 9 mm diameter Aclar plastic (Allied Chemical) cover slips (at approximately 10,000 cells per cover slip) for future immunocytochemical screening of hybridoma supernatants [see Sec.]. Cover slips had been previously coated by immersion in 10 ug/ml poly-lysine solution (Sigma, MO) washed once with distilled water and allowed to air dry [Yavin and Yavin 1984]. Cultures of both glial cells were subsequently maintained from a few days in vitro (DIV) to periods of up to several weeks prior to immunocytochemistry. 2.22.2 Preparation and Maintenance of Whole Brain Cultures. Whole brain cultures of adult and newborn murine CNS material were prepared essentially as described by Cohen 1974, with the modifications implemented by Kim et al. 1985, and has been illustrated in Fig. 2.6. Briefly, cerebral hemispheres from either newborn or adult Balb/C mice were removed and transferred to sterile petri dishes where they were cleaned of any readily identifiable meninges and blood vessels. Material was then washed twice by dipping in CMF-HBSS, minced, and then enzymatically digested for 30 min at 37° C in a solution of 0.25% trypsin-20 ug/ml DNAse in CMF-HBSS. Enzyme digestion was terminated with a serum containing (1% FCS) washing medium. After centrifugation (1,200 rpm for 7 min) mechanical tissue disruption was used to provide single cell suspensions and to concomitantly clear our preparations of larger particles of debris. These 1 0 5 FIG. 2.6: Schematic representation of the preparation of whole-brain cultures. Once tissue is minced and digested, the cellular material is washed, dissociated further with gentle trituration and passed through nylon filters of decreasing size to exclude larger particles. Cell suspensions are seeded onto coverslips for subsequent immunocytochemical assay of hybridoma culture supernatants. 106 methods included repeated trituration (20 to 30 times) with a narrow-bore 5 ml pipette, followed with sequential sieving of the tissues through pre-wetted sterilized nylon bags of 150 |im and 75 ujn pore diameters. Cell suspensions were washed once again (1,200 rpm for 7 min) before being resuspended in feeding medium or seeded directly onto poly-lysine coated cover slips each with approximately 1 to 2 x 10^ cells contained by a single 60 mm dish (approximately 15 cover slips per dish). Once seeded, cells were allowed to adhere onto the cover slips for at least 24 hours before flooding the plates with 2 ml to 3 ml of MEM(+). Cells were maintained at 3 7 ° C in a 5%-C02 humidified atmosphere until needed for immunocytochemistry. 2.12.3 Preparation and Maintenance of Dorsal Root Ganglia (DRG). Cultures of newborn murine (outbred strain) dorsal root ganglia (DRG) were prepared following the technique originally described by Nakai 1956 as illustrated in Fig. 2.7. For most cultures, 4 to 6 newborn mice (12 to 24 hours old) were used. Animals were sacrificed by rapid freezing followed by immersion into 95% ethanol. Specimens were then secured (ventral side down) onto a dissecting board. Under aseptic conditions the apaxial skin was removed with sterile scissors and forceps, exposing the dorsal surface of the vertebral column. The column was then transversed caudally to eliminate the tail followed with bilateral incisions 3 mm to 4 mm on either side of the spinal cord, through the ribs, to approximately the level of the first rib, where the spinal cord was again transversed. The entire spinal column was then freed by slightly lifting and cutting away any adherent tissues and surrounding organs. The column was then transferred to a clean dish and rinsed twice in HBSS. Using watchmaker forceps the spinal column was held securely by its rostal end with the dorsal surface down. Under a stereoscopic microscope the cutting edge of a pair of retina micro-dissection scissors were inserted into the vertebral canal and the column cut bilaterally to remove its ventral surface. The spinal cord, once exposed, was teased out of the 107 Fig. 2.7: The techniques used in establishing DRG cultures from newborn mice. The spinal column is first removed (A). Bilateral incisions are then made (B), under the low magnification of a dissecting microscope, into its ventral surface to expose and subsequently remove (C) the spinal cord. This allows access to the DRG (D). Once removed, DRG are dissected away from any attached neuronal remnants. This is followed with enzymatic digestion, gentle trituration, and washing prior to dispensing onto coverslips. 108 canal with a pair of blunt forceps allowing easy access to the spinal ganglia. Paired thoracico-lumbar DRG were removed from between the segmental intervals by inserting the watchmaker forceps slightly into the intervertebral foramen. Ganglia were then transferred to a 30 mm culture dish containing fresh HBSS. Approximately 25 to 30 DRG could be recovered (in 15 min) per animal. Occasionally these spinal ganglia would be observed, once removed, to still have attached to them remnants of the afferent neurons which constitute the dorsal root. These were removed by scalpel prior to further processing. Once collected, DRG were enzymatically digested with 0.25% trypsin (GIBCO) and 20 u,g/ml DNAse I (Sigma, MO) in CMF-HBSS at 37° C for 30 min. Dissociated cells were then washed twice, each time in 10 ml HBSS containing 1% (v/v) FCS (to terminate digestion) before being resuspended in incubation medium at a density of approximately 1 x 10*> cells/ml. Incubation media consisted of M E M supplemented with 10% FCS, 300 to 600 mg/ml glucose, 100 u.g/ml streptomycin and 100 IU/ml penicillin (GIBCO) and (when required) 100 biological IU/ml nerve growth factor (NGF [Sigma, MO]). Once appropriately diluted, cell suspensions were seeded onto poly-lysine coated aclar cover slips and used shortly thereafter for immunocytochemical analysis of hybridoma culture supernatants. DRG cultures were maintained in a 37° C humidified 5%-C02/95% air atmosphere. 109 3 RESULTS. 3.1 Preparation of Neural Antigens. 3.1.1 Recovery of Human Axolemma-Enriched Fraction. Yields of axolemma-enriched fractions prepared from frozen human material were consistently lower (100 ug to 20 |xg/gm starting material, as determined by the Lowry method) than amounts (approx. 1 mg/gm starting material) recovered by De Vries et al. 1981. Despite providing sufficient amounts of axolemma-enriched fraction for SDS-gel electrophoresis, the method delivered insufficient material to allow for immunization and subsequent micro-ELISA assay. SDS-polyacrylamide gel electrophoresis demonstrated that the majority of the proteins constituting the human axolemma-enriched fraction were between approximately 45,000 and 50,000 daltons. Phase microscopy of crude axolemma-enriched fraction (obtained after density gradient centrifugation) revealed the virtual absence of any identifiable cell structure or organelles. 3.1.2 Human Newborn Brain Particulate Fraction. Phase microscopy of the initial homogenate of whole human newborn cerebellum displayed an absence of intact cells with an abundance of cellular debris including the occasional capillary fragment. Less frequently there was the presence of identifiable organelles (large nuclei). After differential centrifugation, however, high-power phase microscopy did not reveal any identifiable structures or substructures [Fig. 3.1]. Fig. 3.1: Phase contrast microscopy of human newborn brain particulate fraction. Note the homogenous appearance of the field and the absence of any identifiable organelles. 111 The molecular weight distribution of the polypeptides comprising the newborn brain particulate fraction isolated from human cerebellum can be seen in Coomasie blue stained SDS-PAGE [Fig. 3.2]. Although hundreds of protein bands, varying in apparent molecular weight froml0,000 to over 100,000 Da appear to exist after slab gel electrophoresis, only those proteins that most closely approximated the relative migration (M r ) of the major polypeptides from a concomitantly run axolemma-enriched fraction were of concern. It was in this region (35,000 to 55,000 molecular weight) that the major proteins of the axolemma elements were observed [Fig. 3.3]. Excision of this area of gel, its dialysis, and the subsequent concentration of the protein contained within the regions of approximately 34,000 to 36,000 Da, 39,000 to 44,000 Da and 44,000 to 50,000 Da were determined using a protein dye-binding method. They were found to contain 16.5 Ug/ml 18.5 u.g/ml and 17.0 ug /ml , respectively, before subsequent concentration. Re-electrophoresis of from 5 u,g to 10 u.g of these concentrated subfractions (using a small format apparatus) revealed that the proteins constituting the subtraction (b) of approximate molecular weight 44,000 Da to 50,000 Da, best coincided with our axolemma-enriched preparation [Fig. 3.3], and was, therefore, used for subsequent intrasplenic immunization. 3.2.3 Bovine Oligodendrocyte Cell Homogenate. Results concerning the efficacy of the bulk-isolation of oligodendroglia were presented with results describing the characteristics of disaggregated primary cultures [see Sec.]. Bovine oligodendroglial homogenate, prepared in HBSS and used in electroblot characterization of the monoclonality of generated hybridomas, was determined (using the protein-dye binding method) to contain an average of 2 mg to 4 mg of cell protein per tissue culture dish (each containing approximately 1 to 1.5 x 10^  cells), used. 1 12 20.1 1 4 3 •v t* f\ mfty Fig. 3.2: SDS-Polyacrylamide gel electrophoresis of human newborn brain particulate fraction. Coomasie blue was used visualize the protein constituents of whole fraction Fig. 3.3: Electrophoretic mobility of sub-fractionated particulate sample proteins. Note the smaller format gel (used to spare sample) and the representative protein bands of each of the three sections excissed. Again the protein constituents were visualized with coomasie blue. 1 13 32 Characteristics and Growth of Neural Cultures. 3.2.1 Isolation and Culture of Adult Astrocytes and Oligodendrocytes. Glial cultures prepared from adult murine and bovine CNS materials were used in the immunocytochemical screening of our hybridoma supernatants. It is important to note here that as significant as the many recent advances in neural-cell culture techniques are in providing the necessary materials for the present study [see Introduction], their implementation was primarily intended to serve as a tool for establishing the cell-type specificity of our product supernatants, and for providing (in some cases), immunogen for primary intrasplenic immunization. Cell cultures, however, were not so much intended to serve for the study of the specific neural cell-type per se. : this remains to be more fully investigated. Characteristics of The Oligodendrocytes. Typical yields of oligodendrocytes obtained from Percoll density gradients, when whole bovine brain was used as a starting material, was in the order of 5 to 6 x 10^  cells/gm (wet weight). The purity and viability of bulk-isolated bovine oligodendroglia were usually 95% and 98%, determined by GalC immunolabeling and trypan blue exclusion, respectively. "Contaminating" cells were usually red blood cells (RBC). The presence of residual astrocytes, identified by their positive GFAP-immunolabeling, comprised typically less than 5% of the oligodendroglial preparation [Fig. 3.4]. 1 14 The bulk-isolation of oligodendrocytes from whole mouse brain using Percoll density gradients was not as practical as it was cumbersome when considered against the relative quantities of starting material available from the bovine (or Porcine etc.) species. Usually 4 to 5 brains from adult mice (4 to 6[+] weeks old) were required to produce approximately 100 cover slips suitable for immunocytochemistry. Murine oligodendroglial cultures displayed a similar morphological appearance to preparations obtained from bovine CNS. Phase contrast microscopy of bulk oligodendrocyte preparations after initial isolation revealed primary cultures of uniformly smooth, spherical cells, approximately 8 um to 12 um in diameter and possessing a highly refractile (phase-bright) cell surface [Fig. 3.4]. Freshly isolated cells were found to readily attach to the culture dishes and to each other in relatively large clumps. The inherent property of these various cell types to differentially adhere to, and thus stratify the plastic surface (after 1 DIV), necessitated a gentle shaking of the culture plates with sufficient force so as to dislodge the loosely clumped oligodendrocytes. This separated them from a more tightly bound non-oligodendroglial fraction consisting primarily of astrocytes as well as endothelial cells, and fibroblasts [McCarthy and de Vellis 1980; Gebicke-Harter et al. 1984; Kim 1985]. The majority of these cells attached to the aclar cover slips within 24 h after re-plating. Although plating efficiencies were not estimated in the present study, previous work by other investigators has established the value for essentially the same procedure at between 60% [Lisak et al. 1981] to 85% [Gebicke-Harter et al. 1984]. Process outgrowth was observed to begin within 24 h after re-plating and to occur more extensively after 2 to 3 DIV. With increased time in culture, more processes and branching was observed until oligodendrocyte cultures attained their typical interlacing-multipolar morphology [Fig. 3.5]. Positive identification of these cells as oligodendroglia was established by indirect surface immunofluorescence using the cell type-specific marker GalC [Raff et al. 1978b]. 1 15 Fig. 3.4: Phase microscopy of freshly isolated bovine oligodendrocytes. Note the "phase bright" appearance of the much larger glial cells (large arrow) and the presence of residual erythrocytes (small arrows). Bar indicates 50um. Fig. 3.5: Phase microscopy of bovine oligodendroglia maintained in culture for 14 (DIV). Cell processes first become apparent after 2-3 DIV and continue to develop until a typical interlacing-multipolar morphology is attained. Note the abscence of neurons in these cultures. Bar indicates 50 urn. 1 16 Characteristics of the Astrocytes . The astrocyte, which is the second most conspicuous glial cell in these preparations, was isolated and maintained as a by-product of the Percoll enrichment process. Astrocytes, when first isolated, were initially indistinguishable (on the basis of phase microscopy) from oligodendrocytes. However, morphological differences between these cell types became evident once cultures were given an opportunity to fully adhere to the substratum (usually after 2 to 3 DIV). Once attached, astrocytes became primarily flat and fibroblast-like and would occasionally develop a bipolar appearance [Fig.3.6]. Positive identification, however, required GFAP immunostaining as some astroglia may also appear as multipolar process-bearing cells. The purity of astroglial cultures was judged to be in some instances as high as 98% when immunolabeled with the intracytoplasmic marker GFAP. On a morphological basis, no neurons could be identified in our bulk isolated primary cultures of adult oligodendrocytes. 3.2.2 Newborn and Adult Mixed Primary CNS Cultures. Newborn and adult mouse CNS mixed primary cultures were seeded and maintained directly on 9 mm round poly-lysine coated plastic cover slips. 4 to 6 newborn (24 to 48 h) brains (each approximately 0.5 gm wet weight), and 2 to 3 adult (4 to 6[+] week) brains (approximately 1 gm wet weight) would yield on the average of 2 to 6 x 10^  and 1 to 3 x 10^  cells, respectively. Rate of viability for these preparations (as judged by trypan blue exclusion techniques) was in the order of 80% to 90%. Dissociated heterogenous early cultures (approximately 3 DIV) would develop a stratified appearance. Cells which were relatively smaller, darker, and less abundant were judged according to their capacity to bind anti-GalC 1 17 Fig. 3.6: Phase microscopy of an "oligodendrogUal" culture (10 DIV) displaying the characteristics common to astrocytes (arrow). Astrocytes will, in some instances possess, the morphological attributes more typical of an oligodendrocyte. Positive identification, therefore, depends on the cells ultrastructural or, more commonly, on its' immunostaining characteristics. Again note the absence of any identifiable neurons in these preparations. Bar indicates 30 urn. 1 18 to be oligodendroglia. These cells tended to overlay a second more abundant, flat, phase-translucent, polygonal shaped population of cells identified by their positive GFAP immunolabeling as being astrocytes [Fig. 3.7]. The remainder (GalC'/GFAP") were probably ependymal cells and fibroblasts according to the observations of Raffef al. 1979. Regardless of source, however, cultures did display a strong tendency to reaggregate and to subsequently develop an extensive fiber network. 3.2.3 Newborn Dorsal Root Ganglia (DRG). On a morphological basis, neurons could only be sparesly identified in newborn murine whole brain primary cultures. Neurons, however, were not readily detectable (if at all) in cultures prepared from animals greater than 2 days old. These findings are in aggreament with the observations of previous investigations [Sensenbrenner 1977; McCarthy and de Vellis 1980; Banker 1980]. Use of an alternative starting material (DRG) which provided a richer source of viable neurons, was, therefore, considered important. Dissection of 4 to 6 newborn mice (12 h to 24 h) would yield from between 100 to 180 DRG. These would in turn afford adequate material for subsequent culture and immuno-cytochemistry. Cell suspensions at the time of culture were again determined to be (by trypan blue exclusion techniques) approximately 80% to 90% viable. Freshly dissociated cultures of DRG were observed as mainly phase-bright spherical cells, 20 urn to 30 urn in diameter, with little if any distinguishable intracellular detail [Fig. 3.8]. Once plated, up to 50% of the neurons would display rudimentary neurite extensions within 24 h in vitro. Most fiber regeneration was observed in these cultures after 2 to 3 DIV. Degenerating neurons, on the other hand, would appear as possessing a more granular-like, flattened morphology. 1 1 Hi • i F i g . 3.7: A d u l t m i x e d p r ima ry cultures of mur ine C N S . Majo r g l i a l ce l l types, v i r tua l ly indis t inguishable f rom one another us ing phase criteria, are easi ly ident i f ied b y their i m m u n o l a b e l l i n g characteristics us ing rabbit anti-galactocerebroside, a n d a n t i - G F A P serum (also see f ig. 1.3). Bar indicates 50 um. A : Galactocerebroside-fluorescein immunofluorescence. B : G F A P - r h o d a m i n e fluorescence. C : Phase contrast mic roscopy . 120 Fig. 3.8: Phase microscopy of newborn mixed primary cultures of murine CNS. Although the phase characteristics of these cultures appear to be similar to those shown in Fig. 3.7, they now are shown to display the presence of neurons (arrow). Bar indicates 50 urn. 121 Non-neuronal cells in dissociated cultures of DRG include Schwann cells and satellite cells [see Okun 1972]. It was not difficult to identify Schwann cells on the basis of their phase characteristics, displaying a tendency to be darker and more elongated (spindle shaped) than other cell types in DRG cultures [Fig. 3.8]. The unequivocal visualization and identification of a second constituent of DRG cultures, the satellite cell, was difficult as they exist as much smaller spherical elements (5 am in diameter) and are often closely associated to the neuronal perykaria. DRG cultures when needed to screen for anti-neuron immunolabeling were used comparatively soon (24 h to 48 h) after initial plating. 33 Characteristics and Growth of the Myeloma Cell Line. 33.1 Culture and Selectivity of NS-1 to H A T and Azaguanine. The murine myeloma cell line, P3-NS-l-Ag4-l (abbreviated NS-1), displayed good growth characteristics with a doubling time of approximately 10 h and with only a moderate loss (approximately 50%) of cellular viability when reconstituted from frozen stock. Samples aliquoted and tested with fresh aminopterin, however, showed poor sensitivity necessitating the temporary incorporation of 8-azaguanine into the culture medium [see Sec. 2.1.2]. Azaguanine resistant cells, selected for in this manner, ensured that survival and growth of back-revertants (i.e. T K + cells) was adequately suppressed. NS-1 cells appear as moderately large spherical cells (approximately 15 |xm diameter) in culture [Fig. 3.9]. Ficoll density gradient centrifugation proved helpful in enriching the number of viable myeloma cells from non-viable cells and debris after 8-azaguanine pre-treatment. Subsequent trypan blue exclusion studies on azaguanine pretreated NS-1 batch cultures confirmed a complete return of sensitivity to aminopterin. Once desired NS-1 cultures 122 Fig. 3.9: Phase contrast characteristics of a typical field of the myeloma cell line: NS-1 in batch culture prior to fusion. Bar indicates 50 urn. 123 were established, we endeavored to maintain an exponential phase of growth until cells were required for fusion. This necessitated their feeding and subculture every 2 to 3 days. 3.4 Isolation of Sensitized Murine Splenocytes. On average, the amount of viable lymphocytes recovered (after plastic adherence) from 4 to 6 week old Balb/C immunized mouse spleen was approximately 1 to 2 x 10^ cells. Splenocytes prepared in this manner displayed a viability of greater than 90%. Those cells remaining adhered to the plastic culture flask possessed the morphological characteristics of macrophages [Penniline 1981]. 33 Characteristics and Growth of Fusion Products. High viability and exponential growth of myeloma cells prior to fusion was maintained with frequent feeding and subculture. The successful fusion of the murine NS-1 myeloma cell line with splenocytes immunized with a human newborn brain particulate subfraction and of whole living cell cultures of bovine oligodendroglia was demonstrable in 43 and 45 out of the 48 wells in which these fusion products (from 2 separate experiments) were initially distributed, respectively. Fig. 3.10 illustrates a typical low power field of freshly fused splenocytes with the larger myeloma cell just prior to HAT(+) selection. Incorporation of H A T containing medium at 3 days post fusion (depending on initial growth characteristics) resulted in massive cell death but was evidently not cytotoxic for those cells which had successfully undergone hybridization [Fig. 3.10]. 124 Fig. 3.10: Phase microscopy of hybridomas before and subsequent to HAT selection. A: Phase microscopy of freshly fused hybridomas prior to HAT selection. Note that the much larger myeloma cells (large arrow) are now associated with many smaller splenocytes (small arrow). Bar indicates 50 \im. B: Once HAT is incorporated into the culture media, massive cell death can be seen to take place. However, sucessfully fused hybrids will survive and may be identified in these early cultures by their large, spherical, transluscent appearance (large arrow). Bar indicates 50 u.m. 125 3.5.2 Establishment of Anti-Human Newborn Brain Particulate Subfraction Monoclonal Antibody-Secreting Hybridomas. The techniques involved in establishing monoclonal antibody-producing clones directed against our human newborn brain particulate fraction required an initial expansion of viable fusion products followed with primary screening, cloning, and the immunocytochemical determination of the recognized neural cell type (see following sections). Primary Screening (ELISA) of Hybridoma Culture Supernatants. Screening using the micro-ELISA technique described in Section colourimetrically indicated that the concentrated (lOx) culture fluid in 3 out of a total of 43 wells (those displaying viability) contained antibody directed against whole newborn brain particulate fraction. Antibody secreted by hybridomas, however, was not detectable during the first week of fusion but rather appeared at screening after the cells had been maintained for at least 7 days in culture. The number of new antibody producing colonies continued to increase with subsequent testing to the maximum (3) seen at approximately 4 days after antibody first became detectable. The colonies of these positive wells were successfully propagated and prepared for initial cloning using methylcellulose techniques. Cloning in Methylcellulose. Hybridomas appropriately diluted in MCM(+) took an average of 7 days to grow into colonies large enough for selection under the medium-power objective of an inverted phase microscope [Fig. 3.11]. Apparently many hybrids did not tolerate the semisolid media 126 Fig. 3.11: Phase microscopy of newly forming hybrid clone growing in methyl cellulose (2-3 DIV). Clones growing in methyl cellulose were allowed to grow to a size a little larger than what is shown here before being manually selected using a microscope. Such clones, once selected, were expanded in vitro and later tested for antibody activity. Bar indicates 30 \im. 127 effects of dilution. Of the 96 samples selected (under the microscope) from each of the 13 mixed hybridoma cultures growing in MCM(+), a total of 300 clones displayed any indication of growth and survival. Of these, only 8 provided qualitative evidence (as judged by ELISA) in the generation and secretion of monoclonal antibody at second screening. Immunoglobulin Subclass Determination. The immunoglobulin class or subtype being secreted by our ELISA-positive hybridomas was established using a commercially available sub-isotyping system (Biorad, CA). The supernatant of selected hybridoma cultures, once introduced to the panel of ultra pure Rabbit anti-mouse immunoglobulin subtypes, indicated that the single class of immunoglobulin, IgM, was being secreted and that no colourmetric reaction was demonstrable with antisera to mouse IgG2a/ IgG2b> ^8^3 , or IgA. Cell Type Specificity as Determined with Indirect Immunofluoresence. Anticipating a monoclonal antibody directed against a neuronal constituent, immunocytochemistry was first performed on dissociated cultures of newborn mouse DRG [see Introduction]. However, no evidence of surface or intracytoplasmic immunolabeling (using all ELISA positive hybridoma culture supernatants) was apparent. Furthermore, the use of both living and prefixed bulk-isolated cultures of oligodendrocytes (as prepared from both adult murine, and bovine CNS material) did not show any positive immunolabeling. However, when disaggregated cultures of whole newborn murine brain were used for immunocytochemistry, surface labeling was observed with a population of cells after treatment with the product of a single clone (and its duplicate): PF(b)-C3A8. Further 128 immunocytochemical investigation employing dual-immunolabeling techniques, where whole newborn murine brain cultures were reacted with PF(b)-C3A8 concomitantly with the oligodendrocyte-specific surface marker, GalC, or subsequent to the astrocyte-specific intracytoplasmic marker, GFAP, unequivocally identified PF(b)-C3A8 positive neural cells as being comprised of a subpopulation of astroglia [Fig. 3.12]. Demonstrated frequencies of PF(b)-C3A8 positive astrocytes in newborn whole brain cultures were estimated to be in the order of 10% of the total cells present, while immunolabeling of purified/enriched adult murine and bovine astroglia was not apparent. Analysis of Recognized Epitope. The particulate fraction from human newborn brain was subjected to SDS-polyacrylamide gel electrophoresis, the separated fraction elements transferred to nitrocellulose membranes, and the antigenic specificity of PF(b) C3A8 antibody tested. Peroxidase labeled secondary antibody demonstrated that PF(b)-C3A8 antibody specifically recognized a single protein constituent in the particulate fraction of 50,000 molecular weight. Of the 40 other samples tested (including duplicates), 2 gave high nonspecific reaction and were not pursued [Fig. 3.13]. 3.5.2 Establishment of an Anti-Bovine Oligodendrocyte Monoclonal Antibody-Secreting Hybridomas. In our second experiment, of the 48 wells initially seeded after cell fusion, 45 of the hybridoma cultures demonstrated stability in growth and survival after selection. Primary screening of hybridoma culture supernatant raised against cultures of whole bovine oligodendrocytes was performed by first concentrating the immunoglobulins contained by the supernatants, and then using these in an indirect immunofluorescence assay of both 1 2 9 d if Fig. 3.12: Cell-type-specificity of Pf(b)-C3A8. Monoclonal antibody specificity was determined using newborn brain mixed primary murine cultures (4-5 DIV) in conjunction with the immunolabel: GFAP. Bar indicates 20 urn. A: GFAP-rhodamine immunofluorescence; B: Pfft>)-C3A8-fluorescein immunofluorescence; C: Phase contrast microscopy. D: Different field stained in a similar manner as A; E: Similar to B; F: Similar to C. 130 Fig. 3.13: Western blot analysis of the recognized Pf(b)-C3A8 epitope. Whole newborn brain particulate fraction was subfractionated using 10% PAGE. A: Amido black stained nitrocellulose membrane displaying the molecular weight standards as well as the starting edge of the sample containing the pariculate fraction. B: HRP-stained (brown reaction colour product) section initially treated with Pf(b)-C3A8. A single protein band at a relative molecular weight of 50,000 Da can be observed (arrow). C: HRP-stained electroblot treated with NS-1 culture media as control. 131 living and prefixed BOL cultures. Out of the 45 mixed hybrid cultures, 4 supernatants displayed strong immunolabeling of neural cells [Fig. 3.14]. Furthermore, the epitope (or analyte), recognized at primary screening could only be demonstrated on the cell's surface. Cloning Using Micro-manipulative Techniques and Limiting Dilution. Hybridomas (left essentially undisturbed after seeding) formed distinguishable and often separate colonies which could be carefully collected from the original culture vessel [Fig. 3.15]. In order to expedite the successful isolation of desirable clones, a preliminary manual selection of "clones" under the low power objective of an inverted phase microscope was used. From each of the original immunopositive wells were chosen 12 colonies on the basis of morphology (i.e. colony size) and on how physically removed the "clone" appeared to be from other colonies. Although colonies selected in this manner may not necessarily be generated from an individual source or parent, subsequent investigation indicated that the cells constituting a given colony produced antibody with similar immunological properties. Approximately 8% of the manually selected hybridoma colonies displayed relatively good growth characteristics after preliminary cloning. Furthermore, antibody production (as determined by subsequent indirect surface immunofluorescence) was conserved by the majority of viable colonies originally selected. Initial cloning of the cultures I-D4, II-E3, II-F4, and II-G2 provided 3, 8, 1, and 5 immunopositive hybridomas, respectively. Each rudimentary clone was successfully expanded without loss of viability or antibody productivity, and used in further immunochemical characterization. Subclass Determination of Anti BOL Monoclonal Antibody. The immunoglobulin class or subclass produced by each primary clone was ft 1 O * O rv/ i V f -O * * is ' 4 f © lb v . * 1 • Fig.3.14: Positive immunofluorescence of Percoll-enriched bovine oligodendrocytes used for the initial screeing of the presently generated hybridomas. Bar indicates 50 u.m. A: I-D4-fluorescein immunofluorescence. B: Phase contrast microscopy. C: II-E3-fluorescein immunofluorescence. D: Phase contrast microscopy. E: II-F4-fluorescein immunofluorescence. F: Phase contrast microscopy. G: II-G2-fluorescein immunofluorescence. H: Phase contrast microscopy. 133 Fig. 3.15: Typical phase contrast appearance of undisturbed hybridomas growing in selective media. Hybridomas were "cloned" by carefully selecting a few cells from the center of each colony using a pulled pasteur pipet, with the low power of an inverted microscope. Once these cells displayed stability in culture a subsequent cloning using limiting dilution was performed. Bar indicates 50 \im. 134 determined using a dot-immunobinding assay. Supernatants from each immunopositive hybridoma culture (used to treat nitrocellulose membranes saturated with BOL antigen) colourimetrically reacted in almost equal frequency with both the goat anti-mouse IgM or IgG2b subclass of enzyme-immunoconjugate. No reaction, however, to IgG}, IgG23/ IgG3, or IgA was demonstrable [Fig. 3.16]. Once expanded, the most desirable clones (approximately 50% each of the immunoglobulin IgM and IgG2b class) were subcloned into 96-well microtiter plates using limiting dilution. Of the 19 originally cloned samples, 348 viable hybridoma clones were successfully isolated and expanded. Culture supernatants were then concentrated lOx and subsequently tested for cell type-specificity using indirect immunocytochemistry. Of the samples tested, all but one, G2B4-A, did not demonstrate some degree of antibody immunolabeling. Cell type specificity of anti-BOL antibody. Immunocytochemistry of bovine culture preparations with the remaining subclones, displayed varying relative intensities and specificities [Table 3.1]. However, a majority of the antibody produced by our hybridomas appeared to discriminate a specific epitope located on the surface of oligodendroglia as suggested by dual-immunolabeling (using GalC and GFAP staining ) of living BOL cultures [Fig. 3.17]. Analysis of BOL Epitope. Western blotting of whole bovine oligodendroglial homogenate followed by immunolabeling with various hybridoma supernatants revealed that of the 50 or more monoclonal antibodies run on western blot, most specifically recognized an antigenic determinant of approximately 29,000 and/or 59,000 molecular weight (as determined with concomitantly run protein standards). The remaining monoclonal antibodies tested did not Fig. 3.16: Subclass determination of anti-BOL monoclonal antibodies using a dot-immunobinding technique. Samples of hybridoma culture supernatants produced immunostaining with varying intensity and specificity against a panel of class-specific enzyme immunoconjugates. Faint brown circles developed (arrow) only with the IgM and IgG2b subclasses. LEAF 136 OMITTED IN PAGE NUMBERING. FEUILLET 136 NON INCLUS DANS LA PAGINATION. National Library of Canada BibliothSque rationale du Canada Canadian Theses Service Service des thSses canadiennes NO. HYBRID CLONE OUGO ASTRO FB CLASS NO. HYBRID CLONE OUGO ASTRO FB CLASS 1 E3A5 A4 3 - - IgG2b 37 G2B5 B8 3 - - . IgM 2 05 3 - - 38 •i B7 3 - - li 3 " C4 3 - - II 39 " B6 +/- - - " 4 li D5 3 - - it 40 C10 3 - - ti 5 E7 2 - - 41 G3 3 - - ti 6 II E6 2 - - . 42 " D9 3 1 -7 , F10 3 - - n 43 II D3 1 - -8 II F7 3 - - " 44 •i ElO 2 - -9 » 1 F5 4 - - II 45 E9 1 - - " 10 li G6 4 - - 46 " F10 3 - - II 11 It H12 3 - - 47 F9 3 - - •I 12 ti H10 5 - 1 II 48 " G i l - - . - " 13 E3A11 A3 3 - - lgG2b 49 II G9 1 - - " 14 " B5 4 - - 50 n H8 - - - II 15 ti C4 3 - - II 51 E3A1 A8 3 - - lgG2b 16 II D5 3 - - it 52 " A5 2 - - " 17 n F7 - - - " 53 " A4 3 - -18 II G7 - - - 54 " A3 3 - - " 19 E3A12 A7 3 - - IgG2b 55 " B4 3 - -20 II A6 3 2 2 " 56 II C4 2 - -21 " Bll 3 - - li 57 " D6 +/- - - II 22 it B8 3 - - n 58 " DI 3 - -23 " CIO 4 - - n 59 II E6 3 3 - n 24 li C9 3 - - ti 60 E5 3 - -25 D l l 3 - - 61 ti F8 3 - - •I 26 li D9 3 - - ... 62 " F7 3 - - " 27 it D8 4 - - 63 " F6 3 1 -28 tt m 3 - - li 64 " G7 3 - - " 29 li F12 3 - - " 65 " 119 3 - -30 li F l l 3 - - li 66 H8 4 1 -31 II F9 3 - - " 67 H7 3 3 - " 32 " G i l 3 - - 63 E3A7 A4 3 - - IgG2b 33 It H12 3 - - 69 " B4 3 - -34 G2B2 A7 3 - - IgM 70 C4 3 - - " 35 " A6 2 - - 71 " D4 3 - - H 36 II A5 2 - - 72 II Ell 3 - - " Table 1: Characteristics of hybridomas generated against bovine oligodendrocytes. Oligo (oligodendrocytes); Astro (astrocytes); FB (fibroblasts). Numbers in cell columns indicate relative immunofluorescence intensities. NO. HYDRID CLONE OLG AST PB CLASS NO. HYBRID CLONE OLG AST FB CLASS 73 E3A7 E10 3 - - IgG2b 118 E3A6 D7 ' 2 - - . IgG2b 74 " E9 3 - - " 119 - tl D6 3 - - l l 75 " E7 3 - - " 120 E10 3 - -76 re 3 1 - 121 " E6 3 - - II 77 G9 3 - - II 122 P10 3 - - l l 78 " G8 3 3 - " 123 " F9 3 - - • i 79 " • H10 3 - - " 124~ F8 3 - - n 80 •I 117 3 - - " 125 F7 3 - - H 81 • II 116 3 - - " 126 (1 F6 2 - -82 E3A4 A3 3 • - - JgG2b 127 l l G9 3 - -83 II D6 3 - - 128 l l G8 3 - - It 81 " 154 3 - - " 129 l l 1111 3 - . - " 85 H 133 3 - - " 130" " 1110 3 - - n 86 n C 3 - - " 131 M M9 3 ; - - it 87 " 6 3 - - " 132" G2l!2b A4 3 1 -88 C5 3 - - " 133 " m i 3 1 -89 D7 3 - - " 134 ** D8 3 +/- - it 90 " D6 3 - - II 135 II G8 3 1 -100 •I E9 3 - - " 136 " H10 3 +/- -101 •I E8 5 1 - " 137 II H9 3 +/- - M 102 " El 4 1 - 138 " E8 3 1 - •I 103 re 3 - - " 139 " E7 3 +/- - H 104 " 17 3 - - " 140 " 1-10 3 +/- -105 " F6 3 - - " 141 " F9 3 2 -106 " G3 5 1 - " 142 D4B5 A10 2 1 - I R M 107 " G7 3 - - " 143 Bl l 3 +/- -108 C6 3 - - 144 B9 3 1 -109 " G5 3 - - II 145 C12 3 - -110 1110 3 - - " 146 t l CIO 3 - - II 111 119 3 - - " 147 l l E10 3 1 - n 112 118 3 - - II 148 II F12 3 - n 113 E3A6 A4 3 - - IgG2b 149 F10 3 - - t i 114 " D7 3 - - 150 D4116 Bl l 3 - -115 " D5 3 - - 151 D10 +/- - - it 116 B4 3 - - 152 D12 3 - - n 117 CIO 3 - - 153 E12 3 - -138 Table 1 (Cont'd): Characteristics of hybridomas generated against bovine oligodendrocytes. Oligo (oligodendrocytes); Astro (astrocytes); FB (fibroblasts). Numbers in cell columns indicate relative immunofluorescence intensities. NO. HYBRID CLONE OLG AST FB CLASS NO. HYBRID CLONE OLG AST FB CLASS 154 D4B6 G12 2 1 - IgM 186 G2B5 E7 +/- - - IgM 155 D4B5 H12 +/- - - IgM 187 " E5 2 +/- -156 " H10 2 - - it 188 n E4 +/- +/- - " 157 G2B4a D9 - - - IgM 189 M F7 1 +/- - " 158 " F7 +/- - - 190 II F6 +/- +/- - n 159 " H7 - - - 191 II F5 3 1 - •I 160 G2B4b • C12 3 - - IgM 192 II G10 3 1 -161 " E3 - - - it 193 II G9 2 +/- - II 162 F l l - - - 194 " G8 +/- +/- - " 163 it F10 +/- - - 195 G7 +/- +/- - II 164 " G12 - - - 196 " H9 +/- +/- -165 " 1110 - - - II 197 H8 +/- .+/- - " 166 D4A3 B9 - - IgM 198 " H7 +/- - " 167 it B8 - - - 199 E3A2b A10 3 +/- - IgG2b 168 t l Do +/- - - 200 " A9 3 +/- - " 169 l l D4 - - - 201 " A8 3 +/- - " 170 It Fl l - - - " 202 • i BIO 2 +/- - " 171 l l H10 3 1 - 203 n B9 3 - - " 172 G2B5 A4 1 +/- - IgM 204 n B8 3 - -173 n A3 2 1 - 205 C l l 3 - - " 174 n A2 2 - - II 206 n CIO 3 +/- - II 175 " B6 3 - - it 207 " C9 2 2 -176 " B5 +/- - - " 208 D10 +/- - -177 " • B4 2 - - it 209 II E12 4 1 -178 C7 2 +/- - " 210 El l 3 +/- -179 •I C6 1 +/- - " 211 F12 3 - -180 C4 3 +/- -l l 212 F l l 3 - - " 181 " D6 2 - - H 213 " G12 2 2 - " 182 II D4 3 +/- - it 214 " G i l 4 1 -183 " D3 +/- - - 215 H12 3 - - " 184 ti E9 +/- - - " 216 " H l l 3 +/- - " 185 " E8 +/- - -139 Table 1 (Cont'd): Characteristics of hybridomas generated against bovine oligodendrocytes. Oligo (oligodendrocytes); Astro (astrocytes); FB (fibroblasts). Numbers in cell columns indicate relative immunofluorescence intensities. 1 3 9 4 4L ft* * Fig. 3.17: Cell type-specificity as determined through immunocytochemisry. Double immunolabeling of enriched cultures of bovine oligodendrocytes (15 DIV) was demontrated with rabbit anti-galactocerebroside serum and culture supernatants of hybrid II-E3 clone A5 (E3A5/A4), and hybrid II-G2 clone B2 (G2B2/A7). Bar indicates 20 urn. A, D: Galactocerebroside-rhodamine immunofluorescence. B: E3A5/A4-fluorescein immunofluorescence. E: G2B2/A7-fluorescein-immunofluorescence. C, F: Phase contrast microscopy. Negative GFAP-immunofluorescence is not shown. 140 recognize any of the transferred elements adsorbed onto the nitrocellulose strips, and may quite possibly have been directed to oligodendroglial lipid surface determinants [Fig. 3.18]. i 141 Fig. 3.18: Western blot analysis of recognized BOL specificity. Whole bovine oligodendroglial-enriched cultures were homogenized, loaded, and run on a 10% PAGE system. Once transferred onto nitrocellulose strips, membranes were treated using immune and non-immune reagents to visualize antibody specificity and concommitantly run molecular weight standards, respectively. A: Amido black stained section containing markers and a sample of transferred elements. B: Tested hybridoma culture supernatant visualized using the standard "brown" HRP reaction products recognizing a 59,000 and/or 29,000 molecular weight protein constituent. C: Similar results to "B" but instead visualized with the blue HRP reaction product. 142 4 DISCUSSION 4.1 The Need for Cell Type-Specific Imtnunoreagents in Neurobiology. The overwhelming complexity and heterogeneity of the CNS and the increasing use of in vitro methods in neurobiology demands the ability to unambiguously identify individual neural cell types. Immunocytochemical methodologies especially in conjunction with recent advances in tissue culture preparation, have proven to be instrumental in providing sensitive probes necessary for defining differences between neural cell types in the adult and developing animal. Investigations concerning the molecular mechanisms responsible for the formation and function of the nervous system requires the ability to identify specific gene products. The characterization of these molecular components not only helps to identify a cell's unique functional assignment, but will in addition permit the elaboration of these individual determinants to be correlated to a given state of neuronal and glial differentiation; since cellular differentiation is generally succeeded or accompanied by the specific occurrence of molecules related to the performance of a cell's special function. Elucidation of many of these molecules have lead to the definition of novel cell type-specific analytes; essential for identifying neural cell subtypes or for defining previously unrecognized cell lineage even amongst morphologically indistinguishable cells. The antibody provides benefits in determining the physiological distribution of such gene products and also, therefore, a means with which to quantitate the analytes expression. 1 4 3 4.1.1 Advantages offered with the Use of Monoclonal Antibodies. One major recent impetus which has revolutionized neurobiological investigations has been the application of hybridoma technology in the generation of exquisitely specific immunological probes [for rev. see de St. Groth and Scheidegger 1980; Galfre and Milstein 1981; Langone and Van Vunakis 1986; Campbell 1986]. The generation of monoclonal antibodies directed against unique neural cell-surface and intracytoplasmic constituents accentuates positive identification and effective immunoselection of these neural cell types from mixed culture. The use of monoclonal antibodies offers as well, the potential for establishing heretofore uncharacterized glial subtypes with morphological, functional, and developmental subspecialties not yet realized with the use of heteroclonal antisera. For years now investigators have searched for ways of providing homogeneous antibodies of specificity. The overriding technical problems with this objective, however, lay in the relative physical and chemical similarities expressed between various antibodies making attempts at wholesale immunoabsorption of monospecific antibody from heteroantisera an impracticality if not a total impossibility. The selective isolation of sensitized mammalian lymphocytes (which produce individual monospecific antibody) though more pragmatic, suffers from the fact that once isolated, an antibody-producing plasma cell (or its unmaturated B-lymphocyte progenitor) does not have the capacity to be maintained in tissue culture for an extended period of time. The key was, as established in 1975 by Kohler and Milstein, to transform the antibody producing cell so that in addition to producing a single species of antibody of predefined specificity (through conventional immunization), the genetically altered cell could be maintained in vitro indefinitely. They obtained an "immortal" genotype in sensitized murine lymphocytes using somatic cell hybridization techniques, where a suspension of specifically sensitized lymphocytes was 144 brought into close proximity to and subsequently induced to fuse with (using inactivated sendai virus) a murine myeloma cell line. The myeloma cell line (in this case P3 X63-Ag8) apart from having the capacity to be maintained indefinitely in tissue culture and to produce (though not necessarily initially secrete) whole antibody, was also specifically engineered to possess an essential drug marker sensitivity. In this way, non-fused cells could be selected against, ensuring only the survival of fusion products; or hybridomas [Littlefield 1964]. 42 Choice of the Murine System in the Generation of Neural Cell Type-Specific Monoclonal Antibodies. In the present study, the choice of which mammalian system to pursue was primarily based on the availability of starting materials. Despite the nearly 12 years that has passed since the first experiments in hybridoma production, the general methods for developing monoclonal antibodies remain essentially the same. The majority of hybridomas produced thus far and used in the present study were developed from the mouse. Of the currently available compatible murine myeloma cell lines suitable for fusion, all originate from the Balb/C strain. Using an alternative inbred strain of mouse may in some cases be more beneficial as other strains have been shown to display a more vigorous immune response to a given antigen [Kastrukoff et al. 1982; 1986]. To use a mouse strain other than Balb/C necessitates, however, the need for more elaborate methods if the hybridoma is to be grown as ascites [Goding 1986]. 145 4.2.2 The Use of Balb/C Splenocytes. There have been a number of minor improvements which can accentuate the efficiency of hybridoma formation [Feit et al. 1984; Van Mourik et al. 1984; Westerwoudt et al. 1984; Fleit et al. 1984; Westerwouldt 1985]. One such modification requires that splenocytes in a whole cell suspension be selected for and isolated from a plastic-adherent cell population. By doing so, the resulting lymphocyte suspension, in the present study, was found to be largely devoid of fibroblasts and macrophages which interfere with fusion efficiency and which also have the potential for overgrowing hybridomas once in culture. The three most commonly used sources of sensitized splenocytes come from mouse, rat, and human [Goding 1986]. 4.2.2 The Use of the Myeloma Cell Line NS-1. The second most important determinant in our fusion experiment was the choice of a suitable myeloma partner. Rat hybridomas can provide certain definite advantages over those realized with the mouse system. These include quite simply, larger yields of (as high as 10X more) ascites fluid when grown as such [Galfre et al. 1979], possessing a lower frequency of reversion to non-secreting status [Campbell 1986], as well as concurrently expressing higher (as much as 30% higher) spleen immunoglobulins [Clark et al. 1983]. The relative availability and stability of the murine myeloma cell line, however, has made this system by far the most popular for the production of hybridomas. 146 43 Choice of Neural Cell Homogenate and Subfractions in the Production of Monoclonal Antibodies to Neural Cell Types. There are a variety of specialized glial and neuronal cellular elements contained in the myelinated axon region of a nerve cell. Their systematic isolation and characterization should provide a better understanding of the individual functions played by each of the constituents comprising the axon, and those which are responsible for its myelination. 43.1 The Choice of Newborn Brain Particulate Subfraction as an Alternative Immunogen to Human Axolemma-Enriched Fraction. The prime requisite in attempting to establish an anti-human axolemma and anti-bovine oligodendroglial monoclonal antibody was the availability of sufficient immunogen for splenocyte sensitization, and in the case of the anti-newborn brain particulate fraction monoclonal antibody, the availability of immunogen to serve as target analyte in the micro-ELISA screening of hybridoma culture supernatants. The need for such large amounts of human axolemma-enriched fraction considering the relative unavailability of the starting material, the time involved in preparing the fractions compounded with the comparatively poor recovery of the final product (as judged by the amounts delivered employing the techniques of De Vries 1981), prompted us to consider an alternative source of immunogen. A possible explanation for the poor recovery was in implementing alternative methods from those recommended by De Vries; specifically in the hardware used for running discontinuous sucrose density gradients. Despite maintaining relative centrifugal forces, adaptation of the only available smaller-format swing-bucket rotor and accompanying nitrocellulose tubes would have altered the relative gradient height between each interface, thus somewhat affecting relative surface areas which may have been critical for optimal 147 gradient performance. Another factor which could have significantly contributed to such poor yields of human axolemma-enriched fractions may have been associated with the use of starting material that had been maintained frozen for a prolonged period of time. Such storage may have in fact contributed significantly to autolytic changes affecting the integrity of the proteins recovered as suggested by comparable investigations involving frozen and fresh CNS material [Ansari et al. 1975]. Relative yields of human axolemma-enriched fraction, although sufficiently low to preclude its use in immunization and in subsequent micro-ELISA characterization, provided adequate material for SDS-PAGE. From Fig. 3.3 one can see that the major protein constituents of our preparation coincided with those previously reported [De Vries 1981], and that in both cases, the bulk of the elements coinciding with the human axolemma-enriched fraction possessed a molecular weight of approximately 45,000 to 60,000 Da. This latter information was of critical significance in the final decision concerning selection of which newborn brain particulate subfraction to eventually use as immunogen. It is interesting to note that despite the successful generation of a rabbit heteroclonal antiserum directed to human axolemma-enriched fraction using the described preparative techniques [Bigbee et al. 1985], a subsequent preliminary report of the development of an axolemma-specific monoclonal antibody was generated against preparations of non-CNS unmyelinated bovine sciatic nerve [Bigbee et al. 1986]. However, no further information concerning the antibody's characteristics have as yet been made available. CNS fractionation procedures should prove extremely useful in identifying alterations in the basic distribution of specific molecular analytes which are believed to occur with the various degenerating neurological diseases [Matthew et al. 1978]. The use of human newborn brain material for producing a particulate subfraction was chosen as an alternative to human axolemma-enriched fraction because sufficient amounts of starting material were at the time of the present study, made readily available, that this starting material was fresh 148 and not frozen, and that being newborn, little if any, interference from myelin cross contamination would be apparent when used in primary immunization. However, this latter feature (namely the virtual absence of myelin) would preclude the use of De Vries method as the final enrichment process is preceded by a flotation of myelinated axons in a sucrose solution; a feature which is critical to the success of this method. Alternatively, the use of a crude particulate fraction (i.e. a fraction consisting primarily of cellular and synaptic membranes), [Garey et al. 1972; Jorgensen and Bock 1974; Johnson et al. 1978; Chan et al. 1984; Heydorn et al. 1986] has been reported to provide a viable means with which to generate whole panels of monoclonal antibody to neuronal antigens [Ghandour et al. 1984] without the need for a myelinated starting material. The external membrane is perhaps the most interesting and by far the most important cellular component as seen from an immunological perspective because its antigenic specificities are readily accessible when expressed on the surface of an intact cell. These specificities are usually the only functional structures of an otherwise featureless cholesterol-lecithin-protein surface, but are nevertheless primarily responsible for mediating the reactions of a cell with its environment. However, the external membrane is, at the same time, possibly the most difficult part of the cell to recover uncontaminated from other structural material. Recall that the use of somatic cell hybridization techniques abrogates the need for extremely pure immunogen [Kohler and Milstein 1975]. Regardless of this benefit, a specific subfraction (corresponding to the major axolemma proteins as determined by SDS-PAGE) was isolated and subsequently used as immunogen. This was done in order to increase the chance of generating the desired antibody corresponding to (or in fact providing) an anti-axolemma specific immunoreactivity. Amounts of antigen from particulate fraction prepared in this manner proved adequate for immunization as well as for providing sufficient whole unfractionated particulate fraction for micro-ELISA determinations. 149 43.2 The Use of Whole Cultures of Living Bovine Oligodendroglia as an Immunizing Agent. The choice of using whole living oligodendroglial-enriched bovine cultures in the production of monoclonal antibody was prompted by the interesting results obtained from previous studies where similar starting materials were employed to produce heteroclonal antisera. Podulso 1977, and Lisak et al. 1980 produced in individual experiments heteroclonal antiserum (Rabbit and guinea pig, respectively) directed against whole-cell preparations of mammalian oligodendrocytes. In both instances immunological reaction could be removed from the antisera by absorption with isolated oligodendrocytes, but surprisingly enough not with myelin, and in the latter case, not even with GalC [Lisak et al. 1986]. Heteroclonal antisera produced from similar starting materials has also been produced by Abramsky et al. (1978), Traugott et al. (1978), and Kim et al. (1985). Al l were found to immunolabel either surface and/or intracellular oligodendroglial specificities as observed with brain sections or neural cell cultures. Many of these investigations, however, were at the mercy of the technical considerations associated with using heteroclonal antisera, including: variable titers, the expression of questionable specificity, and their limited supply [see Sec. 1.3]. Nevertheless, investigations which have entailed the immunization with whole oligodendroglial preparations have resulted in heteroclonal antisera possessing interesting and unusual characteristics [Poduslo et al. 1977; Lisak et al. 1980]. Relatively similar results have been presently achieved through the implementation of somatic cell hybridization techniques, as modified with the use of intrasplenic injection: but which offer, in addition, the many advantages realized with the generation and use of a monoclonal immunological probe. 150 4.4 The Implementation of Alternative Methods over Conventional Immunization Regimes. 4.4.1 In Vitro Immunization. One recently described method which almost totally circumvents the problems associated with the production and subsequent characterization of monoclonal antibodies to a restricted supply of antigen has been reported, where whole cultures of pre-sensitized murine splenocytes were incubated in the presence of the target antigen supplemented with adjuvant peptide [Boss 1984; Reading 1982]. Adjuvant peptide in this particular application has been reported to facilitate the presentation of the antigen to, and subsequently enhance the specific sensitization of murine lymphocytes in vitro [ Eggers and Tarmin 1984; Specter et al. 1978]. ln vitro immunization not only has the advantage of generating sensitization while only requiring microgram quantities of antigen, but of also producing antibody secreting fusion products in a matter of weeks as compared to the months involved in conventional immunization regimes. There are in fact a number of investigations which have successfully used in vitro methods for producing monoclonal antibodies from mouse [Matthew and Patterson 1983; Pardue et al. 1983; Borrebaeck 1984; Carlsson et al. 1985; Ossendorp et al. 1986], rat [Bodeus et al. 1985], and even specifically sensitized human [Eggers and Tarmin 1984; Cole et al. 1984; Lagace and Brodeur 1985] lymphocytes. In vitro immunization, however, suffers from the need for specifically formulated growth media and the subsequent maintenance of splenocytes in culture; a feature the cells do not readily tolerate. 151 4.4.2 Instrasplenic Immunization. Information concerning the specific efficiency (i.e. number of culture plate wells producing specific antibody divided by the number of culture plate wells exhibiting cell growth X 100) was not provided in the majority of various investigations concerning the production of monoclonal antibody making objective comparisons on the efficiency of fusion between the various methods impossible. However, the numbers of antibody producing primary clones secreting desirable antibody generated in individual investigations (that is when whole murine and rat spleens were used), seems to suggest that both in vitro and intrasplenic immunization techniques worked equally well in the successful generation of usually more antibody producing hybrids than original/conventional schedules In the present study, an alternative though similar approach was employed which again only required microgram quantitites of a given antigenic preparation but which could also adequately generate sensitized lymphocytes in even less time (days rather than weeks). This method has eliminated the need for specifically supplemented culture media, and has been reported in the production of monoclonal antibodies to such specificities as those expressed by human fibrin degradation products, lymphokines, immunoglobulins, and peripheral blood cells [Spitz et al. 1984; Gearing et al. 1985]. In adopting the techniques of Spitz et al. 1984, we were able to demonstrate that this method was not only well suited for the production of a monoclonal antibody to a human newborn brain particulate (gel-excised) subfraction, but also to whole living bovine oligodendroglial cultures. Intrasplenic immunization of the newborn brain particulate subfraction-(b), and of live BOL cells essentially introduced a localized, relatively high concentration of immunogen to the target organ thereby maximizing the frequency of specifically sensitized B-lymphoblasts in the spleen [Luben et al. 1982; Gearing et al. 1985], 152 The results described have substantiated previous investigations illustrating some of the advantages seen in using primary intrasplenic immunization [Thorpe et al. 1984] over those employing several conventional immunization schedules, while maintaining relatively equal if not better success in generating positive fusion products. In using this technique, sensitization was accomplished in 3 to 4 days from primary immunization to the time of fusion without the need for special adjuvants or immunization regimes. Finally, the use of this specific method of generating sensitized splenocytes favours the development of an immunoglobulin class which is peculiar to an early immune response: namely IgM. A n IgM-class monoclonal antibody in turn offers the benefits realized with having and using a multivalent antibody. There are advantages to the use of IgM immunoglobulin, for example, in specific immunoassay systems (cytotoxicity assays) or in studies requiring proteolytic-resistant antibody with relatively low affinity (when not compensated for by multivalence [Goding 1986]). There is also special neuroimmunological implications considering the fact that the IgM class of immunoglobulin (which normally only represents 5% of the total serum globulin) is usually the predominant immunoglobulin class represented by auto-antibodies [Kahn 1985; Ernerudh et al. 1986; Felgenhauer and Schadlich 1987; Hays et al. 1987; Jonsson et al. 1987; Rudnicki et al. 1987] including those reportedly expressed in multiple sclerosis [Pedersen et al. 1983]. 4.5 Generation of an Anti-Newborn Brain Particulate Fraction Monoclonal Antibody. 45.1 The Generation of IgM Class of Monoclonal Antibody to CNS Particulate Fraction. In the first half of the present study, generation of a hybridoma cell line, PF(b)-C3A8, was demonstrated as producing an IgM class of antibody. The generation of an IgM class of 153 monoclonal antibody would normally be a relatively unexpected occurrence. Adopting the methodologies used for intrasplenic immunization (and the relatively short period of time necessary for sensitization to take place) produced, however, splenocytes typical of an "early" immune response. The subsequent use of these cells for fusion, therefore, tends to favour the generation of hybridomas secreting an antibody class associated with such an early response, and with it provides the many potential benefits realized with having an IgM class of monoclonal antibody. 4.5.2 Cell type Specificity Demonstrated by Pf(b)-C3A8. The reactivity of the antibody PF(b)-C3A8 with human newborn brain particulate fraction was initially demonstrated by micro-ELISA and examined concomitantly with, and subsequent to, initial screening of antibody. Although primary screening did not demonstrate overwhelming numbers of antibody producing hybridomas, the final isolation of a monospecific antibody which discriminately labeled newborn astroglial subpopulations, though not entirely anticipated, is not a novel occurrence when using newborn brain material as primary immunizing agent [see sec. 4.5.4]. Furthermore, the use of gel subfractionation techniques in the successful preparation of a variety of both heteroclonal [Yen and Fields 1981; Dunbar 1981; Bravo et al 1983; Krol 1981] and monoclonal antigen-specific antibody [Tracy et al. 1983] have been described. The present use of gel excised particulate subfraction from human newborn brain did not (as originally thought) provide an antibody specific for axolemma but instead resulted in the generation of an antibody-secreting clone which could discriminate between newborn astroglial subclasses, and which was almost certainly generated against an astroglial "contaminant". This "contaminating" element evidently possessed a similar relative mobility as adult human axolemma-enriched fraction. Furthermore, the use of SDS-PAGE subfractionation appears to have resulted in a greater restriction of the varieties of monoclonal antibody developed [Tracy et al. 1983] other 154 than which would have probably been realized had the whole particulate fraction been used for injection. This would possibly explain the lack of immunolabeling of other various glial elements which would have been evidenced by the presence of more than one type of antibody secreting hybridoma at primary screening. 45.3 The Electroblotting Characteristics of the Recognized Analyte. The monoclonality of the Pf(b)-C3A8 hybridoma was determined by its Western blot characteristics in that of all the proteins transferred, only a single protein band (of apparent molecular weight 50,000 Da) was recognized. 4-5.4 Comparison of Pf(b)-C3A8 to Other Similarly Raised Monoclonal Antibodies. There exists previous other examples in the literature where crude homogenates have been used as the immunizing agent in the production of heteroclonal [Schachner et al. 1975; 1982a] and monoclonal antibodies directed against novel neural epitopes [Lagenaur et al. 1980]. Homogenates of whole embryonic rat brain [Yamamoto et al. 1986], corpus callosum [Sommer et al. 1981] and preparations of rat synaptosomal membranes [Hawkes et al. 1982a; De Bias 1984] and/or particulate fractions from newborn mouse brain cerebellum [Lagenaur et al. 1980; Ghandour et al. 1984], have for example, led to the development of the monoclonal antibodies 8-9/G7 and 8-6/A2, the monoclonal antibody C l , the monoclonal antibodies H8 to HI2, and the monoclonal antibody M l , respectively. These monoclonal antibodies have in fact been shown to recognize specific glial and neuronal structures, whole cell populations, as well as their subtypes. In fact, M l and C l , not unlike PF(b)-C3A8, recognize different subclasses of specific astroglial cells in the murine system [Lagenaur et al. 1980; Sommer et al. 1981]. These monoclonal antibodies are distinct, however, from PF(b)-C3A8 in a few important primary characteristics. First, M l and C l discriminate only 155 intracellular analytes and thereby display a similar localization as that of, for example, GFAP The PF(b)-C3A8 determinant, on the other hand, is only immunocytologically expressed on the surface of astrocytes. Furthermore, whereas M l and C l discriminate adult murine astroglial subclasses, PF(b)-C3A8 could only be demonstrated on cultures originating from newborn material. The observation that the expression of PF(b)-C3A8 antigen is modulated during the normal development of specific astrocyte subclasses, may represent recognition of a reactive or proliferative phenotype expressed only in unmaturated astroglial cells [see Introduction]. PF(b)-C3A8, as suggested by other antibodies which discriminate among subclasses of astrocytes in normal cultures of astroglia [Lagenaur et al. 1980], may also provide an indication of an astrocyte's response to a mechanically induced pathological condition (Latov et al. 1979]. Until recently, the specific origin of the elements comprising particulate (crude membrane) fractions could only be inferred on the basis of indirect lines of evidence. The occurrence of antigenic modulation peculiar to a proliferative response in normal ontogeny or to that which may be induced through mechanical injury, has not only been indicated by the presently generated monoclonal antibody (and the monoclonal antibodies M l and Cl) , but has also been suggested in a study by Seyfried et al. 1982, where it was demonstrated that the ganglioside Gr_)3 is preferentially expressed on a subclass of (reactive) astrocytes. A similar situation exists with respect to differential immunolabeling of the ganglioside G Q as demonstrated with the monoclonal antibody A2B5 [Abney et al. 1981; Raff et al. 1983a, b; Miller and Raff 1984; Raff and Miller 1984; Temple and Raff 1985,1986; Ffrench-Constant and Raff 1986]. The possibility that the analyte recognized in the present study is of a particular class of ganglioside (as suggested by surface immunolabeling and by studies indicating an ontogenically restricted expression of specific classes of the glycolipids G Q and Grj)3 previously mentioned), however, is not in agreement with the capacity of the present analyte to be electroblotted; which is, in fact, characteristic of a protein or protein-complex. 1 5 6 The monoclonal antibody (SSEA-1), generated to a specific glycolipid of a teratocarcinoma (F9) cell line [Solter and Knowles 1978], has been described as having the capacity of discriminately labeling developmentally restricted expression of normal fetal antigens on specific subclasses of whole newborn cerebellum [Lagenaur et al. 1982b]. Despite the selective immunolabeling demonstrated by SSEA-1 for only a specific astrocyte subclass, its non-protein nature negates the possibility that a similar analyte to that which was recognized in the present study had been identified. In an investigation where a human tumor cell line was used as the primary immunogen (in this case the neuroblastoma-IMR8), monoclonal antibody PI-153/3, which in addition to recognizing an analyte common to retinoblastoma, neuroblastoma, and to a lesser degree glioblastoma, was also shown to specifically recognize brain tissue homogenates of human fetal origin. The specific fetal brain constituent(s) immunolabeled by PI-153/3, however, were not identified [Kennett and Gilbert 1979]. One final example of monospecific antibodies which have been generated with the capacity to recognize either fetal of specific subclasses of astroglia have been reported in a recent study by Dickson et al. 1983. These investigators have used two monoclonal antibodies: M l / N l of Kemshead et al. 1981,(and not to be confused with M l of Lagenaur et al. 1982a, and N l of Schnitzer et al. 1984) and "308", which detect cell surface antigens present exclusively on a human fetal astroglial subclass [Dickson et al. 1983]. In this way M l / N l and 308 posses almost identical immunocytochemical characteristics as those exhibited by PF(b)-C3A8. The primary immunizing agent in the development of M l / N l (and similar to the studies by Kennett and Gilbert, 1979) was the human neuroblastoma cell line, CHP-100. In contrast, however, the independently developed monoclonal antibody, 308, was generated against whole fetal brain homogenate, and in this respect, resembles most closely the preparative and subsequent immunocytochemically defined characteristics of PF(b)-C3A8. 157 A further comparison of the generated immunoglobulin subclass as well as the analyte(s) recognized by both M l / N l and 308 monoclonal antibody, however, is not possible as this information has not as yet been made available [Dickson et al. 1983]. PF(b)-C3A8, as a surface specific antibody would allow a means for discriminately immunoselecting from whole cultures of fetal CNS material the recognized subclass of living astroglia [for rev. see Nicola 1982; Basch et al. 1983] without the need for alternative, more elaborate methodologies involved with the immunoselection of viable cells when based on the expression of an intracytoplasmic specificity [Grabman 1970; Graessmann and Graessmann 1976; Graessmann et al. 1977;Floros et al. 1981; Arnheiter et al. 1984]. The clone PF(b)-C3A8, unfortunately has since stopped producing antibody, making further characterizations at the present time impossible. IgM class specific monoclonal antibody producing clones have been described in general as being relatively susceptible to the adverse conditions encountered as when in preparing the cells for freezing or in thawing, as well as to culture conditions anything other than optimal. Approximately 40% of all IgM producing hybridoma producing clones will, for example, spontaneously stop producing antibody. As well, only 10% to 20% of IgM clones will demonstrate viability after thawing frozen stock [Campbell 1986]. This is one reason why it is recommended that multiple/duplicate cultures be maintained. During the present study, however, a particularly toxic lot of fetal calf serum (Celled Silver, GIBCO) was used to supplement hybridoma feeding medium when other suitable sources became depleted. The clones died or alternatively ceased antibody production soon thereafter. Initial efforts to recover specific antibody producing hybridomas from the available frozen stocks have been initially unsuccessful. However, many more frozen samples remain to be thawed and tested. The outcome of fusion experiments involving BOL sensitized splenocytes, has proved much more successful. 158 4.6 Generation of an Anti-Bovine Oligodendrocyte Monoclonal Antibody. Oligodendrocytes have been found responsible for the formation and maintenance of the myelin sheath in the central nervous system [Raine 1981; Norton 1983; Pfeiffer 1984] and, therefore, are believed to be a primary target in some of the human demyelinating diseases including multiple sclerosis [Traugott and Raine 1981; McFarlin and McFarland 1982; Raine 1983; Bologa 1985]. Several recent investigations have demonstrated that oligodendroglia (and astrocytes [see Sec. 1.6.1] ) may be induced to express M H C class I antigens (H-2, but not Ia) on their surface in response to exogenous factors (lymphokines, concanavalin A, or even specific viruses) and as such may present themselves as possible target cells in M H C -restricted T-cell dependent cytotoxicity [Wong et al. 1984; Suzumara and Silberberg 1985; Suzumura et al. 1986; Hirayama et al. 1986]. Whether they can demonstrate or be induced to express class II or Ia surface glycoproteins, seems to be as yet undecided [Lisak et al. 1983; Kim et al. 1985]. However, in unraveling the mysteries associated with the pathogenicity of the many neurological disorders, the ability to generate pure preparations of oligodendrocytes will be of great value. Several methods have been reported in which adult mammalian oligodendrocytes suitable for long term culture were enriched from CNS material containing non-oligodendroglial constituents [Lisak et al. 1981; Hirayama et al. 1983; Kim et al. 1983; Gebicke-Harter et al. 1984]. We have recently described the methods (using both heteroclonal antisera that can specifically recognize GalC and monoclonal antibody directed to MAG) which can provide adult mammalian oligodendroglia exceeding 98% purity [Smyrnis et al. 1986; Kim et al. 1987]. It has been established that in culture, conventionally raised antisera directed against CNPase [Sprinkle et al. 1983], GalC [Raff et al 1978b], MBP [Mikoshiba et al 1985], M A G [Inuzuka et al. 1985], and Sulphatide [Raff et al 1979] specifically immunolabels both surface 159 and/or intracytoplasmic constituents of oligodendroglia. 4.6.2 Subclass Determination and Immunolabeling Characteristics of Anti-BOL Antibody. In the second half of the present study, whole cultures of living BOL were used for intrasplenic immunizations in the development of a panel of hybridoma clones secreting monospecific (IgM and IgG2b class) antibody. The cell type-specific reactivity of these antibodies (as determined by indirect dual-immunolabeling of bulk-isolated BOL cultures) was found to consist primarily of anti-oligodendrocyte monospecificity. It was further determined that all element(s) immunolabeled were surface components and that the surface analyte (a protein or protein-complex) as demonstrated with electroblotting was of apparent molecular weight 29,000 and 59,000 Da. The surface epitope(s) as recognized by our panel of monoclonal antibodies remain to be fully characterized. 4.6.2 Comparison of Anti-BOL Antibodies to Other Similarly Raised Monoclonal Antibodies. The anti-oligodendrocyte specific monoclonal antibodies generated in the present study, to a great extent, displayed similar immunocytochemical characteristics consistent with other monoclonal antibodies raised against this glial cell type. To date, many of the monoclonal antibodies generated against oligodendrocytes have been raised to a purified pre-defined specificity. Biochemical and immunocytochemical characteristics, however, have distinguished many of these monoclonal antibodies as recognizing a specificity different from those developed in the present study. 160 In separate recent studies initiated by Fujishiro et al. 1986, and Brenner et al. 1986a, oligodendroglial specific immunolabeling could be demonstrated by monoclonal antibodies (M-12 and anti-CNP mAb, respectively), generated against ammonium acetate-extracted phenyl sepharose CL-4b-purified bovine CNPase. In contrast to our antibodies, however, these hybrid products could only recognize intracytoplasmic specificities, which incidentally, corresponded to the Wla and Wlb components of CNPase [Fujishiro et al. 1986; Brenner et al. 1986a]. Similarly studies which have generated monoclonal antibodies that immunolabel oligodendrocytes by specifically recognizing the immunogenic protein (or polypeptide) constituents of MBP are not demonstrable on the surface of these glial cells [for rev. see Day and Potter 1986] and for this reason were not considered as recognizing similar specificities as those which were immunolabeled by our anti-BOL antibodies. Sommer and Schachner 1981, using white matter from bovine corpus callosum (a starting material which contains the highest relative amount of oligodendroglia in whole brain [Raff et al. 1979]), have generated a panel of monoclonal antibodies (Ol to O i l ) directed against oligodendrocytes [Schachner et al. 1975; Kettenmann et al. 1985]. However, subsequent characterization of these oligodendrocyte-specific "O" epitopes has revealed that these IgM-class of monoclonal antibodies are directed against glycolipid constituents (presumably anti-GalC [Ol, 02 and 07], anti-suphatide [03 to 06 and 08, 09], and to an as yet undefined glycolipid: O10 [Kettenmann et al. 1985]), and not directed against an oligodendrocyte-specific surface protein (except O10 on which there is no additional information) as was demonstrated by our antibodies in their ability to immunolabel electroblotted oligodendroglial constituents [Leoni et al. 1986]. In a similar manner, the H8/H9 monoclonal IgG3 class antibody of Ranscht et al. 1982, and the IgM-class M-anti-GalC monoclonal antibody as generated by Rotsammi et al. 1983 (which were initiated against bovine hippocampus synaptic plasma membranes and lower spot bovine cerebroside, respectively), were also demonstrated (again using high-performance thin-layer chromatography [HPTLC]) to be directed against the surface glycolipid GalC, and are therefore immunochemically distinct from the antibodies presently generated. 161 Furthermore, monoclonal antibodies directed against the surface sialic acid bearing glycosphingolipids such as G J 3 3 as recognized by the IgG3 class monoclonal antibody AbR24 [Pukel et al. 1982], and G Q as recognized by the IgM-class monoclonal antibody A2B5 [Kasai and Yu 1983], must be immunolabeling oligodendroglial cell surface specificities necessarily different from those recognized by our monoclonal antibodies. The recent generation of a monoclonal antibody (10F6) which is directed to a specific class of functionally similar sialoglycoproteins or cell adhesion molecules [Grumet et al. 1984] has also been demonstrated on the surface of these neural cells. However, evidence suggests that these C A M molecules, in spite of being surface proteins, appear to be much larger than what was recognized by our monoclonal antibodies [Grumet et al. 1985; Kruse et al. 1985; Thor et al. 1986; Pollerberg et al. 1986] as suggested by immunoblot. Furthermore, immunocytochemical characterization has demonstrated that N-CAM} 8o/D2 and the Gjyi glycoprotein (as recognized by the monoclonal antibodies N - C A M 4 and 5 [Grumet et al. 1984], and L2 [Kruse et al. 1985], respectively), immunolabel oligodendrocytes as well as astrocytes which is not in total agreement with that which was immunolabeled by the monoclonal antibodies generated in the present study [see Results]. There was evidence, however, to suggest that some of the monoclonal antibodies developed in the present study demonstrated similar properties to those which have been generated to specificities expressed by the myelin associated glycoprotein. The apparently high molecular weight (100 KDa) characteristic of intact M A G precludes the suggestion that HNK-1 (Leu-7) monoclonal antibody may be recognizing an identical specificity on the surface of oligodendroglia as those which were labeled with our antibodies. M A G (expressing both polypeptide and carbohydrate antigenic moieties) however, when treated with trifluoromethanesulphonic acid (TFMS) becomes deglycosylated [Ilyas et al. 1984], and that deglycosylation of M A G , will decrease its relative 162 molecular weight from 100 KDa to about 70 KDa. In fact, it has been reported by Inuzuka et al. 1984, that monoclonal antibody Leu-7 (which is equivalent to anti-MAG antibody and HNK-1 monoclonal antibody) specifically binds to both a carbohydrate epitope of M A G as well as to a group of lower molecular weight (20K Da to 26K Da) glycoproteins; possibly recognizing specificities which represent autolytic changes or products similar to those which have been demonstrated to occur with the N - C A M molecule [Edelman 1985]. In addition, the monoclonal HNK-1 has been also found to react with several other lower molecular weight glycoproteins present in mammalian nervous system [O'Shannesy et al. 1985]. Lithium-diiodosalicylate-phenol extraction was used to purify this quantitatively minor glycoprotein (MAG) for subsequent use in the generation of XV1-2, GPD3, and D7E10; all independently generated murine anti-MAG monoclonal antibodies [Tanaka et al. 1985; Dobersen et al. 1985b; Nishizawa et al. 1986]. Some are in fact able to discriminate between carbohydrate and protein analytes suggesting that immuno-discrimination of M A G in the PNS [Frail et al. 1985] may be attributed to the expression of a common carbohydrate moiety [Nishizawa et al. 1986]. However, no mention is made in these studies on the ability of the independently developed monoclonal antibodies to immunolabel electroblots or anything other than either the intact [Tanaka et al. 1985; en et al. 1985b; Nishizawa et al. 1986] or degly cosy lated [Nishizawa et al. 1986] forms of M A G . The neurobiological significance of possibly possessing an anti-MAG monoclonal antibody, apart from those examples discussed in the Introduction [see Sec.], comes from immunocytochemical investigations which demonstrate monoclonal antibodies raised to this glycoconjugate as specifically recognizing a carbohydrate specificity that is concomitantly expressed on a subset of a human lymphoctyes; including those with natural killer activity [Kruse et al. 1984; Tanaka et al. 1985; Dobersen et al. 1985b; Nishizawa et al. 1986; Ilyas et al. 1986]. Alternatively, the murine monoclonal antibodies HNK-1 and Leu-7 [Sato et al. 1983], raised against a lymphoblastoma T-cell line differentiation antigen; HSB-2 [Abo and 163 Balch 1981], has been demonstrated to not only specifically discriminate a surface carbohydrate determinant on a population of N K cells but to also discriminately immunolabel oligodendroglia in mixed culture [Schuller-Petrovic et al. 1983; McGarry et al. 1983; Kruse et al. 1984; Kim et al. 1984a; Tanaka et al. 1985]. The Leu 7 / H N K - l monoclonal antibody is routinely used to identify natural killer T lymphocytes in peripheral blood [for rev. see Porwit-Ksiazek 1983a, b]. We have recently substantiated findings from previous investigations demonstrating high intensity HNK-1 immunofluorescence labeling of oligodendrocyte-enriched neuron-free cell suspensions [Smyrnis et al. 1986]. The significance of demonstrating molecular specificities common to both the immune and central nervous system has been previously addressed. 4.7 Summary and Conclusion. The development and use of monoclonal antibodies directed against specificities associated with normal and abnormal nervous system tissue will augment our basic understanding of how the nervous system operates, and may form the basis for identifying the circumstances inherent to its pathogenesis. With the advent of monoclonal antibody technology the ability to generate monospecific antibody to both simple and complex neural antigens becomes universally unrestricted, providing with it the ability with which to demonstrate specific distribution of various analytes within the nervous system. The identification and manipulation of cell type-specific antigens by monoclonal antibodies have already been extensively applied in investigations concerning the cellular immunology of distinct immune-cell subpopulations. Comparable efforts in neurobiology are yielding a growing number of immunological probes which can efficiently discriminate between the traditional classes of neural cells in culture. Some have even used the fruits of somatic cell hybridization to detect unique specificities among morphologically, functionally, and biochemically indistinguishable cell types, and thus more clearly define cell lineage as well as 1 6 4 establishing previously unrecognized neural subclasses. In summary, we have combined the methods for intrasplenic primary immunization with the established techniques of somatic cell hybridization to successfully generate two families of monoclonal antibody-producing hybridoma cell lines. By fusing myeloma cells to lymphocytes sensitized in this manner, we have shown that it is possible to develop hybridomas of specific immunoglobulin class and subclass. 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