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The application of immunology to food science, two studies : production of monoclonal antibodies (Mabs)… Jarvis, Sandra Marie 1989

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THE APPLICATION OF IMMUNOLOGY TO FOOD SCIENCE: TWO STUDIES: PRODUCTION OF MONOCLONAL ANTIBODIES (Mabs) SPECIFIC FOR AN ENTEROPATHOGENIC E.COLI (EPEC); DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR 6-N-ACETYLGLUCOSAMINIDASE (NAGase) by Sandra Marie Jarv i s B . S c . ( A g r ) , Un ivers i ty of B r i t i s h Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Food Science) We accept t h i s thes is as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1989 © Sandra Marie J a r v i s , 1989 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. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Two hybridoma clones, labelled 4D10 c l and 2H4 H12, produced monoclonal antibodies which recognized the outer membrane of an enteropathogenic Escherichia c o l i (EPEC) 0142:K86:H6 in an enzyme-linked immunosorbent assay (ELISA) and the whole c e l l in an immunofluorescence assay. Large scale production of the monoclonal antibodies was accomplished through ascites production In balb/c mice. P u r i f i c a t i o n of the ascites f l u i d was achieved by gel f i l t r a t i o n and ion exchange chromatography. Isotyping of the p u r i f i e d fractions showed 4D10 Cl to be an lgG2 and 2H4 H12 an IgM. These monoclonal antibodies were screened by immunofluorescence assay against several pathogenic and non-pathogenic strains of E . c o l i in addition to other Enterobacteriaciae. Results of the screening showed these antibodies to be s p e c i f i c for the E . c o l i serotype to which they were raised. Minimal c r o s s - r e a c t i v i t y with other Enterobacteriaceae was observed. In a separate and concurrent project, the use of an ELISA capable of detecting G-N-acetylglucosaminidase (NAGase) was examined. White Leghorn hens were injected with commercially prepared bovine NAGase. Eggs were collected and the immunoglobulin f r a c t i o n separated from the egg yolk by polyethylene glycol p r e c i p i t a t i o n followed by ion exchange on a DEAE-Sephacel column. The use of the p u r i f i e d immunoglobulins was examined in a sandwich, double-sandwich and a competitive ELISA. A s t a t i s t i c a l l y s i g n i f i c a n t i i standard curve for the d e t e c t i o n of NAGase was s u c c e s s f u l l y derived using a double-sandwich ELISA when r a b b i t immunoglobulin was used to coat the microwell p l a t e s . This assay was used to measure the NAGase co n c e n t r a t i o n i n press j u i c e and f i s h e x t r a c t of f r e s h and frozen salmon muscle samples. The r a t i o of the NAGase concent r a t i o n i n the press j u i c e to the t o t a l NAGase concentration was compared. No s i g n i f i c a n t d i f f e r e n c e was found between the c a l c u l a t e d c o n c e n t r a t i o n r a t i o s of the fr e s h muscle samples and samples frozen for 1 week at -20°C. i i i TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i i LIST OF FIGURES i x ACKNOWLEDGEMENTS x i i i INTRODUCTION 1 LITERATURE REVIEW A. Antibodies and the Immune Response 3 1. The Immune System 3 2. Antibody Production 4 3. A n t i b o d i e s : Structure and Function 4 4. Antigens and Antigen-Antibody I n t e r a c t i o n 8 5. Chickens as a Source of Antibody 9 B. Monoclonal Antibodies 10 1. H i s t o r y 10 2. Monoclonal Antibody Production 11 a. Basic P r i n c i p l e 11 b. Immunization 11 c. Myeloma C e l l s and the HAT S e l e c t i o n System ...13 d. Fusion 14 3. Advantages of Monoclonal Antibodies 14 4. A p p l i c a t i o n s to Food Science 15 C. E s c h e r i c h i a c o l i 17 1. E . c o l i and the Incidence of I n f a n t i l e D i a r r h e a l Disease 17 2. Types of E . c o l i 18 3. Mode of P a t h o g e n i c i t y of EPEC 19 4. Detection of EPEC 21 D. Immunoassays 22 1. I n t r o d u c t i o n 22 2. Enzyme-Linked Immunosorbent Assay 23 3. A p p l i c a t i o n s of ELISA to Food Science 24 4. Use of ELISA to detect 6-N-Acetylglucosaminidase (NAGase) 27 i v MATERIALS AND METHODS PART I: MONOCLONAL ANTIBODY (Mab) STUDY A. Hybridoma Production 31 1. Outer Membrane Preparation 31 2. Immunization of Mice 33 3. Growth of Myeloma C e l l Line 33 4. Fusion 34 B. Expansion 35 C. Recloning 36 D. Freezing for Long Term Storage 37 E . Asc i tes Production 37 F . P u r i f i c a t i o n of Asc i tes F l u i d 38 1. Gel F i l t r a t i o n Chromatography 38 2. Ion-Exchange Chromatography 38 G. Detection of Antibody A c t i v i t y Towards E . c o l i 39 1. Enzyme-Linked Immunosorbent Assay (ELISA) 39 2. Immunofluorescence 40 H. SDS-Polyacrylamide Gel Electrophores is 42 1. Method A 42 2. Method B 43 I . Character izat ion of Mabs 44 1. Isotyping of Mabs 44 2. Immunoblot Assay 45 J . Preparation of Po lyc lona l Antiserum to E .co l1 47 1. E . c o l i Sample Preparation 47 2. Immunization of Chickens 47 3. I so la t ion and P u r i f i c a t i o n of Chicken IgY 47 K. Ammonium Sulphate P r e c i p i t a t i o n 49 L . Prote in Determination 49 PART I I : DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) A. Immunization Procedures 50 1. Immunization of Chickens 50 2. Immunization of Rabbits 50 v B. Immunoglobulin P r e p a r a t i o n 51 1. I s o l a t i o n and P u r i f i c a t i o n of chicken igY 51 2. i s o l a t i o n of Immunoglobulins from Rabbit Blood Serum 51 C. P r e p a r a t i o n of Anti-NAGase - A l k a l i n e Phosphatase Conjugate 52 1. Glutaraldehyde Method 52 2. Periodate Oxidation Method 53 D. Pre p a r a t i o n of Press J u i c e and F i s h E x t r a c t s from F i s h Muscle 53 E. Enzyme-Linked Immunosorbent Assays 55 1. I n d i r e c t ELISA 55 a. Detection of Antibody A c t i v i t y Towards NAGase 55 b. Determination of the Working D i l u t i o n of A n t i -NAGase - A l k a l i n e Phosphatase Conjugates ....56 2. Competitive ELISA 56 3. Double-Sandwich ELISA ...57 F. R a d i a l Immunodiffusion 57 G. SDS-Polyacrylamide Gel E l e c t r o p h o r e s i s 58 H. S t a t i s t i c a l A n a l y s i s 58 RESULTS AND DISCUSSION PART I: PRODUCTION OF MONOCLONAL ANTIBODIES (Mabs) SPECIFIC FOR AN ENTEROPATHOGENIC E.COLI (EPEC) A. Production of S p e c i f i c Mab Secr e t i n g Hybridomas 60 1. Hybridoma Production 60 2. Recloning 64 3. Immunofluorescence Screening 65 B. Batch Production and P u r i f i c a t i o n of Mabs 70 C. C h a r a c t e r i z a t i o n of the Mabs and t h e i r Antigens 89 1. Immunoreactivity 89 2. Isotype A n a l y s i s 91 3. Immunofluorescence Screening 93 4. Immunoblot A n a l y s i s 96 D. Conclusion 105 v i PART I I : USE OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) TO DETECT fl-N-ACETYLGLUCOSAMINIDASE (NAGase) A. Production of Antibodies S p e c i f i c f o r NAGase 110 B. Prepa r a t i o n of Antl-NAGase - A l k a l i n e Phosphatase (ALP) Conjugates 115 C. A p p l i c a t i o n of an ELISA to Detect NAGase 121 1. Competitive ELISA 121 2. Double-Sandwich ELISA 125 D. A p p l i c a t i o n of a Double-Sandwich ELISA to Detect NAGase i n F i s h Muscle 128 E. Conclusion 134 BIBLIOGRAPHY 136 v i i LIST OF TABLES Tab le I Number and Percentage of Wel l s C o n t a i n i n g S p e c i f i c A n t i b o d y P r o d u c i n g Hybridomas 63 Tab le II Numbers of P o s i t i v e Wel l s S e l e c t e d for R e c l o n i n g and Numbers of R e s u l t i n g P o s i t i v e S i n g l e Clones 66 Table I I I R e s u l t s of the P r e l i m i n a r y S c r e e n i n g of S e l e c t e d Mabs A g a i n s t a Pane l of E n t e r o b a c t -e r i a c e a e by Immunofluorescence Assay u s i n g Supernatant F l u i d from Hybridoma C u l t u r e s . . . . 6 8 Table IV R e s u l t s of I s o t y p i n g of Mabs 92 Tab le V R e s u l t s of the Second S c r e e n i n g of S e l e c t e d Mabs A g a i n s t a Pane l of E n t e r o b a c t e r i a c e a e by Immunofluorescence Assay u s i n g P u r i f i e d Mabs 94 Table VI R e s u l t s of Immunoblott ing v s . Immunofluor-escence Assay 104 Tab le VII L e v e l s of 6 - N - a c e t y l g l u c o s a m i n i d a s e (NAGase) i n F r e s h and F r o z e n Salmon Samples as Determined by a Double - sandwich ELISA 129 Table V I I I C o n c e n t r a t i o n R a t i o s of 6 - N - a c e t y l g l u c o s a m i n -idase (NAGase) i n F r e s h and F r o z e n Salmon Muscle Samples ( c a l c u l a t e d from T a b l e V I I ) . . 131 v i i i LIST OF FIGURES F i g u r e 1 F i g u r e 2 F i g u r e 3 F i g u r e 4 F i g u r e 5 F i g u r e 6 F i g u r e 7 F i g u r e 8 F i g u r e 9 B a s i c s t r u c t u r e o£ an a n t i b o d y molecule (adapted from C a l v a n i c o , 1984 ) 5 O u t l i n e of a common p r o t o c o l used to produce hybridomas (Bankert et a l . , 1984 ) 12 P r o t o c o l f or Mab p r o d u c t i o n 32 Hybridoma c l o n e s as seen under a l i g h t m i c r o -scope ( b r i g h t f i e l d ) in a s i n g l e w e l l of a micro w e l l p l a t e a f t e r a p p r o x i m a t e l y 10 days of growth i n AHAT media (400X m a g n i f i c a t i o n ) 62 E l u t i o n p r o f i l e of Mab 2E1 H6 s e p a r a t e d by g e l f i l t r a t i o n chromatography. Immunoreac t iv i ty as determined by an i n d i r e c t ELISA i s a l s o shown. Column: S e p h a c r y l S-300 (1.8 X 34 cm); E l u t i n g b u f f e r : 0.1 M T r i s - H C l , pH 8, w i t h 0.5 M N a C l ; Flow r a t e : 2 m l / h ; F r a c t i o n s : 2 m l . . . . , 71 E l u t i o n p r o f i l e of Mab 2F9 B3 s e p a r a t e d by g e l f i l t r a t i o n chromatography. I m m u n o r e a c t i v i t y as determined by an i n d i r e c t ELISA i s a l s o shown. Column: S e p h a c r y l S-300 (1.8 X 34 cm); E l u t i n g b u f f e r : 0.1 M T r i s - H C l , pH 8, w i t h 0.5 M N a C l ; Flow r a t e : 2 m l / h ; F r a c t i o n s : 2 m l . . . . 72 E l u t i o n p r o f i l e of Mab 4D10 CI s e p a r a t e d by g e l f i l t r a t i o n chromatography. Immunoreac t iv i ty as de termined by an i n d i r e c t ELISA i s a l s o shown. Column: S e p h a c r y l S-300 (1.8 X 34 cm); E l u t i n g b u f f e r : 0.1 M T r i s - H C l , pH 8, w i t h 0.5 M N a C l ; Flow r a t e : 2 m l / h ; F r a c t i o n s : 2 ml 73 E l u t i o n p r o f i l e of Mab 2H4 H12 s e p a r a t e d by g e l f i l t r a t i o n chromatography. Immunoreac t iv i ty as de termined by an i n d i r e c t E L I S A i s a l s o shown. Column: S e p h a c r y l S-300 (1.8 X 34 cm); E l u t i n g b u f f e r : 0.1 M T r i s - H C l , pH 8, w i th 0.5 M N a C l ; Flow r a t e : 2 m l / h ; F r a c t i o n s : 2 ml 75 SDS-PAGE p r o f i l e s of 2 -ME-reduced samples from g e l f i l t r a t i o n p u r i f i c a t i o n . Lanes 1 and 6: m o l e c u l a r weight s t a n d a r d s ; Lanes 2-5: 2E1 H6 f r a c t i o n s 16, 20, 22, 24; Lanes 7-10: 2F9 B3 f r a c t i o n s 16, 20, 22, 24 77 i x F i g u r e 10 SDS-PAGE p r o f i l e s of 2 -ME-reduced samples from g e l f i l t r a t i o n p u r i f i c a t i o n . Lanes 1 and 6: m o l e c u l a r weight s t a n d a r d s ; Lanes 2-5: 4D10 C l f r a c t i o n s 16, 20, 22, 24; Lanes 7-10: 2H4 H12 f r a c t i o n s 16, 20, 22, 24 78 F i g u r e 11 E l u t i o n p r o f i l e of Mab 4D10 C l s e p a r a t e d by anion-exchange chromatography. Immuno-r e a c t i v i t y as determined by an i n d i r e c t ELISA i s a l s o shown. Column: DEAE-Sephace l (1 .8 X 7 cm); E l u t i n g b u f f e r : 10 mM T r i s - H C l , pH 8, NaCl c o n c e n t r a t i o n g r a d i e n t ; F low r a t e : 5 m l / h; F r a c t i o n s : 1 ml . . . . 8 2 F i g u r e 12 E l u t i o n p r o f i l e of Mab 2F9 B3 s e p a r a t e d by an ion-exchange chromatography. Immuno-r e a c t i v i t y as determined by an i n d i r e c t ELISA i s a l s o shown. Column: DEAE-Sephace l (1.8 X 7 cm); E l u t i n g b u f f e r : 10 mM T r i s - H C l , pH 8, NaCl c o n c e n t r a t i o n g r a d i e n t ; Flow r a t e : 5 m l / h; F r a c t i o n s : 1 ml 83 F i g u r e 13 E l u t i o n p r o f i l e of Mab 2E1 H6 s e p a r a t e d by anion-exchange chromatography. Immuno-r e a c t i v i t y as determined by an i n d i r e c t ELISA i s a l s o shown. Column: DEAE-Sephace l (1.8 X 7 cm); E l u t i n g b u f f e r : 10 mM T r i s - H C l , pH 8, NaCl c o n c e n t r a t i o n g r a d i e n t ; Flow r a t e : 5 m l / h; F r a c t i o n s : 1 ml 84 F i g u r e 14 SDS-PAGE p r o f i l e s of 2 -ME-reduced samples from anion-exchange p u r i f i c a t i o n . Lane 1: m o l e c u l a r weight s t a n d a r d s ; Lanes 2-4: 2F9 B3 f r a c t i o n s 40, 45, 51; Lanes 6-9: 2E1 H6 f r a c t i o n s 41, 46, 56 86 F i g u r e 15 SDS-PAGE p r o f i l e s of 2 -ME-reduced samples from anion-exchange p u r i f i c a t i o n . Lane 1: m o l e c u l a r weight s t a n d a r d s ; Lanes 3-6: 4D10 C l f r a c t i o n s 40, 46, 50, 53 88 F i g u r e 16 A n t i g e n b i n d i n g a c t i v i t y of Mabs as de termined by an i n d i r e c t E L I S A . Mab 2E1 H6 ( • ) ; 2F9 B3 ( X ) ; 4D10 C l ( A ) ; 2H4 H12 (O) 90 x Figure 17 SDS-PAGE p r o f i l e s of 2-ME-reduced bacter ia samples. Lanes 1 and 9: molecular weight standards; Lanes 2 and 10: EPEC Ol42:K86:H6; Lane 3: EPEC Ol28:K67; Lane 4: EPEC 055:K59; Lane 5: EPEC 044:K74; Lane 6: E . c o l i 0157: H7; Lane 7: E . c o l i 0157:K88:H19; Lane 8: E . c o l i , non-EPEC; Lane 11: C. freundi i ; Lane 12: E .c loacae; Lane 13: K.pneumoniae; Lane 14: S.marcescens; Lane 15: E.hermani i ; Lane 16: P . m i r a b i l i s 97 Figure 18 Immunoblots of EPEC with Mabs 2E1 H6 and 2F9 B3. Lane 1: EPEC 0142:K86:H6 (contro l ) ; Lane 2: EPEC 0128:K67; Lane 3: EPEC 055:K59; Lane 4 : EPEC 044 :K74 98 Figure 19 Immunoblots of non-EPEC with Mabs 2E1 H6 and 2F9 B3. Lane 1: EPEC 0142:K86:H6 (contro l ) ; Lane 2: E . c o l i 0157:H7; Lane 3: E . c o l i 0157:K88:H19; Lane 4: E . c o l i , non-EPEC 99 Figure 20 Immunoblots of Enterobacteriaceae with Mabs 2E1 H6 and 2F9 B3. Lane 1: EPEC 0142:K86: H6 ( contro l ) ; Lane 2: C. freundi i ; Lane 3: E .c loacae: Lane 4: K.pneumoniae  100 Figure 21 Immunoblots of Enterobacteriaceae with Mabs 2E1 H6 and 2F9 B3. Lane 1: EPEC 0142:K86: H6 (contro l ) ; Lane 2: S.marcescens; Lane 3: E.hermani i ; Lane 4: P . m i r a b i l i s  102 Figure 22 Immunoblots of EPEC and other Enterobacter-iaceae with po lyc lonal antiserum. Lanes 1 and 5: EPEC 0142:K86:H6 (contro l ) ; Lane 2: EPEC 0128:K67; Lane 3: EPEC 055:K59; Lane 4 EPEC 044:K74; Lane 6: C . f r e u n d i i ; Lane 7: E .c loacae; Lane 8: K.pneumoniae  106 Figure 23 SDS-PAGE p r o f i l e s of 2-ME-reduced p u r i f i e d IgY f r a c t i o n s . Lane 1: IgY standard; Lanes 3, 5 and 7: IgY fract ions from t r i a l s 1, 2 and 3 . . . I l l Figure 24 SDS-PAGE p r o f i l e s of 2-ME-reduced p u r i f i e d rabbi t IgG f r a c t i o n s . Lane 1: molecular weight standards; Lanes 3 and 5: IgG fract ions from rabbits 1 and 2 112 Figure 25 Antigen binding a c t i v i t y of IgY fract ions as determined by an i n d i r e c t ELISA. T r i a l 1 ( X ) ; T r i a l 2 (O); T r i a l 3 ( • ) 114 x i Figure 26 Antigen binding a c t i v i t y of rabbit IgG fractions as determined by an indire c t ELISA. Rabbit 1 ( x ) ; Rabbit 2 (•) 116 Figure 27 Diagram of the sandwich ELISA 117 Figure 28 Determination of the working d i l u t i o n of the glutaraldehyde prepared anti-NAGase - ALP conjugate. ( X ) with NAGase coating; (•) without NAGase coating 118 Figure 29 Determination of the working d i l u t i o n of the IgY fr a c t i o n used in the preparation of a n t i -NAGase - ALP conjugates. ( X ) with NAGase coating; ( D ) without NAGase coating 120 Figure 30 Determination of the working d i l u t i o n of the periodate-oxidation prepared anti-NAGase - ALP conjugate. ( X ) with NAGase coating; ( Q) without NAGase coating 122 Figure 31 Diagram of the competitive ELISA 123 Figure 32 Relationship between NAGase concentration and absorbance at 405 nm in a competitive ELISA...124 Figure 33 Diagram of the double-sandwich ELISA 126 Figure 34 A t y p i c a l standard curve for NAGase as deter-mined by a double-sandwich ELISA {rz = 0.96; SEE = 0.066) 127 x i i ACKNOWLEDGEMENTS I would l i k e to extend my g r a t i t u d e to my advisor Dr. s . Nakai for h i s guidance throughout the course of t h i s p r o j e c t . I would a l s o l i k e to thank the members of my committee, Dr. Skura, Dr. vanderstoep and Dr. Fitzsimmons for t h e i r valuable input. A very s p e c i a l thanks i s a l s o extended to Mr. Andrew Wieczorek of the A g r i c u l t u r e Canada Research S t a t i o n i n Vancouver, BC, who took a great deal of time from h i s own work to teach me the techniques of monoclonal antibody production, answer my many questions and give me a s s i s t a n c e i n the l a b o r a t o r y . x i i i INTRODUCTION The immune system is a complex defense mechanism with an evolut ionary h i s tory spanning 400 m i l l i o n years (Weir, 1988). This system includes a l l those phys io log ica l mechanisms that enable an animal to recognize materials that are foreign to i t s e l f , such as b a c t e r i a , and to neutra l i ze or el iminate them without in jury to i t s e l f ( B e l l a n t i and Kadlec, 1985). Recognition of, and u l t imate ly protect ion against , foreign substances i s accomplished by immunoglobulins. Immunoglobulins are glycoproteins that are capable of binding to the in fec t ive agent, or antigen (Weir, 1988). Immunoglobulins produced as a re su l t of s t imulat ion of the immune system are termed "polyclonal" due to the i r d i v e r s i t y in c l a s s , s p e c i f i c i t y for the antigen and b i o l o g i c a l function (Bankert et a l . , 1984). One of the most important advances in immunology has been the development of current monoclonal antibody production techniques. Monoclonal antibodies are homogeneous, having ar i sen from the clone of a s ingle plasma c e l l (Bankert et a l . , 1984). These antibodies offer many advantages over conventional po lyc lona l a n t i s e r a , inc luding improved s p e c i f i c i t y for the antigen. Monoclonal antibodies may be useful in the detect ion of enteropathogenic E . c o l i (EPEC). EPEC represent one of the major causative agents of d iarrhea among infants in developing countr ies , r e s u l t i n g in a high incidence of morbidity (Black et a l . , 1981). While monoclonal antibodies have been raised against the v iru lence-assoc iated antigens of 1 enterotoxigenic E . c o l i (Svennerholm et a l . , 1986), th i s remains to be done with EPEC s t r a i n s . If developed, such a monoclonal antibody may have potent ia l use in an immunoassay for EPEC in immunoglobulin infant feeding s tudies , food systems, and in diagnost ic and epidemiological studies of causes of diarrhea in developing countr ies . An immunoassay is based on the in terac t ion between an antigen and i t s s p e c i f i c antibody (Pesce et a l . , 1978). The most popular immunoassay used today is the Enzyme-Linked Immunosorbent Assay (ELISA) f i r s t described by Engval l and Perlmann in 1971. The use of enzyme labels in immunoassays has allowed them to become more economical, safe and simple (Monroe, 1984). Due to the i r v e r s a t i l i t y , ELISAs are being used more widely among the s c i e n t i f i c d i s c i p l i n e s , inc luding food sc ience . Attempts to develop a simple and r e l i a b l e method to d i s t i n g u i s h fresh from frozen/thawed f i s h f i l l e t s have not been success fu l . The use of an ELISA to detect the release of the lysosomal enzyme (3-N-acetylglucosaminidase during freezing of f i sh muscle may have po tent ia l use for th i s purpose. The object ives of t h i s research were to: 1. gain a knowledge of monoclonal antibody techniques. 2 . produce a monoclonal antibody s p e c i f i c for enteropathogenic E . c o l i . 3 . develop an ELISA for 6-N-acetylglucosaminidase which could be used to d i f f e r e n t i a t e between fresh and frozen/thawed f i sh muscle. 2 LITERATURE REVIEW A. Antibodies and the Immune System 1. The Immune System The immune systems' responses can be narrowed down into two categories, the "Innate" or nonspecific responses and Spe c i f i c Aquired Immunity. Nonspecific responses, while being capable of d i f f e r e n t i a t i n g s e l f from nonself, are not dependent on the s p e c i f i c recognition of a foreign configuration. Nonspecific immunity includes, for example, those f i r s t l i n e barriers against i n f e c t i o n such as the skin, phagocytosis of c e l l s by macrophages and the inflammatory response (Roltt, 1988; B e l l a n t i and Kadlec, 1985). Sp e c i f i c Aquired Immunity is mediated by two types of mechanisms. They are cell-mediated Immunity and Humoral Immunity. Cell-mediated immunity is mediated by s p e c i f i c a l l y sensitized lymphocytes that d i f f e r e n t i a t e under the influence of the thymus and are c a l l e d T-lymphocytes (B e l l a n t i and Rocklin, 1985) Humoral Immunity is mediated by a group of lymphocytes that are derived from the bone marrow (or bursa of Fabricus in birds) and are therefore c a l l e d B-lymphocytes ( B e l l a n t i and Kadlec, 1985). The products of the humoral immune response are antibodies. Antibodies are complex proteins known as immunoglobulins which are capable not only of act i v a t i n g the complement system and stimulating phagocytic c e l l s (Roitt, 1988), but also binding to and neutralizing substances foreign to the body. These substances are c a l l e d 3 a n t i g e n s . 2. A n t i b o d y P r o d u c t i o n While the bone marrow i s the s o u r c e of B-lymphocytes, once produced t h e y w i l l t r a v e l t o p e r i p h e r a l lymphoid t i s s u e s such as t h e lymph nodes, s p l e e n and lymphoid t i s s u e of the g a s t r o i n t e s t i n a l and r e s p i r a t o r y t r a c t s ( B e l l a n t i and K a d l e c , 1985). Each B-lymphocyte i s g e n e t i c a l l y programmed t o produce o n l y one a n t i b o d y s p e c i e s . T h i s a n t i b o d y i s p r e s e n t on the o u t e r s u r f a c e of the lymphocyte and a c t s as a r e c e p t o r 5 ( R o i t t , 1988). Each lymphocyte has on the o r d e r of 10 a n t i b o d y m o l e c u l e s on i t s s u r f a c e . When a f o r e i g n s u b s t a n c e ( a n t i g e n ) e n t e r s the body, i t w i l l b i n d t o t h o s e a n t i b o d y r e c e p t o r s which p r o v i d e a "good f i t " . Once t h i s b i n d i n g t a k e s p l a c e , the B-lymphocytes a r e s t i m u l a t e d t o d i f f e r e n t i a t e and p r o l i f e r a t e i n t o plasma c e l l s . These plasma c e l l s t hen s e c r e t e a n t i b o d i e s which a r e i d e n t i c a l t o the r e c e p t o r a n t i b o d y on the B - c e l l from which i t was d e r i v e d ( R o i t t , 1988), and t h e r e f o r e a r e c a p a b l e of b i n d i n g t o the a n t i g e n . 3. A n t i b o d i e s : S t r u c t u r e and F u n c t i o n A n t i b o d i e s b e l o n g t o a c l a s s of p r o t e i n s known as i m m u n o g l o b u l i n s . They a r e composed of f o u r p o l y p e p t i d e c h a i n s bound by d i s u l p h i d e bonds (Guttman e t a l . , 1981) as i n d i c a t e d i n F i g u r e 1. i n t r a - c h a i n d i s u l p h i d e bonds i n f l u e n c e the shape of the i n d i v i d u a l c h a i n s (Weir, 1988). The m o l e c u l e c o n s i s t s of two i d e n t i c a l l i g h t c h a i n s , each of 4 Figure 1: Basic s t r u c t u r e of an antibody molecule (adapted from Calvanico, 1984) 5 which is 214 amino acids long, and two Identical heavy chains which are between 450 and 700 amino acids long (Guttman et a l . , 1981). Both the l ight chains and the heavy chains have variable and constant regions (Bellanti and Kadlec, 1985). In the l ight chains, the constant sequence of amino acids is located at the C terminal end of the chain. The variable region exists in the f i r s t 107 amino acids at the N terminal. Up to 50% of the positions in the N terminal have been found to be variable (Weir, 1988). This var iab i l i ty tends to be concentrated in three areas known as the hyperviariable regions (Weir, 1988) (Figure 1). Consequently, a large number of permutations are possible in sequence and therefore antibody spec i f ic i ty . Similar variation is seen in the N terminal of the heavy chain (Figure 1). It is in these variable portions of the heavy and light chains that the antigen-binding site is found (Weir, 1988). There are two antigen-binding sites per immunoglobulin molecule (Figure 1). In humans, five different classes of immunoglobulins are known to exist (this differs from species to species) (Weir, 1988). These can be differentiated from one another on the basis of s ize, amino acid sequence, biological function and biochemical properties. These classes are known as IgG, IgA, IgM, IgD and IgE (Bellanti and Kadlec, 1985). IgG is the major immunoglobulin in serum, accounting for 70-75% of the serum immunoglobulin. This immunoglobulin has the common structure already defined and has a molecular weight of approximately 150,000 daltons (Bernier, 1985). IgG can also be broken down into subclasses based o differences in the Fc regions (Calvanico, 1984). There are four IgG subclasses: IgGl, IgG2, IgG3, and IgG (Spiegelberg, 1974) . IgA is the second most abundant immunoglobulin in the serum and is important in the external secretory system There are two IgA subclasses (IgAl and IgA2) (Calvanico, 1984). IgA has a molecular weight of approximately 170,000 daltons (Bernier, 1985). IgM is the largest of the immunoglobulin molecules. Five of the 4-chain structures common to immunoglobulin structure are joined together to form a large molecule of approximately 900,000 daltons (Bernier, 1985). There are two subclasses of IgM (igMl and lgM2) in man (Calvanico, 1984). This class of immunoglobulins is most important in the f i r s t few days of the primary immune response (Bernier, 1985). IgD has a molecular weight of approximately 150,000 daltons (Bernier, 1985) and is commonly present in only trace amounts in serum. The biological function of IgD is not exactly known (Weir, 1988). IgE has a molecular weight of approximately 196,000 daltons and is also present in only trace amounts in serum. IgE is responsible for triggering a l lergic reactions (Weir, 1988) . Antibodies can be broken down into three pieces by digestive enzymes. Two pieces are identical and carry the 7 antigen-binding s i t e . They are c a l l e d the Fab fragments (Figure 1). The t h i r d fragment i s c a l l e d the Fc p o r t i o n . This fragment lacks the antibody binding s i t e , but has many other important functions such as complement f i x a t i o n (Weir, 1988) . 4. Antigens and Antigen-Antibody I n t e r a c t i o n Substances which are capable of t r i g g e r i n g an immune response are c a l l e d immunogens. Antigens are substances which are capable of binding with a s p e c i f i c antibody (Pesce et a l . , 1978). An antigen alone may not be immunogenic. For example, some low molecular weight substances, c a l l e d haptens, cannot s t i m u l a t e the immune system unless f i r s t coupled to a l a r g e r c a r r i e r molecule (Weir, 1988). Areas on the antigen such as a c e r t a i n amino a c i d sequence or a sugar s i d e chain are c a l l e d a n t i g e n i c determinants or epitopes. I t i s these s i t e s that the antibody binds t o . The part of the v a r i a b l e region on the antibody which binds with the epitope i s c a l l e d the paratope ( B e r n i e r , 1988) . An antibody i s able to recognize an antigen based on i t s conformation (Weir, 1988). This i s s i m i l a r to the "lock and key" arrangement i n enzyme-substrate i n t e r a c t i o n s . The forces that bind the antigen and antibody together are common inte r m o l e c u l a r forces such as e l e c t r o s t a t i c , hydrophobic and hydrogen bonding, as w e l l as Van der Waal's f o r c e s . No covalent bonding i s involved (Weir, 1988). 8 The s p e c i f i c i t y of an a n t i b o d y for an a n t i g e n i s not a b s o l u t e . In o ther words, an a n t i b o d y r a i s e d a g a i n s t a s p e c i f i c a n t i g e n may c r o s s - r e a c t w i th a r e l a t e d a n t i g e n which e i t h e r has an i d e n t i c a l or s i m i l a r a n t i g e n i c d e t e r m i n a n t . When the a n t i g e n i s s i m i l a r , the " f i t " between a n t i g e n and a n t i b o d y w i l l not be e x a c t . T h i s w i l l r e s u l t i n weaker b i n d i n g . 5. Ch ickens as a Source of A n t i b o d y C o n v e n t i o n a l sources of a n t i b o d i e s or a n t i s e r a i n c l u d e such an imal s as r a b b i t s and mice . More r e c e n t l y , i n t e r e s t has been shown i n the use of immunoglobul ins from a v i a n sources such as c h i c k e n s and , i n p a r t i c u l a r , c h i c k e n egg y o l k . Three major c l a s s e s of a n t i b o d y have been i d e n t i f i e d i n b i r d s . They are IgM, IgA and IgG (Rose and O r l a n s , 1981) . Most r e s e a r c h e r s agree t h a t y o l k c o n t a i n s o n l y IgG (Rose and O r l a n s , 1981). The IgG found i n c h i c k e n egg y o l k i s commonly r e f e r r e d to as IgY due to d i f f e r e n c e s from IgG i n s e v e r a l c h a r a c t e r i s t i c s , i n c l u d i n g m o l e c u l a r we ight , s e d i m e n t a t i o n c o n s t a n t and i s o e l e c t r i c p o i n t ( L e s l i e and Clem, 1969; A l t s c h u h et a l . , 1984) . I t has a l r e a d y been demonstrated t h a t hens immunized w i t h an immunogenic substance l a y eggs c o n t a i n i n g l a r g e amounts of IgY s p e c i f i c f o r t h i s substance (Po i son et a l . , 1980; Berger e t a l . , 1985) and tha t t h i s immunoglobul in can be e a s i l y i s o l a t e d from the egg y o l k i n r e l a t i v e l y h i g h y i e l d s (Burger et a l . , 1 9 8 5 ) . In a d d i t i o n , there i s i n c r e a s e d convenience in 9 the c o l l e c t i o n of eggs over the bleeding of animals. For t h i s reason, the use of chickens represents a v i a b l e a l t e r n a t i v e for production of a n t i s e r a . B. Monoclonal Antibodies 1. H i s t o r y Because of the heterogeneity or d i v e r s i t y among immunoglobulins i n t h e i r s t r u c t u r e , s p e c i f i c i t y and b i o l o g i c a l f u n c t i o n , antiserum derived from an immunized animal i s termed " p o l y c l o n a l " (Bankert et a l . , 1984). The c l o n a l s e l e c t i o n theory (Burnet, 1957) po s t u l a t e s that each B - c e l l or plasma c e l l produces a n t i b o d i e s with a s i n g l e s p e c i f i c i t y . Therefore, a clone from a s i n g l e c e l l would secrete a n t i b o d i e s of uniform s p e c i f i c i t y . The term monoclonal i s used to define a population of immunoglobulins that are i d e n t i c a l ; meaning that they have a r i s e n from a s i n g l e clone of a plasma c e l l . This property could not be u t i l i z e d i_n v i t r o i n i t i a l l y because B - c e l l s and plasma c e l l s cannot be maintained i n t i s s u e c u l t u r e f o r any length of time . In 1975, Kohler and M i l s t e i n devised a method whereby they could i s o l a t e a s i n g l e plasma c e l l from an immunized mouse and make i t "immortal" by f u s i n g i t with a myeloma c e l l . Since that time, the technology has come so far that i t i s reasonable to say that i t i s p o s s i b l e to r a i s e monoclonal a n t i b o d i e s against any antigen that can st i m u l a t e an immune response. 10 2. Monoclonal Antibody Production a. Basic P r i n c i p l e When two c e l l s are brought i n t o c l o s e contact with one another and t h e i r membranes are caused to fuse together, the fu s i o n product w i l l c o n t a i n both n u c l e i . E v e n t u a l l y the n u c l e i w i l l fuse producing a hyb r i d ; a c e l l which contains a s i n g l e nucleus yet r e t a i n s the genetic information from both of the o r i g i n a l c e l l s (Zola and Brooks, 1982). This p r i n c i p l e forms the ba s i s of the Kohler and M i l s t e i n (1975) monoclonal methodology which involves the f u s i o n of murine myeloma c e l l s and spleen c e l l s derived from an immunized mouse. An example of present day monoclonal antibody production methodology i s summarized i n Figure 2. b. Immunization The f i r s t step i n hybridoma production i s the immunization of a s u i t a b l e animal. The animals most commonly used f o r t h i s purpose are r a t s and mice (Waldmann, 1986), since myeloma c e l l l i n e s for these species are a v a i l a b l e commercially (Eshhar, 1985) and they are e a s i l y immunized. I n t e r s p e c i e s hybridomas, such as rodent-human hybrids (Westerwoudt, 1986) or rodent-bovine hybrids (Guidry et a l . , 1986), can be produced. However, l o s s of chromosomes u s u a l l y occurs as a r e s u l t of f u s i o n . This r a r e l y occurs with rodent-rodent hybrids (Westerwoudt, 1986). While i n vivo immunization i s most common, immunization of the spleen i n v i t r o i s a l s o p o s s i b l e (Sirvaganian et a l . , 1983). This approach can be used i f the immunogen i s t o x i c to the animal. IMMUNIZED ANIMAL SPLEEN CELLS MYELOMA CELLS FREEZE HYBRIOOMAS igure 2: Outline of a common p r o t o c o l used to produce hybridomas (Bankert et a l . , 1984) 12 c. Myeloma C e l l s and the HAT S e l e c t i o n System In order to be s u i t a b l e f o r monoclonal work, the myeloma c e l l l i n e s e l e c t e d must possess a c h a r a c t e r i s t i c which makes i t p o s s i b l e to s e l e c t i v e l y separate hybrids from the parent myeloma c e l l s . A s e l e c t i v e media i s used which r e s u l t s l n the death of myeloma c e l l s , but allows hybrids to grow. The most commonly used media i s HAT media. The HAT media system was developed by L i t t l e f i e l d , 1964. HAT media contains hypoxanthine, aminopterin and thymidine. Aminopterin i s a f o l i c a c i d analog which blocks the main b i o s y n t h e t i c pathway of n u c l e i c a c i d s y n t h e s i s (Bankert et a l . , 1984). C e l l s can continue to grow by a salvage pathway i f hypoxanthine and thymidine are present i n the media. This salvage pathway can be u t i l i z e d only i f the c e l l s c o n t a i n the enzymes hypoxanthine guanine phosphoribosyltransferase (HGPRT) (Bankert et a l . , 1984) and thymidine kinase (Eshhar, 1985). In the most common technique of monoclonal antibody production, myeloma c e l l mutants are s e l e c t e d which are d e f i c i e n t i n e i t h e r of these enzymes. Therefore, they cannot grow i n the presence of aminopterin, even i f hypoxanthine and thymidine are present i n the media (Bankert et a l . , 1984). Hybrids formed between a spleen c e l l and a myeloma c e l l can grow because the spleen c e l l parent contains the necessary enzymes to u t i l i z e the salvage pathway. As was mentioned p r e v i o u s l y , spleen c e l l s cannot be maintained i n t i s s u e c u l t u r e ; t h e r e f o r e , spleen c e l l s and hybrids formed between 13 spleen c e l l s d i e o f f n a t u r a l l y , d. Fusion The frequency of f u s i o n of c e l l membranes i s increased by the a d d i t i o n of f u s i n g agents, of t e n c a l l e d fusogens (Zola and Brooks, 1982). The Sendai v i r u s was the fusogen used i n the f i r s t f u s i o n experiments (Kohler and M i l s t e i n , 1975). This has given way to the use of more p r a c t i c a l agents, the most popular being polyethylene g l y c o l (PEG) (G a l f r e et a l . , 1977). The exact mechanism of a c t i o n of the PEG i s not known; however, i t i s l a r g e l y thought to cause a g g l u t i n a t i o n of the c e l l s , thereby i n c r e a s i n g the area of plasma membrane contact (Knutton and Pasternak, 1979). Other methods of f u s i o n have been explored, i n c l u d i n g the a p p l i c a t i o n of e l e c t r i c f i e l d pulses to induce f u s i o n of c e l l s (Vienken and Zimmerman, 1985). 3. Advantages of Monoclonal Antibodies The success of monoclonal a n t i b o d i e s has a r i s e n out of t h e i r many advantages over p o l y c l o n a l a n t i s e r a . Large amounts of monoclonal a n t i b o d i e s can be generated very e a s i l y by batch c u l t u r e or a s c i t e s production (Yelton et a l . , 1981). To generate large amounts of p o l y c l o n a l a n t i s e r a , many animals are needed and/or re-immunization i s requ i r e d . Because monoclonal a n t i b o d i e s are a s i n g l e antibody s p e c i e s , whose p r o p e r t i e s remain constant, they can be used as a standard reagent i n immunoassays. This i s not the case with p o l y c l o n a l a n t i s e r a since the p r o p e r t i e s may change from one 14 immunization to the next. A l s o , the supply of p o l y c l o n a l a n t i b o d i e s ends when the animal d i e s . Hybridomas can be stored i n l i q u i d n itrogen i n d e f i n i t e l y , thus p r o v i d i n g an endless supply of monoclonal a n t i b o d i e s (Eshhar, 1985). Another advantage i s t h e i r m a n i p u l a b i l i t y , since hybridoma c e l l l i n e s can often be mutated to produce a n t i b o d i e s not found i n nature (Kohler, 1986). Although c r o s s - r e a c t i o n s are not completely e l i m i n a t e d with the use of monoclonal a n t i b o d i e s , they are minimized since a s i n g l e antibody species i s u t i l i z e d . L a s t l y because one i s s e l e c t i n g a s i n g l e antibody species from a whole po p u l a t i o n , the antigen p r e p a r a t i o n does not have to be pure provided the assay method can d i s t i n g u i s h between a n t i b o d i e s to the antigen and a n t i b o d i e s to the i m p u r i t i e s (Bankert et a l . , 1984). 4. A p p l i c a t i o n s to Food Science While monoclonal a n t i b o d i e s have been used e x t e n s i v e l y i n the medical f i e l d , uses r e l a t e d to food science have been l i m i t e d . C u r r e n t l y t h e i r use i s expanding, p a r t i c u l a r l y i n areas where immunoassays have already been a p p l i e d . S k e r r i t t (1985) i s o l a t e d two antibody clones which bound s p e c i f i c a l l y to c e r t a i n l o w - m o b i l i t y prolamins i n wheat, rye, b a r l e y and oats. These p r o t e i n s were s t a b l e to heating so they could p o s s i b l y be u s e f u l i n an assay for g l u t e n i n cooked or processed foods. In a previous paper ( S k e r r i t t et a l . , 1984) used s i m i l a r monoclonal a n t i b o d i e s to examine the homology between the storage p r o t e i n s of d i f f e r e n t c e r e a l g r a i n s , since each monoclonal could be h i g h l y s p e c i f i c for a 15 g i v e n amino a c i d sequence . Kaminogawa et a l . (1987) produced a monoc lonal a n t i b o d y which c o u l d b ind both n a t i v e and u n f o l d e d B - l a c t o g l o b u l l n . These r e s e a r c h e r s suggested t h a t t h i s monoc lonal a n t i b o d y would be u s e f u l i n i d e n t i f y i n g tha t p a r t of the molecule which s t i m u l a t e s a l l e r g i e s . In a d d i t i o n , the a n t i b o d y c o u l d be used to s t u d y the u n f o l d i n g and r e f o l d i n g of the p r o t e i n and i t s s u r f a c e s t r u c t u r e . The m a j o r i t y of work w i t h monoclonals i n food s c i e n c e i s wi th food pathogens and t h e i r t o x i n s . In 1983, Robison et a l . r e p o r t e d on a monoc lonal a n t i b o d y c a l l e d MOPC 467 which was a b l e to s p e c i f i c a l l y d e t e c t S a l m o n e l l a b a c t e r i d i n mixed c u l t u r e and at the same time d i d not c r o s s r e a c t w i th o ther e n t e r i c b a c t e r i a . T h i s a n t i b o d y was found to be s p e c i f i c f o r a f l a g e l l a r d e t e r m i n a n t . Butman et a l . (1988) r a i s e d genus-s p e c i f i c monoclonal a n t i b o d i e s f o r L i s t e r i a which showed no c r o s s - r e a c t i o n s w i th a pane l of n o n - L i s t e r i a s p e c i e s , i n c l u d i n g those to which i t i s a n t i g e n i c a l l y r e l a t e d . The r e s e a r c h e r s i d e n t i f i e d the a n t i g e n as a h e a t - s t a b l e p r o t e i n wi th a m o l e c u l a r weight of 30,000 - 38,000 d a l t o n s . F a r b e r and S p e i r s (1987) r a i s e d monoclonal a n t i b o d i e s a g a i n s t a f l a g e l l a r a n t i g e n common to L i s t e r l a s p e c i e s . These a l s o d i d not c r o s s r e a c t w i th any of the 30 n o n - L i s t e r i a s t r a i n s t e s t e d . These r e s e a r c h e r s s u c c e s s f u l l y demonstrated the use of these monoclonal a n t i b o d i e s i n an immunoassay of mi lk and cheese samples . Monoc lona l a n t i b o d i e s have a l s o been d e r i v e d for 16 b a c t e r i a l t o x i n s . One example of t h i s a p p l i c a t i o n i s work by Shone et a l . (1985) who produced a monoclonal antibody s p e c i f i c f o r Type A neurotoxin of C l o s t r i d i u m botulinum. When t h i s monoclonal antibody was used i n an Immunoassay of salmon and corned beef samples, d e t e c t i o n l i m i t s were cl o s e to that of the mouse bioassay which i s commonly used for d e t e c t i o n of t h i s t o x i n . Other examples include work by Wnek et a l . (1985) who derived a monoclonal antibody s p e c i f i c for C l o s t r i d i u m perfrlngens Type A en t e r o t o x i n and Edwin et a l . (1984) who produced monoclonal a n t i b o d i e s s p e c i f i c f o r sta p h y l o c o c c a l e n t e r o t o x i n A. C. E s c h e r i c h i a c o l i 1. E^ c o l l and the Incidence of I n f a n t i l e D i a r r h e a l Disease While the incidence of i n f a n t i l e d i a r r h e a i n developed c o u n t r i e s i s q u i t e low, i t i s , not s u r p r i s i n g l y high i n underdeveloped c o u n t r i e s where hygiene standards are poor and the c h i l d r e n are most often malnourished. In developing c o u n t r i e s , i t i s reported that d i a r r h e a l diseases cause 3 to 5 m i l l i o n c h i l d deaths per year (WHO, 1987a) and i s estimated to be res p o n s i b l e f o r 25% of a l l deaths i n c h i l d r e n under 5 years of age (WHO, 1987b). These numbers are expected to d e c l i n e i n coming years due to the implementation of a D i a r r h e a l Disease C o n t r o l Program which makes use of o r a l r e h y d r a t i o n therapy and vaccines s p e c i f i c to some causative agents. Due to problems i n t r a i n i n g and l o g i s t i c s , the success of t h i s program has been l i m i t e d to only a few areas and therefore morbidity from d i a r r h e a remains high. 17 The major e t i o l o g i c a l agents of d i a r r h e a i n developing c o u n t r i e s include S h i g e l l a dysenterlae and 5 . f l e x n e r 1 , V l b r t o c h o l e r a , Salmonella t y p h i , r o t a v i r u s and others (WHO, 1987b). In cases where the causative organism was i d e n t i f i e d , the d i f f e r e n t s t r a i n s of E . c o l i are together the most common cause of acute d i a r r h e a l i l l n e s s (Black et al.,1981; Guerrant et a l . , 1983) . 2. Types of E.col1 There are p r e s e n t l y four known c l a s s e s of E . c o l l that cause d i a r r h e a . They are: (1) enterohemorrhagic; (2) e n t e r o i n v a s i v e ; (3) e n t e r o t o x i g e n i c and (4) enteropathogenic E . c o l i . In a d d i t i o n to c l i n i c a l symptoms, epidemiology and 0 - antigen serogroups, each c l a s s can be d i s t i n g u i s h e d by i t s pathogenic mechanism (Rennels and Levine, 1986). Enterohemorrhagic E . c o l l (EHEC) exerts i t s p a t h o g e n i c i t y through a c y t o t o x i n which appears to be i d e n t i c a l to Shiga t o x i n (O'Brien et a l . , 1983). En t e r o i n v a s i v e E . c o l i (EIEC) invades the e p i t h e l i a l c e l l s of the i n t e s t i n e and causes h i s t o l o g i c a l damage le a d i n g to d i a r r h e a l symptoms ( E d i t o r i a l , 1983). E n t e r o t o x i g e n i c E.col1 (ETEC) i s best known as the cause of t r a v e l l e r ' s d i a r r h e a , but i t i s a l s o a major cause of i n f a n t i l e d i a r r h e a i n developing c o u n t r i e s . These E . c o l i are c h a r a c t e r i z e d by the presence of adhesion o r g a n e l l e s by which the b a c t e r i a adhere to the i n t e s t i n e i n a d d i t i o n to e n t e r o t o x i n production. The t o x i n s produced are e i t h e r heat l a b i l e (LT) or heat-stable (ST) (Rennels and Levine, 1986). Enteropathogenic E . c o l i (EPEC) were the f i r s t c l a s s of 18 E . c o l i to be recognized, although t h e i r exact mode of pa t h o g e n i c i t y remains unknown. The World Health Organization E . c o l i Centre recognizes 14 O serogroups as enteropathogenic i n c l u d i n g O20, 026, 086, 0142, 055, 0111, 0114, 0119, 0125, 0126, 0127, 0128, 044 and 0158 (Back et a l . , 1980). I t i s l a r g e l y thought that these b a c t e r i a cause d i a r r h e a without t i s s u e damage and without producing t o x i n ( M o r i a r t y and Turnberg, 1986). 3. Mode of P a t h o g e n i c i t y of EPEC Considerable research has gone i n t o the search f o r the mode of p a t h o g e n i c i t y of enteropathogenic E . c o l 1 . During examination of EPEC i n f e c t i o n i n i n t e s t i n a l b i o p s i e s of animal models and i l l i n f a n t s , i t was found that there was a h i s t o p a t h o g i c l e s i o n i n the small bowel (Rennels and Levine, 1986). EPEC have been shown to adhere to the i n t e s t i n a l mucosa and produce an "at t a c h i n g and e f f a c i n g " (AE) l e s i o n i n the brush border m i c r o v i l l o u s membrane. This l e s i o n involves l o c a l i z e d d e s t r u c t i o n of m i c r o v i l l i and attachment of the b a c t e r i a to the a p i c a l enterocyte membrane (Knutton et a l . , 1989). I t i s p o s s i b l e that t h i s damage leads to a decrease i n absorptive surface area, r e s u l t i n g i n di a r r h e a (Rothbaum et a l . , 1983). This l e s i o n has been shown to occur i n i n f e c t i o n s of a l l serotypes of EPEC t e s t e d so f a r (Rennels and Levine, 1986). I t has a l s o been shown that of a l l the c l a s s i c EPEC s t r a i n s , 80% are able to adhere to HEp-2 (human e p i t h e l i a l ) c e l l s i n t i s s u e c u l t u r e i n the presence of D-mannose. This 19 c h a r a c t e r i s t i c i s not seen l n other s t r a i n s of E . c o l l (Cravloto et a l . , 1979). The a b i l i t y to adhere to HEp-2 c e l l s i j i v i t r o has been shown to be plasmid-mediated. The genes encoding t h i s adhesiveness are located on a 50-70 megadalton plasmid (Levine et a l . , 1985). This plasmid-mediated adhesion has been termed the EPEC adherence f a c t o r or EAF (Rennels and Levine, 1986). Studies i n Peru showed that HEp-2 adhesiveness i s more commonly found i n those EPEC serotypes i m p l i c a t e d i n epidemic d i a r r h e a (Class I s t r a i n s ) rather than those that cause infrequent outbreaks (Class I I s t r a i n s ) (Nataro et a l . , 1985). Levine et a l . (1985) reported t h a t while the EAF plasmid i s necessary for f u l l expression of p a t h o g e n i c i t y of Class I s t r a i n s , Class I I s t r a i n s do not have t h i s plasmid yet can s t i l l cause d i a r r h e a . In a d d i t i o n , Class II s t r a i n s e i t h e r do not adhere to HEp-2 c e l l s i r i v i t r o or show d i f f u s e adherence (Levine, 1987). Therefore another mechanism of p a t h o g e n i c i t y must occur. Levine et a l . (1985) a l s o showed that volunteers given a s t r a i n of E . c o l l c o n t a i n i n g EAF developed a n t i b o d i e s to a 94 k i l o d a l t o n plasmid-associated outer membrane p r o t e i n that i s found i n other Class I EPEC but not i n enterotoxogenlc E . c o l i . These researchers suggested that t h i s p r o t e i n might play a r o l e i n adherence of these b a c t e r i a i n the gut. Despite these observations, i t has been postulated that t h i s adherence i n i t s e l f i s not what causes d i a r r h e a l disease (Rothbaum et a l . , 1983). Rothbaum et a l . (1983) suggested 20 that at l e a s t one serogroup of EPEC may produce a c y t o t o x i n that a f f e c t s p r o t e i n s y n t h e s i s , e v e n t u a l l y r e s u l t i n g i n c e l l death and l o s s . O'Brien et a l . (1982) and Cleary et a l . (1985) found that many EPEC serotypes produce a t o x i n s i m i l a r to that of S h i g e l l a dysenteriae Type 1 (Shiga). Cleary et a l . (1985) found that t h i s S h i g a - l l k e c y t o t o x i n was detected more oft e n and i n l a r g e r amounts i n EPEC than i n other f e c a l E . c o l i . Therefore, t h i s t o x i n may pl a y a r o l e i n the pathogenisis of EPEC-related g a s t r o e n t e r i t i s . 4. Detection of EPEC The study of b a c t e r i a l e n t e r i c pathogens t y p i c a l l y i nvolves f i r s t i s o l a t i n g them on s e l e c t i v e media from the t o t a l b a c t e r i a l population present i n the feces, then t e s t i n g i n d i v i d u a l c o l o n i e s i n biochemical r e a c t i o n s followed by a g g l u t i n a t i o n i n s p e c i f i c a n t i s e r a (Echeverria et a l . , 1985). U n t i l r e c e n t l y , t h i s was the only way researchers could i d e n t i f y EPEC s t r a i n s . This method i s expensive, labourious and somewhat u n r e l i a b l e (Robins-Browne, 1987). Most r e c e n t l y Nataro et a l . (1985) developed a DNA h y b r i d i z a t i o n probe from a 1-kilobase segment of the EAF plasmid. They found t h i s probe to be h i g h l y s e n s i t i v e and s p e c i f i c i n d e t e c t i n g EPEC s t r a i n s that e x h i b i t HEp-2 l o c a l i z e d adhesiveness. However, t h i s probe i s incapable of d e t e c t i n g Class I I EPEC which e i t h e r do not adhere to HEp-2 c e l l s or adhere d i f f u s e l y . Therefore, the development of a more p r a c t i c a l technique such as using a n t i s e r a to EPEC v i r u l e n c e - a s s o c i a t e d antigens i s d e s i r e d . Such a technique 21 might Involve the use of monoclonal a n t i b o d i e s . Numerous researchers have used monoclonal an t i b o d i e s i n t h e i r study of the E . c o l i b a c t e r i a . Monoclonal a n t i b o d i e s have been s u c c e s s f u l l y r a i s e d against the h e a t - l a b l e (LT) and heat s t a b l e (ST) enterotoxins of e n t e r o t o x i g e n i c E . c o l i (Svennerholm et a l . , 1986) i n a d d i t i o n to the c o l o n i z a t i o n f a c t o r antigens (CFA) which a l l o w some s t r a i n s of ETEC to c o l o n i z e the i n t e s t i n e (Lopez-vidal et a l . , 1988). These monoclonal a n t i s e r a can be used to detect the presence of ETEC s t r a i n s . However, a p r a c t i c a l technique for the d e t e c t i o n of EPEC s t r a i n s using monoclonal a n t i s e r a remains to be developed. Such a technique would be u s e f u l to both researchers and d i a g n o s t i c i a n s . D. Immunoassays 1. I n t r o d u c t i o n C l a s s i c a l methods f o r the a n a l y s i s of foods often involve lengthy e x t r a c t i o n procedures using organic s o l v e n t s and/or chromatographic techniques. The use of an immunoassay o f f e r s a simple and economical a l t e r n a t i v e with o f t e n improved s e n s i t i v i t y over conventional a n a l y t i c a l methods. Immunoassays are based on the a b i l i t y of an t i b o d i e s to bind to a s p e c i f i c antigen (Pesce et a l . , 1978). Chemical measurements based on t h i s i n t e r a c t i o n have been c a r r i e d out i n a c l i n i c a l s e t t i n g f o r the past 20 years. However, i t i s j u s t r e c e n t l y that t h i s methodology has begun to have an impact i n food a n a l y s i s due to an increased demand f o r a n a l y t i c a l methods with lower d e t e c t i o n l i m i t s . This i s , i n 22 p a r t , due to increased concern over food s a f e t y and r e g u l a t i o n . The development of methods which use l a b e l l e d a n t i b o d i e s or antigens has r e s u l t e d i n assays with high l e v e l s of s e n s i t i v i t y and s p e c i f i c i t y . Labels used i n immunoassays have included f l u o r e s c e n t l a b e l s i n fluorescence immunoassay (FIA) and isotopes i n radioimmunoassay (RIA) ( V o l l e r et a l . , 1978). However, these l a b e l s have l i m i t a t i o n s . FIA i s time consuming and not e a s i l y automated while RIA depends on isotopes as reagents which have a short s h e l f l i f e and are under r e g u l a t o r y c o n t r o l . In a d d i t i o n , expensive equipment and s k i l l e d personnel are required (Monroe, 1984). To date, use of antigens or a n t i b o d i e s l a b e l l e d with enzymes such as horse r a d i s h peroxidase or a l k a l i n e phosphatase has proven to be most s u c c e s s f u l i n immunoassays ( V o l l e r , 1980). Enzyme Immunoassays (EIA) are comparable to FIA and RIA i n s e n s i t i v i t y , o f t e n d e t e c t i n g substances i n the nanogram to picagram range. In a d d i t i o n , not only can EIAs often be completed i n only a few hours, but a l s o minimal equipment and t r a i n i n g i s r e q u i r e d . A l s o , enzymes have a longer s h e l f - l i f e , than f l u o r e s c e n t l a b e l s or isotopes (Monroe, 1984). 2. Enzyme-Linked Immunosorbent Assay In 1971, E n g v a l l and Perlmann described an EIA i n which the analyte to be detected binds e i t h e r to the antigen or antibody which i s coated on a s o l i d s u r f a c e . Molecules that do not bind are washed away from the s o l i d surface and the 23 adsorbed enzyme conjugate measured by chemical react ion with substrate (Monroe, 1984). Engval l and Perlmann (1971) c a l l e d th i s immunoassay an Enzyme-Linked Immunosorbent Assay or ELISA. This assay could be used to detect e i ther antibodies or antigens (Engvall and Car lsson , 1976). The s o l i d phase in an ELISA can be in the form of p a r t i c l e s of c e l l u l o s e , polyacrylamide or agarose, or i t can be preformed into d i s c s , tubes, or beads ( V o l l e r , 1980). The most popular form of ELISA involves passive adsorption of the reactant to polystyrene microplates ( V o l l e r , 1980). ELISAs can be broken down into three types: competi t ive , sandwich and i n d i r e c t . In a competitive ELISA, the test sample containing the antigen is mixed with a known concentration of enzyme-labelled antigen. Both then compete for a l imi t ed number of binding s i t e s on antibodies adsorbed on the microplate . In the sandwich ELISA, the antigen being assayed is held between antibody adsorbed on the microplate and a second antibody which contains the enzyme l a b e l . The i n d i r e c t ELISA is general ly used for antibody detec t ion . In th i s assay the antigen is adsorbed on the microplate . Sample containing antibody is then added, followed by enzyme-labe l l ed a n t i g l o b u l i n (Monroe, 1984). 3. Appl icat ions of ELISA to Food Science ELISA has been used success fu l ly for both q u a l i t a t i v e and quant i ta t ive detect ion of food analytes , contaminants and disease agents. Saunders and C l i n a r d (1984) developed an ELISA that could detect T r i c h i n a s p i r a l i s i n 30-60 minutes. Ruitenberg et a l . (1983) demonstrated that ELISA can detect as l i t t l e as 1 larva/lOOg of muscle versus 1 l a r v a / g by d i g e s t i o n methods and 3 l a r v a / g by trichonoscopy. ELISA has a l s o been used to detect molds and mycotoxins in foods. Notermans et a l . (1986) i s o l a t e d a heat s t a b l e mold s p e c i f i c antigen from a mold c u l t u r e . This antigen was s p e c i f i c to molds and was produced by both P e n l c i l l l u m and A s p e r g i l l u s species which are the predominant molds i n foods. Using t h i s a n t i g e n , these researchers developed an ELISA f o r the d e t e c t i o n of these molds i n foods. With respect to mycotoxins, as l i t t l e as 50 pg of a f l a t o x i n can be detected i n peanut butter using a monoclonal antibody i n an ELISA (Ram et a l . , 1986). Patterson and Jones (1983) were able to d i s t i n g u i s h meat of d i f f e r e n t species using an ELISA which detected d i f f e r e n t serum albumins. A s i m i l a r ELISA was used to detect soya p r o t e i n among meat p r o t e i n s (Crimes et a l . , 1984). The m a j o r i t y of a p p l i c a t i o n s of ELISA to food science have been for the d e t e c t i o n of b a c t e r i a and t h e i r t o x i n s i n foods. L i t t o n B i o t e c h n i c s s e l l s a "Bioenzabead Diagnostic K i t " which i s an ELISA for the d e t e c t i o n of Salmonella i n foods. This k i t provides a time saving of 1 to 2 days over o f f i c i a l c u l t u r e methods. The k i t employs a monoclonal antibody which i s capable of d e t e c t i n g 94% of a l l Salmonella s t r a i n s (Eckner et a l . , 1987). 25 One of the most p r o m i s i n g a r e a s where ELISA might be a p p l i e d i s i n the d e t e c t i o n of L i s t e r l a . As was mentioned p r e v i o u s l y i n t h i s paper, F a r b e r and S p e i r s (1987) d e v e l o p e d a monoclonal a n t i b o d y a g a i n s t L i s t e r i a which c o u l d have p o t e n t i a l use i n an ELISA. B a c t e r i a l t o x i n s have a l s o been d e t e c t e d u s i n g ELISA, f o r example S t a p h y l o c o c c u s e n t e r o t o x i n A (Saunders and B a r t l e t t , 1977) and E . c o l i e n t e r o t o x i n s (Svennerholm e t a l . , 1986) . ELISA has a l s o had a p p l i c a t i o n i n the d e t e c t i o n of enzymes. Vaag (1985) d e s c r i b e d the use of an ELISA t o c o n t r o l the a d d i t i o n of enzyme p r e p a r a t i o n s such as p a p a i n and a m y l o g l u c o s i d a s e t o beer. In a d d i t i o n , S t e p a n i a k e t a l . (1987) used an ELISA t o m o n i t o r the p r o d u c t i o n of heat s t a b l e p r o t e i n a s e s and l i p a s e from Pseudomonas i n o r d e r t o e v a l u a t e the q u a l i t y of c o l d s t o r e d m i l k . Compared w i t h agar d i f f u s i o n methods f o r p r o t e o l y t i c and l i p o l y t i c a c t i v i t y , d e t e c t i o n l i m i t s f o r p u r i f i e d p r o t e i n a s e P I were 0.25 ng/ml and 120 ng/m of m i l k by ELISA and agar d i f f u s i o n r e s p e c t i v e l y . D e t e c t i o n l i m i t s f o r p u r i f i e d l i p a s e P I were 0.25 ng/ml of m i l k by ELISA and 1900 ng/ml of m i l k by agar d i f f u s i o n . Another p o t e n t i a l a p p l i c a t i o n of the d e t e c t i o n of enzymes by ELISA was r e p o r t e d by McCannel (1988) and Y o s h i o k a (1988) . They suggested t h a t an ELISA c o u l d be used t o d e t e c t the enzyme 6 - N - a c e t y l g l u c o s a m i n i d a s e i n f i s h muscle. They su g g e s t e d t h a t t h i s a s s a y c o u l d be used as a means of 26 d i f f e r e n t i a t i n g between f r e s h f i s h and f i s h t hat had been frozen then thawed. 4. Use of ELISA to Detect G-N-acetylglucosaminidase (NAGase) P h y s i c a l , chemical and o r g a n o l e p t i c changes i n f i s h are of great commercial importance (Quaranta and Perez, 1983). In Europe, i t has been reported that sea-frozen f i l l e t s are often thawed i n the f i s h shops and s o l d as f r e s h f i l l e t s (Rehbein, 1979). Because i t i s not always v i s u a l l y apparent whether f i s h has been frozen, a d e t e c t i o n method based on p h y s i c a l or chemical changes would be u s e f u l i n q u a l i t y c o n t r o l . A wide v a r i e t y of methods have been proposed f o r the measurement of q u a l i t y changes i n f i s h during frozen storage. In a d d i t i o n to sensory a t t r i b u t e s , they include assays to measure changes i n the nature and composition of free f a t t y a c i d s , production of carbonyls from l i p i d s and development of r a n c i d i t y , measurement of e x t r a c t a b l e p r o t e i n s , trimethylamine content and enzyme p r o p e r t i e s (Quaranta and Pere z ) . However, use of any one of these methods may not n e c e s s a r i l y be i n d i c a t i v e of the f r e e z i n g process alone. Despite the extensive s t u d i e s done on the q u a l i t y of frozen f i s h , attempts to d i s t i n g u i s h between frozen/thawed and f r e s h f i s h f i l l e t s have been l a r g e l y unsuccessful. Yoshioka and Kitamikado (1988) showed that erythrocytes i n f i s h were destroyed when frozen and that t h i s d e s t r u c t i o n could be detected by microscopic examination of blood samples. This method was a p p l i e d to d i f f e r e n t i a t e between 27 f r e s h and frozen/thawed f i s h . Y o s h i o k a (1983a) a l s o used a h e m a t o c r i t v a l u e as an i n d e x of b l o o d c e l l d e s t r u c t i o n f o r the same purpose. The same r e s e a r c h e r d i s t i n g u i s h e d f r o z e n / t h a w e d f i s h from f r e s h f i s h by e x a m i n a t i o n of the m e d u l l a of the l e n s of the f i s h , s i n c e i t was found t h a t i t became opaque d u r i n g f r o z e n s t o r a g e ( Y o s h i o k a , 1983b). These methods were found t o be time-consuming and i m p r a c t i c a l . Some r e s e a r c h has i n v o l v e d the use of enzymes f o r d e t e c t i o n of frozen/thawed f i s h . The r e l e a s e of the m i t o c h o n d r i a l form of g l u t a m a t e - o x a l o a c e t a t e - t r a n s a m i n a s e (E.C. 2.6.1.1) by f r e e z i n g and thawing was used s u c c e s s f u l l y f o r t he d e t e c t i o n of frozen/thawed beef and pork (Vandekerckhove e t a l . , 1972) y e t was found t o be inadequate f o r f i s h f i l l e t s s i n c e m i t o c h o n d r i a were a l s o found t o be d e s t r o y e d by a u t o l y s i s d u r i n g i c e d s t o r a g e of f r e s h f i l l e t s (Hamm and M a s i c , 1971). Another d i s a d v a n t a g e of u s i n g m i t o c h o n d r i a l enzymes as an i n d i c a t o r of f r e e z i n g i s the p o s s i b l e e x i s t e n c e of isozymes i n the c y t o p l a s m (Rehbein e t a l . , 1978). For t h i s r e a s o n , e l e c t r o p h o r e s i s or some o t h e r s e p a r a t i o n method i s r e q u i r e d . S u b s e q u e n t l y , Rehbein e t a l . (1978) and Rehbein (1979) found t h e use of l y s o s o m a l enzymes t o be more p r o m i s i n g . F i s h muscle c o n t a i n s v a r i o u s l y s o s o m a l enzymes, many of which a r e r e l e a s e d d u r i n g f r e e z i n g and subsequent t h a w i n g of a f i l l e t or the i s o l a t e d l y s o s o m a l f r a c t i o n . These r e s e a r c h e r s found 6 - N - a c e t y l g l u c o s a m i n i d a s e (NAGase) (E.C. 3.2.1.30) and a - g l u c o s i d a s e (E.C. 3.2.1.20) t o show the most promise. 28 Using a spectrophotometric method to determine enzyme a c t i v i t y , they found that the press j u i c e / e x t r a c t a c t i v i t y r a t i o increased 6-9 times for a-giucosidase and 3-5 times for NAGase during f r e e z i n g and thawing of f i l l e t s from cod, s a i t h , red f i s h and haddock. Yoshioka (1988) and McCannel (1988) proposed the use of an ELISA for the d e t e c t i o n of NAGase as a means of d i s t i n g u i s h i n g between f r e s h and frozen/thawed f i s h f i l l e t s . They s u c c e s s f u l l y i s o l a t e d anti-NAGase immunoglobulins from the eggs of chickens immunized with commercially prepared bovine kidney NAGase. Using t h i s antibody, an i n d i r e c t ELISA was developed to measure the NAGase co n c e n t r a t i o n i n the press j u i c e and e x t r a c t of both f r e s h and frozen f i s h f i l l e t s . The r e s u l t s obtained were ambiguous. The higher the d i l u t i o n of sample a p p l i e d , the higher the c o n c e n t r a t i o n of NAGase (based on a standard curve with bovine kidney NAGase). McCannel (1988) suggested that t h i s may have been due to the presence of other substances i n the j u i c e and e x t r a c t which i n t e r f e r e d with NAGase binding to the p l a t e . P atterson and Jones (1985) reported a s i m i l a r phenomenon i n t h e i r ELISA for species i d e n t i f i c a t i o n of meat. They o f f e r e d a s i m i l a r e x p l a n a t i o n . In a d d i t i o n to these r e s u l t s , Yoshioka (1988) found that for salmon, samples frozen for one week showed c o n s i s t e n t l y lower NAGase concentrations than the f r e s h samples. However, examination of the same samples for enzyme a c t i v i t y showed increased NAGase a c t i v i t y i n frozen samples. S i m i l a r r e s u l t s 29 were found when the same t e s t s were performed on f r e s h and f r o z e n bovine k i d n e y samples. McCannel (1988) suggested t h a t t h i s may be due t o a p o s s i b l e change i n the a n t i g e n i c s t r u c t u r e of the NAGase as a r e s u l t of f r e e z i n g . 3 0 MATERIALS AND METHODS PART 1: MONOCLONAL ANTIBODY (Mab) STUDY A. Hybridoma Production The p r o t o c o l used for hybridoma production i s that o u t l i n e d by Kannangara et a l . (1989) with m o d i f i c a t i o n s (Figure 30). A l l aspects of hybridoma production were c a r r i e d out under s t e r i l e c o n d i t i o n s . 1. Outer Membrane P r e p a r a t i o n Cultures of E . c o l l 0142:K86:H6 (ATCC 23985) were donated by Dr. B. Skura. A l o o p f u l of c e l l s was t r a n s f e r e d to each of 5 i n d i v i d u a l 250 ml f l a s k s c o n t a i n i n g 150 ml of t r y p t i c soy broth (TSB) (Difco Labs., D e t r o i t , MI) and incubated i n a C o n t r o l l e d Environment Incubator Shaker (NB S c i e n t i f i c Co. Inc., Edison, NJ) at 80 rpm and 37°C for 24 hr. C e l l s were harvested by c e n t r i f u g a t i o n at 12,000 X g for 10 min at 5°C. C e l l s harvested from approximately 200 ml of c u l t u r e were resuspended i n 10 ml of breaking b u f f e r (10 mM T r i s - H C l , 20% sucrose (w/v), 5 mM magnesium c h l o r i d e (MgC^)) and d i s r u p t e d using a French Press (Loomis Engineering and Mfg. Co., C a l d w e l l , NJ) at 10,000 - 12,000 p s i . Unbroken c e l l s were removed by c e n t r i f u g a t i o n at 5000 X g for 10 min at 5°C. The broken c e l l s were loaded i n t o Sw-41 tubes (Beckman Instruments Inc., Toronto, ON) with the f o l l o w i n g sucrose step g r a d i e n t : 2 ml of 70% sucrose (w/v) overlayed with 7 ml of 60% sucrose (w/v). The tubes were spun at 200,000 X g for 3 hr at 5°C i n a Beckman L8-80 u l t r a c e n t r i f u g e (Beckman 31 OUTER MEMBRANE ISOLATION IMMUNIZATION OF MICE 1 BOOST 3-5 DAYS PRIOR TO REMOVAL OF SPLEEN I AT THE SAME TIME GROW UP MYELOMA CELL LINE SCREEN FOR ANTIBODY PRODUCTION SCREEN RECLONES FOR Ab PRODUCTION FUSION EG. ELISA SELECTION AND EXPANSION OF POSITIVE CLONES FREEZE FOR LONG TERM STORAGE RECLONE SELECTION AND EXPANSION OF POSITIVE RECLONES FREEZE FOR LONG TERM STORAGE SCREEN AGAINST -SELECTED BACTERIA IMMUNOFLUORESCENCE ASCITES PRODUCTION PURIFICATION SCREEN AGAINST -SELECTED BACTERIA IMMUNOFLUORESCENCE CHARACTERIZATION gure 3: Protocol for Mab product ion. instruments Inc., Toronto, ON). The layer at the boundary of the 60% and 70% sucrose gradient was harvested with a pasteur p i p e t t e . The r e s u l t i n g sample of approximately 6 ml was s p l i t between two Ti-70 tubes (Beckman Instruments Inc., Toronto, ON) and the remainder of the space f i l l e d with 0.01 M phosphate buffered s a l i n e (PBS), pH 7.4. The tubes were spun at 265,000 X g for 2.5 hr at 5°C. Each p e l l e t was r e d i s s o l v e d i n 200 >ul of PBS and stored at 4°C. 2. Immunization of Mice Four 3 month o l d balb/c mice ( A g r i c u l t u r e Canada Research S t a t i o n , Vancouver, BC) were i n j e c t e d subcutaneously with 50 /ug of the outer membrane preparation mixed 1:1 with Freund's incomplete adjuvant (Difco Labs., D e t r o i t , MI) using a 22G1/2 needle. The mice were then rested f o r one month. Four days p r i o r to f u s i o n the mice were boosted i n t r a p e r i t o n e a l l y with 20 /ag of the outer membrane preparation without any adjuvant. 3. Growth of Myeloma C e l l Line Five days p r i o r to f u s i o n , one v i a l of Fox-NY myeloma c e l l s (Hyclone Labs., Logan, UT) was removed from a l i q u i d o n i t r o g e n f r e e z e r (-150 C) and allowed to thaw by holding the v i a l under running lukewarm tap water. The c e l l s were resuspended i n 10 ml of Dulbecco's Modified Eagle's medium (DME) (Gibco/BRL, B u r l i n g t o n , ON) and c e n t r i f u g e d at 800 X g for 10 min at room temperature (RT). The p e l l e t was resuspended i n 10 ml DME c o n t a i n i n g 20% f e t a l c a l f serum 33 (FCS) (Hyclone Labs., Logan, UT) i n a s t e r i l e P e t r i p l a t e . o This was incubated at 37 C i n an incubator with an atmosphere of 10% carbon d i o x i d e (CO2). When the c e l l s covered approximately 50 - 70% of the surface of the p l a t e , the c u l t u r e was s p l i t i n t o two p l a t e s and f r e s h medium added. This was repeated u n t i l s i x or more p l a t e s c o n t a i n i n g c e l l s c overing 50 - 70% of the p l a t e were obtained. 4. Fusion Resuspended c e l l s from s i x P e t r i p l a t e s of myeloma c e l l s were c e n t r i f u g e d at 800 X g for 10 min at RT. C e l l s were harvested and washed by resuspending the p e l l e t i n 10 ml DME followed by c e n t r i f u g a t i o n . Five days f o l l o w i n g boosting, one of the immunized mice was s a c r i f i c e d and the spleen removed. The spleen was placed i n a s t e r i l e P e t r i p l a t e c o n t a i n i n g 10 ml DME and a one inch square of s t e r i l e guaze placed over i t . The spleen was minced through the gauze using the end of a s t e r i l e syringe plunger. Spleen c e l l s were harvested by c e n t r i f u g a t i o n at 800 X g f o r 10 min at RT. The p e l l e t was resuspended i n 10 ml DME and t r a n s f e r e d to a clean t e s t - t u b e . The c e n t r i f u g a t i o n step was repeated. The p e l l e t s of myeloma and spleen c e l l s were resuspended together i n 10 ml DME followed b y . c e n t r i f u g a t i o n at 800 X g for 10 min at RT. The supernatant was a s p i r a t e d o f f and the bottom of the test-tube c o n t a i n i n g the p e l l e t was immersed i n a 37°C water bath. While immersed, 1 ml of fusogen (50% polyethylene g l y c o l 4000 (w/v) (Sigma, St. L o u i s , MO), 10% 34 dimethyl s u l f o x i d e (DMSO) i n DME) was added s l o w l y over 1 min while g e n t l y s t i r r i n g with a p i p e t t e . One m i l l i l i t r e of DME was then added s l o w l y over 1 min while s t i r r i n g , followed by 2 ml of DME over 1 min and an a d d i t i o n a l 6 ml over 3 min. This mixture was c e n t r i f u g e d at 800 X g for 10 min at RT and the p e l l e t resuspended i n 5 ml of AHAT medium (DME c o n t a i n i n g 20% FCS and 5 u l of a stock s o l u t i o n c o n t a i n i n g 2.58 mg/ml adenine (Sigma, St. L o u i s , MO), 1.36 mg/ml hypoxanthine (Sigma, St. Lo u i s , MO), 0.176 mg/ml aminopterin (Sigma, St. Lo u i s , MO) and 0.776 mg/ml thymidine (Sigma, St. L o u i s , MO)). Two 4 - 6 week balb/c mice, which were not immunized, were s a c r i f i c e d and the thymuses removed. The organs were minced and the thymocytes c o l l e c t e d using the same procedure as for the spleen c e l l s . The thymocytes were mixed with the fused c e l l mixture and d i l u t e d to 50 ml with DME. This mixture was plat e d out at 100 u l per w e l l i n t o 5 s t e r i l e Nunclon 96 microwell p l a t e s (Gibco/BRL, B u r l i n g t o n , ON). The p l a t e s were incubated at 37°C i n 10% CO2. A f t e r 3 - 4 days 100 >ul of AHT medium (AHAT medium without aminopterin) was added to each w e l l . A f t e r 7 days 180 /il of medium was removed from each w e l l and replaced with f r e s h AHT medium. Three days l a t e r , hybridoma clones were t e s t e d f o r antibody production. Selected p o s i t i v e s were expanded and recloned. B. Expansion C e l l s i n we l l s c o n t a i n i n g s e l e c t e d p o s i t i v e hybridoma clones were resuspended i n 1 ml of AHT media i n a Nunclon 24 w e l l t i s s u e c u l t u r e p l a t e (Gibco/BRL, B u r l i n g t o n , ON). The 35 plates were incubated at 37°C in 10% CO2. Three days later the wells were retested for antibody production. C e l l s from selected positive wells were expanded into 10 ml of DME containing 20% FCS and Incubated as above. Once c e l l s reached a high c e l l density they were expanded to a 10 times larger volume of medium. C. Recloning Recloning was performed to ensure homogeneity of hybridoma clones. Thymocytes from two young balb/c mice were obtained using the method described in the fusion protocol. The p e l l e t was resuspended in 10% DME and the suspension added to approximately 60 ml of HAT media. The number of c e l l s in the culture of clones to be recloned was counted using a heaemocytometer (CanLab, Richmond, BC) Based on the number obtained, the o r i g i n a l stock of c e l l s was di l u t e d with AHAT media containing the thymocytes such that 50, 20, and 5 c e l l s per ml were obtained. The d i l u t i o n s were plated out at 100 / i l per well in a Nunclon microwell plate (32 wells per clone per d i l u t i o n ) . The plates were incubated at 37°C in 10% CO2. Reclones were allowed to grow u n t i l they were large enough to score (observed v i s u a l l y using a l i g h t microscope with a magnification of 200X - 400X). Those wells containing only one clone were recorded and tested for antibody production. Selected positives were expanded as described previously. 36 D. Freezing for Long Term storage Selected hybridoma cultures, both before and after recloning, were frozen for long term storage. Cultures in two 10 ml P e t r i plates with a high c e l l density and exhibiting logarithmic growth were centrifuged at 800 X g for 10 min at RT. The p e l l e t was resuspended in 3 - 5 ml of a cryoprotectant solution containing 20% FCS and 10% DMSO in DME. One m i l l i l i t r e volumes were transfered to Nunc Freezer v i a l s (Gibco/BRL, Burlington, ON) and placed in an insulated cardboard box (approximately 30 cm lined with 2.5 cm of styrofoam in s u l a t i o n ) . This was placed in a -80°C freezer for 24 hr then transfered to l i q u i d nitrogen. E. Ascites Production Large scale production of monoclonal antibodies was accomplished through ascites production in mice. Eight 3 - 4 month old balb/c mice were injected i n t r a p e r i t o n e a l l y with 0.5 ml prlstane (Sigma, St, Louis, MO) using a 21G1 needle. One week l a t e r , 2 P e t r i plates of hybridoma cultures with a high c e l l density in log phase of growth were centrifuged at 800 X g for 10 min at RT and the p e l l e t resuspended in 0.5 ml DME. This was injected i n t r a p e r i t o n e a l l y using a 22G1/2 needle into each pristane treated mouse (2 mice per hybridoma clone). Seven days later the ascites f l u i d was c o l l e c t e d . This was done by inserting an 18G1/2 needle into the swollen abdomen of the mice and holding the mice over a 10 ml t e s t -tube u n t i l the f l u i d flow stopped. The f l u i d c o llected was centrifuged at 800 X g for 15 min at RT. The supernatant was 37 d i v i d e d i n t o 1 ml a l i q u o t s and frozen at -20°C. F. P u r i f i c a t i o n of A s c i t e s F l u i d 1. Gel F i l t r a t i o n Chromatography P u r i f i c a t i o n of a s c i t e s f l u i d by g e l f i l t r a t i o n chromatography was performed on a column packed with Sephacryl S-300 SF (Pharmacia, Dorval, PQ), using a 0.1 M T r i s - H C l b u f f e r , pH 8, c o n t a i n i n g 0.5 M sodium c h l o r i d e (NaCl) as the e l u t i n g agent. Column dimensions were 1.8 X 34 cm. A s o l u t i o n of 10 mg/ml Blue Dextran 2000 (Pharmacia, Dorval, PQ) was a p p l i e d i n order to check the column packing and determine the void volume ( V Q ) . A 1 ml sample of a s c i t e s f l u i d f i l t e r e d through a Millex-GS 0.22 um f i l t e r u n i t ( M i l l i p o r e Corp., Bedford, MA) was a p p l i e d under eluent flow. Eluent flow rate was 2 ml h * from top to bottom. Two m i l l i l i t r e f r a c t i o n s were c o l l e c t e d and the p r o t e i n c o n c e n t r a t i o n monitored by measuring the absorbance at 280 nm using a Shimadzu U V - V i s i b l e Recording Spectrophotometer (Shimadzu Corp., Kyoto, Japan). Immunoreactivity of s e l e c t e d f r a c t i o n s was monitored by i n d i r e c t ELISA. C o l l e c t e d f r a c t i o n s were concentrated to 1 ml using a Centriprep-30 concentrator (Amicon, Danvers, MA) and sodium azide (NaN^) added to 0.02%. 2. Ion-Exchange Chromatography P u r i f i c a t i o n of a s c i t e s f l u i d by ion-exchange chromatography was performed on a 10 ml p l a s t i c syringe (Becton-Dickson, Rutherford, NJ) packed with DEAE-Sephacel 38 (Sigma, St. Lo u i s , MO) using a 10 mM T r i s - H C l b u f f e r , pH 8.0 as the e l u t i n g agent. Column dimensions were 1.8 X 7 cm. To apply the samples, eluent flow was stopped and the buf f e r allowed to d r a i n to the top of the column bed. One m i l l i l i t r e of f i l t e r e d a s c i t e s f l u i d was a p p l i e d to the top of the column bed using a pasteur p i p e t t e and eluent flow resumed. Eluent flow r a t e was 5 ml h 1 from top to bottom. One m i l l i l i t r e f r a c t i o n s were c o l l e c t e d and the p r o t e i n c o n c e n t r a t i o n and immunoreactivity monitored as described i n gel f i l t r a t i o n . C o l l e c t e d f r a c t i o n s were concentrated to 1 ml and NaN 3 added to a concentration of 0.02%. A l i n e a r e l u t i o n gradient was created by using two c y l i n d e r s of l i k e dimensions connected with tubing. The f i r s t c y l i n d e r contained 20 ml of the e l u t i n g b u f f e r while the second c y l i n d e r contained an equal volume of 1 M NaCl. During eluent flow the two were mixed with a magnetic s t i r bar as g r a v i t y caused the saLt s o l u t i o n i n the second c y l i n d e r to move i n t o the f i r s t . G. Detection of Antibody A c t i v i t y Towards E. col1 1. Enzyme-Linked Immunosorbent Assay (ELISA) An i n d i r e c t ELISA described by Kannangara et a l . (1989) was used to screen hybridoma c u l t u r e s for antibody production. Unless otherwise s t a t e d , volumes of samples and reagents added were 100 jil per w e l l . A L i n b r o / T i t e r t e k EIA m i c r o t i t r a t i o n p l a t e (Flow L a b o r a t o r i e s , McClean, VA) was coated with 10/ug/ml of outer membrane preparation i n PBS. P l a t e s were incubated e i t h e r for 1 hr at 37°C or overnight at 39 4 C. Fo l l o w i n g incubation the coating s o l u t i o n was removed by shaking the pl a t e contents i n t o a s i n k . B l ocking s o l u t i o n ( 2 % B l o t t o : 1 0 % skimmilk powder i n deionized d i s t i l l e d water c o n t a i n i n g 0.02% NaN 3 i n PBS) (PBS B l o t t o ) was added ( 2 0 0 T U I per w e l l ) . For each sample w e l l r e q u i r e d , a second w e l l c o n t a i n i n g no coating was prepared as a c o n t r o l . Incubation was c a r r i e d out for 30 min at 37°C and the b l o c k i n g s o l u t i o n removed. F l u i d from each hybridoma c u l t u r e was added and the p l a t e s incubated for 1 hr at 37°C. F o l l o w i n g washing with tap water, r a b b i t anti-mouse a l k a l i n e phosphatase (ALP) conjugate (Bio/Can S c i e n t i f i c Inc., Mississauga, ON) d i l u t e d 1/3000 with PBS B l o t t o was added and the p l a t e s incubated for 1 hr at 37°C. A f t e r a f i n a l wash, 0 . 0 5 % p-nitrophenyl phosphate (Sigma, St. L o u i s , MO) i n 1 0 % diethanolamine b u f f e r , pH 9.8, c o n t a i n i n g 0 . 0 1 % MgCl 2 and 0.0 2 % NaN 3 was added and the p l a t e s incubated u n t i l a colour r e a c t i o n was observed. The absorbance of each w e l l at 4 0 5 nm was read using a T i t e r t e k M u l t i s c a n MCC ELISA reader (Flow Labs., McClean, VA). Con t r o l values were subtracted from sample values. A s i m i l a r ELISA was used to monitor the immunoreactivity of the g e l f i l t r a t i o n and ion-exchange f r a c t i o n s . Instead of adding hybridoma c u l t u r e f l u i d to the w e l l s , 1 00 A i l of each column f r a c t i o n was added. 2. Immunofluorescence An immunofluorescence assay was performed to t e s t the monoclonal an t i b o d i e s produced for c r o s s - r e a c t i v i t y with 40 other bacteria. Cultures of the following bacteria were obtained from Mr. J. Jessop of the B.C. Pr o v i n c i a l Health Laboratory (Enterics): 5 serotypes of non-enteropathogenic E . c o l i : 0157:H7, 0157:K88:H19 and three for which no serotype information was given (labelled #1,2 and 3); five serotypes of enteropathogenic E . c o l i : 0128:K67, 055:K59, 044:K74, 0112:K68, 018:K77, and thirteen other Enterobacteriaciae: Serratla marcescens, Citrobacter  freundi i , Enterobacter cloacae, Edwardsiella tarda,  K l e b s i e l l a pneumoniae, Proteus m i r a b l l i s , Proteus morgani1,  Hafnia a l v e i , Escherichia fergosonii, Escherichia hermanli,  Proteus r e t t g e r i , Kluyvera ascorbita, and Alkalescens-dispar-I_. Slants of the above bacteria were prepared on t r y p t i c soy agar (TSA) (Difco Labs., Detroit, MI) and incubated for 24 hrs at 37°C. To each slant 3 ml of deionized d i s t i l l e d water f i l t e r e d through a Millex-GS 0.22 /am f i l t e r unit (Mill i p o r e Corp., Bedford, MA) were added. The slant was then agitated using a vortex mixer. The re s u l t i n g c e l l suspension was added to a 50 ml centrifuge tube. F i l t e r e d water was added to approximately 40 ml followed by centrifugation at 15,000 X g for 20 min. The p e l l e t was resuspended in 40 ml of the f i l t e r e d water and the centrifugation step repeated. The p e l l e t was resuspended in 2 ml f i l t e r e d water and a d i l u t i o n series of 10 ^ to 10~4 prepared. Twenty m i c r o l i t r e s of each d i l u t i o n was placed in a well on a toxoplasmosis s l i d e (Bellco Glass Inc., Vineland, 41 NJ) and allowed to a i r dry. B a c t e r i a were heat f i x e d by passing the s l i d e s through a flame 4 times. Once the s l i d e s cooled, 20 yul of c u l t u r e f l u i d from each hybridoma or 20 yul of p u r i f i e d Mab d i l u t e d 1/500 were placed i n the w e l l s . Negative c o n t r o l s contained no Mab s o l u t i o n . S l i d e s were placed i n a sealed p l a s t i c container and incubated f o r 30 min at 37°C then washed with d i s t i l l e d water and a i r dryed. Twenty m i c r o l i t r e s of f l u o r e s c e in-conjugated anti-mouse antibody conjugate (Bio/Can S c i e n t i f i c Inc., Mississauga, ON) d i l u t e d 1/1000 i n PBS c o n t a i n i n g 5% B l o t t o were added, o followed by a 30 min incubation at 37 C. A f t e r a f i n a l wash and a i r dry, one drop of mounting f l u i d ( g l y c e r o l c o n t a i n i n g 10% PBS and 0.1% p-phenylenediamine (w/v)) was added to each w e l l and the c o v e r s l i p s a p p l i e d . S l i d e s were observed using a fluorescence microscope (model no. 64881, C a r l Z e i s s , W. Germany) equipped with a mercury vapour l i g h t source and set up for e p i - i l l u m i n a t i o n with a f i l t e r set for f l u o r e s c e i n fluorescence ( e x c i t a t i o n at 495 nm and emission at 520 nm). A 100X o i l immersion Neofluar o b j e c t i v e was used for observation. H. SDS-Polyacrylamide Gel E l e c t r o p h o r e s i s Sodium dodecyl s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o -phoresis (SDS-PAGE) was performed using two methods. I. Method A The f i r s t method used was described by Weber and Osborn (1969). Ten percent acrylamide g e l s were prepared by mixing 42 the following: 4.8 ml of a 0.2 M sodium phosphate buffer, pH 7.0, containing 0.2% SDS (w/v) (Sigma, St. Louis, MO), 4.2 ml of a 22.2% acrylamide (w/v) (Sigma, St. Louis, MO) solution containing 0.6% bisacrylamide (w/v) (Sigma, St. Louis, MO), and 6 O O / 1 I of a 1.2% ammonium persulfate (w/v) (Sigma, St. Louis, MO) solution. To i n i t i a t e polymerization, 15 Ail of N, N,N',N'-tetramethylethylened iamene (TEMED) (Bio-Rad, Richmond, CA) were added. Samples of eluted fractions from gel f i l t r a t i o n and ion-exchange were mixed 1:1 with sample buffer containing 4% SDS in sodium phosphate buffer, containing 10% g l y c e r o l , 1% 2-mercaptoethanol and 0.01% malachite green (Fischer S c i e n t i f i c , Ottawa, ON). Ten mi c r o l i t r e samples were applied to the gels. 2. Method B The second method used was described by Laemmli (1970). Twelve percent acrylamide separating gels were prepared by mixing the following: 3.35 ml d i s t i l l e d water, 2.5 ml of a 1.5 M Tris-HCl buffer, pH 8.0, 0.1 ml of a 10% SDS stock solution, 4 ml of a 30% stock solution of acrylamide/bis (Bio-Rad, Richmond, CA), and 25 /ul of a 10% ammonium persulfate solution. Four percent stacking gels were prepared by mixing the following: 3.05 ml d i s t i l l e d water, 1.25 ml of a 0.5 M Tris-HCl buffer, pH 6.8, 50/al of the SDS stock solution, 0.67 ml of the acrylamide/bis stock solution and 50 ul of the ammonium persulfate solution. Five m i c r o l i t r e s of TEMED was added to each of the above mixtures to i n i t i a t e polymerization. Samples were mixed 1:1 with 43 sample bu f f e r c o n t a i n i n g 12.5% of a 0.5 M T r i s - H C l b u f f e r , pH 6.8, 10% g l y c e r o l , 2% SDS, 5% 2-mercaptoethanol and 0.01% bromophenol blue (Bio-Rad, Richmond, CA). Gel mixtures from both methods were poured i n t o a B i o -Rad s l a b type V e r t i c a l Gel E l e c t r o p h o r e s i s Unit (Bio-Rad Richmond, CA) with dimensions of 7.2 X 10 cm and a g e l thickness of 0.75 mm. Samples used i n both methods were placed i n a b o i l i n g water bath for approximately 5 min p r i o r to a p p l i c a t i o n to the g e l s . Standards used were Bio-Rad Molecular Weight Standards (Bio-Rad, Richmond, CA). Gels prepared by method A were run at 90 mA and gels prepared by method B were run at 25 mA u n t i l the t r a c k i n g dye reached the s t a c k i n g g e l and then the amperage was increased to 50 mA. In both methods the gels were run u n t i l the t r a c k i n g dye reached the bottom of the g e l s . S t a i n i n g of gels was accomplished with 0.05% Coomassie Blue R250 (ICN, Cleveland, OH) and 0.05% Coomassie Blue G250 (Bio-Rad, Richmond, CA) i n a s o l u t i o n of 30% methanol and 10% a c e t i c a c i d for 1 - 2 hr. Destaining was done using a s o l u t i o n of 30% methanol and 10% a c e t i c a c i d . I... Character i z a t ion of Mabs 1. I s o t y p i n g of Mabs Determination of the immunoglobulin isotype of the monoclonal a n t i b o d i e s was done using a Mouse Hybridoma Sub-Isot y p i n g K i t (Behring d i a g n o s t i c s . La J o l l a , CA) . The procedure u t i l i z e s an ELISA s i m i l a r to the one already described. P l a t e s were coated with a 1/1000 d i l u t i o n of goat 44 anti-mouse Ig and incubated for 1 hr at 37°C. A f t e r removal of the p l a t e contents, b l o c k i n g s o l u t i o n was added and the pl a t e s incubated for another 30 min. P l a t e contents were removed and monoclonal antibody samples, d i l u t e d 1/500 i n PBS B l o t t o , were added. Con t r o l w e l l s contained no antibody samples. Fol l o w i n g incubation f o r 1 hr, p l a t e s were washed with tap water. For each antibody sample, the f o l l o w i n g r a b b i t anti-mouse immunoglobulins (Ig) d i l u t e d 1/1000 i n PBS B l o t t o were added: Ig G l , IgG2a, IgG2b, IgG3, IgM and IgA. P l a t e s were incubated f o r 1 hr and then washed with tap water. This was followed by a d d i t i o n of anti-mouse ALP conjugate and incubation for 1 hr. Enzyme substrate was added a f t e r a f i n a l wash. A f t e r incubation for approximately 1 hr, absorbance of the w e l l s was read at 405 nm. Cont r o l values were subtracted from sample values. 2. Immunoblot Assay An Immunoblot Assay of the monoclonal antibody samples was performed as described by Dunn (1986) with m o d i f i c a t i o n s . TSA s l a n t s of s e l e c t e d b a c t e r i a were prepared and a b a c t e r i a l p e l l e t obtained as was described i n the Immunofluorescence assay procedure. The p e l l e t was resuspended i n 2 ml of deionized d i s t i l l e d water. SDS-PAGE p r o f i l e s were prepared using the method B described p r e v i o u s l y . For b l o t s probed with Mabs 4D10 CI and 2H4 H12, b a c t e r i a l suspensions were prepared i n sample bu f f e r both with and without mercaptoethanol. B a c t e r i a l suspensions were mixed 1:1 with e l e c r o p h o r e s i s sample bu f f e r p r i o r to a p p l i c a t i o n to the g e l . Seven m i c r o l i t r e samples were a p p l i e d . G e l p r o f i l e s were t r a n s f e r e d e l e c t r o p h o r e t i c a l l y t o an Immobilon-P ( p o l y v i n y l i d e n e d i f l u o r i d e ) membrane ( M i l l i p o r e Corp., B e d f o r d , MA) w i t h a pore s i z e of 0.45/um u s i n g a * TE S e r i e s Transphor E l e c t r o p h o r e s i s U n i t (Hoefer S c i e n t i f i c I n s t r u m e n t s , San F r a n c i s c o , CA). The t r a n s f e r b u f f e r used c o n t a i n e d 25 mM T r i s , 192 mM g l y c i n e , pH 8.3, i n 20% methanol ( v / v ) . B l o t t i n g was c a r r i e d out f o r 2 hr a t 0.5 A. The temperature was kept c o o l by r u n n i n g c o l d water t h r o u g h the c o o l i n g c o i l . F o l l o w i n g t r a n s f e r , the g e l s were s t a i n e d w i t h a Coomassie Blue s t a i n f o r 1 - 2 hr f o l l o w e d by d e s t a i n i n g . The membranes were p l a c e d i n P e t r i p l a t e s and 10 ml of 5% B l o t t o added. P l a t e s were i n c u b a t e d f o r 30 min a t room temperature w i t h s t i r r i n g . The s o l u t i o n was decanted and 10 ml of monoclonal or p o l y c l o n a l a n t i b o d y sample, d i l u t e d 1/500 i n PBS B l o t t o , were added. I n c u b a t i o n was r e p e a t e d f o r 1 hr and the a n t i b o d y s o l u t i o n s d e c a n t e d . The membranes were washed by i n c u b a t i n g them i n 10 ml of PBS f o r 10 min. T h i s was r e p e a t e d 3 t i m e s w i t h f r e s h PBS. A f t e r washing, 10 ml of r a b b i t anti-mouse I g - ALP c o n j u g a t e d i l u t e d 1/3000 or r a b b i t a n t i - c h i c k e n IgG - ALP c o n j u g a t e (Sigma, S t . L o u i s , MO) ( f o r p o l y c l o n a l samples) d i l u t e d 1/2000 w i t h PBS B l o t t o was added. Membranes were i n c u b a t e d f o r an a d d i t i o n a l hour. The c o n j u g a t e s o l u t i o n s were decanted and the washing s t e p r e p e a t e d w i t h 50 mM T r i s - H C l b u f f e r , pH 8, c o n t a i n i n g 50 mM Na C l . Ten m i l l i l i t r e s of s u b s t r a t e (0.33 mg/ml N a p t h o l AS-MX phosphate d i s o d i u m s a l t (Sigma, S t . L o u i s , MO) and 3 mg/ml 46 Fast Red TR s a l t (Sigma, St. L o u i s , MO) i n the above b u f f e r ) was added. Incubation was c a r r i e d out i n the dark. A f t e r approximately 30 min, the membranes were r i n s e d with PBS then a i r dryed. j . P r e p a r a t i o n of P o l y c l o n a l Antiserum to E. c o l i 1. E . c o l i Sample Prep a r a t i o n E . c o l i 0142:K86:H6 (ATCC 23895) c e l l s which had been for m a l i n t r e a t e d (Shimizu et a l . , 1988a) and f r e e z e - d r l e d were a g i f t from Dr. S. Shimizu. C e l l s (2.2 mg) were dispersed i n PBS p r e v i o u s l y s t e r i l i z e d by f i l t r a t i o n through a Millex-GS 0.22 um f i l t r a t i o n u n i t . This was mixed with an equal volume g of adjuvant to y i e l d approximately 10 c e l l s / m l . An emulsion was formed by repeatedly drawing i n and e j e c t i n g t h i s mixture from a 5 ml syringe with a 20G3/2 needle. 2. Immunization of Chickens I n i t i a l i n j e c t i o n s contained Freund's complete adjuvant, while subsequent i n j e c t i o n s contained Freund's incomplete adjuvant. Three l a y i n g White Leghorn hens obtained from the U n i v e r s i t y of B r i t i s h Columbia P o u l t r y Unit were given intramuscular i n j e c t i o n s of 0.25 ml of c e l l p reparation to each of four s i t e s (one i n each breast and thigh) using a 26G3/8 needle. This i n j e c t i o n was repeated approximately 2 weeks l a t e r . 3. I s o l a t i o n and P u r i f i c a t i o n of Chicken IgY Eggs from the immunized hens were c o l l e c t e d and stored at 4°C. The chicken IgY f r a c t i o n was separated using a 47 method described by Poison et a l . (1980) with m o d i f i c a t i o n s . Egg yolks were separated from the white and r i n s e d with d i s t i l l e d water. Yolks were punctured and the contents allowed to d r a i n i n t o a graduated c y l i n d e r . PBS (0.01M, pH 7.4) e q u a l l i n g 4 times the volume of the yolk was added followed by the a d d i t i o n of polyethylene g l y c o l (PEG) (Sigma, St. L o u i s , MO) with a molecular weight of 8000 daltons to a f i n a l c o n c e n t r a t i o n of 3.5% (w/v). This mixture was s t i r r e d u n t i l the polymer d i s s o l v e d . The mixture was then c e n t r i f u g e d at 14,500 X g for 20 min at 4°C. The supernatant was decanted over cheesecloth to remove the l i p i d l a y e r then f i l t e r e d through Whatman No. 1 f i l t e r paper (Whatman L t d . , England). Twelve grams of PEG per 100 ml of f i l t r a t e was added. A f t e r s t i r r i n g at RT for approximately 10 min, c e n t r i f u g a t i o n was repeated. The supernatant was discarded and the p r e c i p i t a t e d i s s o l v e d i n 200 ml of 0.02 M PBS, pH 7.2. Twelve percent PEG was added and the s o l u t i o n s t i r r e d u n t i l the PEG d i s s o l v e d . C e n t r i f u g a t i o n was repeated and the p r e c i p i t a t e c o l l e c t e d and d i s s o l v e d i n 50 ml of a 25 mM potassium phosphate b u f f e r , pH 8.0, per 100 ml of s t a r t i n g yolk. Further p u r i f i c a t i o n of the chicken IgY was accomplished by ion-exchange chromatography. A 10 ml syringe was packed with DEAE-Sephacel e q u i l i b r a t e d with 25 mM potassium phosphate b u f f e r , pH 8. Twenty f i v e m i l l i l i t r e s of sample was a p p l i e d under flow. The column was washed with 10 bed volumes of e q u i l i b r a t i n g b u f f e r , then e l u t e d with 250 mM 48 potassium phosphate b u f f e r , pH 8. Flow rate was maintained at 0.75 ml min-'1' from top to bottom. s i x m i l l i l l t r e f r a c t i o n s were c o l l e c t e d and absorbance at 280 nm monitored. F r a c t i o n s from the peak eluted with the 250 mM phosphate buf f e r were pooled and concentrated by ammonium sulphate p r e c i p i t a t i o n . K. Ammonium Sulphate P r e c i p i t a t i o n Ammonium sulphate was added to chicken a n t i - E . c o l i IgY f r a c t i o n s to give h a l f s a t u r a t i o n at RT (31.3 g/100 ml). This was s t i r r e d at RT for 30 min then c e n t r i f u g e d at 12,000 X g for 10 min at RT. The r e s u l t i n g p r e c i p i t a t e was d i s s o l v e d i n 2 ml of PBS and d i a l y s e d against the same buf f e r (X2 changes) overnight at 4°C. L. P r o t e i n Determination Unless otherwise s t a t e d , p r o t e i n determinations were done using the Bio-Rad P r o t e i n Assay (Bio-Rad, Richmond, CA). A standard curve was prepared using e i t h e r bovine serum albumin (Sigma, St. Lo u i s , MO) or chicken IgG (Sigma, St. Lo u i s , MO) ranging i n concentration from 0.2 to 1.2 mg/ml i n PBS. Blanks contained only b u f f e r . To each tube, 100 u l of sample or standard and 5 ml of dye reagent ( d i l u t e d f i v e times with d i s t i l l e d water) was added. The mixture was vortexed and allowed to stand f o r 15 min. Absorbance at 595 nm was read and the p r o t e i n concentrations of the samples c a l c u l a t e d from the standard curve. 49 PART I I : DEVELOPMENT OF AN ENZYME-LINKED IMMUNOSORBANT ASSAY (ELISA) A. Immunization Procedures 1. Immunization of Chickens Pr e p a r a t i o n of immunogen and immunization of chickens was performed by Anne McCannel. 6-N-acetylglucosamlnidase (NAGase) p u r i f i e d from bovine kidney (Sigma, St. L o u i s , MO) i n a 3.2 M ammonium sulphate s o l u t i o n , pH 6, with a p r o t e i n c o n c e n t r a t i o n of 5 mg/ml was mixed 1:1 with adjuvant i n order to give a f i n a l p r o t e i n c o n c e n t r a t i o n of 2.5 mg/ml. Laying White Leghorn hens were obtained from the U n i v e r s i t y of B r i t i s h Columbia P o u l t r y U n i t . Chickens were immunized using the procedure already described i n PART I f o r preparation of a n t i - E . c o l l antiserum. 2. Immunization of Rabbits Two New Zealand White r a b b i t s were housed at the U n i v e r s i t y of B r i t i s h Columbia Animal Care Centre. A l l i n j e c t i o n s and blood c o l l e c t i o n s were done by Animal Care Centre personnel. S i x t y s i x m i c r o l i t r e s of the NAGase s o l u t i o n used above were suspended i n 1 ml of a s a l i n e s o l u t i o n (0.85% sodium c h l o r i d e (w/v)). This was mixed 1:1 with Freund's complete adjuvant to give a f i n a l p r o t e i n concentration of 165 ug/ml. To each r a b b i t , 0.1 ml of t h i s preparation was i n j e c t e d subcutaneously i n t o each of s i x s i t e s using a 22G1/2 needle for a t o t a l of lOO^Aig p r o t e i n . Three weeks l a t e r , a second i n j e c t i o n was performed using an immunogen preparation 50 prepared i n the f o l l o w i n g manner. F o r t y m i c r o l l t r e s of NAGase s o l u t i o n were suspended i n 200/ul of s a l i n e s o l u t i o n . This was mixed 1:1 with Freund's incomplete adjuvant to give a f i n a l p r o t e i n concentration of 0.5 mg/ml. Each r a b b i t received 0.1 ml of t h i s preparation i n t r a m u s c u l a r l y i n t o each of 2 s i t e s f o r a t o t a l of 100/ug p r o t e i n . Three weeks l a t e r the animals were bled by c a r d i a c puncture. The blood was allowed to coagulate at RT, then c e n t r i f u g e d at 5000 X g for 30 min. The serum was c o l l e c t e d and stored at 4°C with 0. 02. NaN 3. B. Immunoglobulin P r e p a r a t i o n 1. I s o l a t i o n and P u r i f i c a t i o n of Chicken IgY Eggs from immunized chickens were donated by Anne McCannel. I s o l a t i o n and p u r i f i c a t i o n of chicken IgY from the egg y o l k s was c a r r i e d out as described i n PART I. 2. I s o l a t i o n of Immunoglobulins from Rabbit Blood Serum I s o l a t i o n of the immunoglobulin f r a c t i o n from the blood serum of immunized r a b b i t s was c a r r i e d out by ammonium sulphate p r e c i p i t a t i o n as described by Garvey et a l . (1977). The pH of a 25 ml a l i q u o t of a saturated (RT) ammonium sulphate s o l u t i o n was adjusted to pH 7.8 by a d d i t i o n of 5 N sodium hydroxide. A 50 ml serum sample was added dropwise to t h i s s o l u t i o n with constant s t i r r i n g . The mixture was s t i r r e d f o r an a d d i t i o n a l 3 hrs at RT followed by c e n t r i f u g a t i o n at 1400 X g for 30 min at RT. The r e s u l t i n g p r e c i p i t a t e was d i s s o l v e d i n s u f f i c i e n t s a l i n e s o l u t i o n to 51 r e s t o r e the volume of s o l u t i o n to that of the o r i g i n a l serum sample. A second and t h i r d p r e c i p i t a t i o n step was c a r r i e d out. The f i n a l p r e c i p i t a t e was d i s s o l v e d i n 5 ml of PBS and 0. 02% NaN 3 added. C. Pr e p a r a t i o n of Anti-NAGase - A l k a l i n e Phosphatase  Conjugates Prepared anti-NAGase chicken IgY was conjugated to a l k a l i n e phosphatase (ALP) Type VII-S from bovine i n t e s t i n a l mucosa (Sigma, St. L o u i s , MO) using two d i f f e r e n t methods. A 2:1 molar r a t i o of ALP to chicken IgG was used i n each method. 1. Glutaraldehyde Method. The glutaraldehyde method used was described by E n g v a l l and Perlmann (1972) and modified by Shimizu (1988b). Three hundred and s i x t y s i x m i c r o l i t r e s of ALP s o l u t i o n were c e n t r i f u g e d at 1100 X g for 15 min at 4°C. The r e s u l t i n g p r e c i p i t a t e was d i s s o l v e d i n 1.5 ml of PBS c o n t a i n i n g 1 mM MgCl2 and d i a l y s e d against the same buf f e r (X2 changes) overnight at 4°C. Ten m i c r o l i t r e s of a 25% glutaraldehyde s o l u t i o n (BDH Chemicals, Toronto, ON) were added and the mixture s t i r r e d f o r 50 min at RT. A 0.2 ml s o l u t i o n c o n t a i n i n g 3 mg p r e v i o u s l y prepared anti-NAGase chicken IgY i n PBS was added and s t i r r i n g continued for 75 min. The s o l u t i o n was cooled i n an ice bath then a p p l i e d to a Sephacryl S-300 SF column e q u i l i b r a t e d with 50 mM T r i s -HCl b u f f e r , pH 8, c o n t a i n i n g 1 mM MgCl 2 and 0.1% NaN^• Column dimensions were 1.3 X 50 cm. Eluent flow rate was 9 52 ml min from top to bottom. One and a h a l f m i l l i l i t r e f r a c t i o n s were c o l l e c t e d and the p r o t e i n concentration monitored as p r e v i o u s l y described. F r a c t i o n s i n the f i r s t e l u t e d peak were pooled and stored at 4°C. 2. Periodate Oxidation Method Anti-NAGase IgY was a l s o conjugated to ALP by periodate o x i d a t i o n . The method used was based on methods described by Williams (1984) and Tussen and Kurstak (1984) with m o d i f i c a t i o n s . Three hundred and s i x t y s i x m i c r o l i t r e s of ALP s o l u t i o n were c e n t r i f u g e d at 1100 X g for 15 min at 4°C. The r e s u l t i n g p r e c i p i t a t e was d i s s o l v e d i n 0.5 ml of a 0.3 M sodium carbonate s o l u t i o n and d i a l y s e d against the same overnight at 4°C with two s o l u t i o n changes. To t h i s s o l u t i o n , 0.5 ml of a 40 mM sodium periodate s o l u t i o n was added and the r e a c t i o n allowed to proceed f o r 4 hr i n the dark at RT with a g i t a t i o n . A 0.5 ml s o l u t i o n c o n t a i n i n g 3 mg anti-NAGase chicken IgY, p r e v i o u s l y d i a l y s e d overnight against a 0.01 M sodium carbonate b u f f e r , pH 9.5, was added. The r e a c t i o n was continued f o r 24 hr under the same c o n d i t i o n s . To the r e a c t i o n mixture, 40 / u l of a 2 M ethanolamine s o l u t i o n , pH 9.5, was added, and the mixture d i a l y s e d overnight against PBS. Gel f i l t r a t i o n chromatography was c a r r i e d out as i n the previous method. D. Pre p a r a t i o n of Press J u i c e and F i s h E x t r a c t s from F i s h  Muscle Fresh Sockeye salmon (not p r e v i o u s l y frozen) was obtained i n chunks from a l o c a l seafood r e t a i l s t o r e . The 53 time the f i s h were stored on ice was not known. Samples were cut i n t o 2.5 cm t h i c k steaks and analysed on the day of c o l l e c t i o n and a f t e r one week of frozen storage. Steaks that were frozen were placed i n i n d i v i d u a l f r e e z e r bags and then put i n a cardboard box with dimensions of 10 X 30 cm. Storage was at -20°C fo r one week. These samples were thawed overnight at 4°C p r i o r to a n a l y s i s . P r e p a r a t i o n of press j u i c e and f i s h e x t r a c t f r a c t i o n s from f i s h muscle was done according to Yoshioka (1988) with m o d i f i c a t i o n s . F i s h muscle was ground using a Kitchen Aid meat grinder (Hobart Manufacturing Co., Troy, OH) by f o r c i n g the muscle through holes with a diameter of 5 mm. Five grams of the ground sample were placed i n a f i l t e r - f u g e tube (model 2427, I n t e r n a t i o n a l Equipment Co., Needham Heights, MA) and c e n t r i f u g e d at 9,600 X g for 30 min at 5°C. J u i c e c o l l e c t e d at the bottom of the f i l t e r - f u g e tube was d i l u t e d to 2.5 ml with 1% NaCl and f i l t e r e d using a Millex-HA 0.45 um f i l t e r u n i t ( M i l l i p o r e Corp., Bedford, MA). This was termed the press j u i c e ( P J ) . The residue was weighed and a volume of 1% NaCl of 10 times the residue weight was added. This mixture was homogenized for 1 min at 12,000 rpm using an U l t r a - T u r r a x Ika-Tron (Janke and Kunkel, Staufen, W. Germany) with an 18N probe. A f t e r e q u i l i b r a t i n g for 30 min at 4°C , the mixture was c e n t r i f u g e d at 22,500 X g for 10 min at 5°C. The supernatant was d i l u t e d to 20 ml with 1% NaCl then f i l t e r e d through a Millex-HA 0.45 um f i l t e r u n i t . This was termed the f i s h e x t r a c t (FE). 54 E. Enzyme-Linked Immunosorbent Assays A l l ELISAs were performed on Immulon II m i c r o t i t r e p l a t e (Dynatech Labs., C h a n t i l l y , VA). Bovine kidney NAGase used for animal i n j e c t i o n s was a l s o used for c o a t i n g p l a t e s and f o r standards. Unless otherwise s t a t e d , sample and reagent volumes added were 100>ul per w e l l and incubation was f o r 1 hr at 37°C. Standard curves were derived using NAGase concentrations ranging from 0 to 20/jg/ml i n the competitive ELISA and 0.031 to 20 ug/ml i n the double-sandwich ELISA. 1. I n d i r e c t ELISA a. Detection of Antibody A c t i v i t y Towards NAGase An i n d i r e c t ELISA was performed f o r the d e t e c t i o n of antibody a c t i v i t y of chicken IgY and r a b b i t IgG towards NAGase. A m i c r o t i t r e p l a t e was coated with 20 Xig/ml NAGase i n 0.05 M sodium carbonate b u f f e r , pH 9.6, and stored overnight at 4°C. F o l l o w i n g incubation the c o a t i n g s o l u t i o n was removed by shaking the p l a t e contents i n t o a s i n k . PBS B l o t t o (200 u l per w e l l ) was added and the p l a t e s incubated f o r 30 min. B l o c k i n g s o l u t i o n was removed and the antibody samples d i l u t e d with PBS B l o t t o were added to the w e l l s . PBS B l o t t o c o n t a i n i n g no sample was added to 8 w e l l s f o r blanking ( c o n t r o l ) . P l a t e s were incubated then washed with tap water. A n t i - c h i c k e n - ALP conjugate d i l u t e d 1/2000 i n PBS B l o t t o was added and the p l a t e s incubated. F o l l o w i n g a f i n a l wash, enzyme substrate (0.1% p-nitrophenyl phosphate i n diethanolamine b u f f e r ) was added and incubated f o r 30 min. The absorbance of each w e l l at 405 nm was read using an SLT 55 L a b i n s t r u m e n t ( A u s t r i a ) ELISA r e a d e r . C o n t r o l v a l u e s were s u b t r a c t e d from sample v a l u e s . b. D e t e r m i n a t i o n of the Working D i l u t i o n of Anti-NAGase -A l k a l i n e Phosphatase Conjugates An i n d i r e c t ELISA, s i m i l a r t o the one d e s c r i b e d above, was used t o d e t e r m i n e the w o r k i n g d i l u t i o n s of p r e p a r e d a n t i -NAGase ALP c o n j u g a t e s . The f o l l o w i n g e x c e p t i o n s were made. F o l l o w i n g the b l o c k i n g and i n c u b a t i o n s t e p s , d i l u t i o n s of -1 -5 anti-NAGase - ALP c o n j u g a t e s r a n g i n g from 10 t o 10 i n PBS B l o t t o were added. C o n t r o l w e l l s c o n t a i n e d no c o n j u g a t e . F o l l o w i n g i n c u b a t i o n the enzyme s u b s t r a t e was added. 2. C o m p e t i t i v e ELISA A c o m p e t i t i v e ELISA s i m i l a r t o t h a t d e s c r i b e d by Huang e t a l . (1987) was p e rformed, w i t h m o d i f i c a t i o n s , f o r the d e t e c t i o n of NAGase. A m i c r o t i t r e p l a t e was c o a t e d w i t h 20 /ug/ml NAGase i n 0.5 M sodium c a r b o n a t e b u f f e r , pH 9.6, and s t o r e d o v e r n i g h t a t 4°C. W e l l s f o r the c o n t r o l were not c o a t e d . C o a t i n g s o l u t i o n was removed and 250/Ul PBS B l o t t o added t o each w e l l . A f t e r i n c u b a t i o n f o r 30 min, the s o l u t i o n was removed and t o each w e l l p r e p a r e d c h i c k e n a n t i -NAGase (0.1 mg/ml i n PBS B l o t t o ) added. Immediately f o l l o w i n g , NAGase s t a n d a r d s were added. F o l l o w i n g i n c u b a t i o n and washing, 200 ./ul of a n t i - c h i c k e n IgG - ALP c o n j u g a t e d i l u t e d 1/2000 i n PBS B l o t t o was added and i n c u b a t i o n r e p e a t e d . P l a t e s were washed and the s u b s t r a t e added. A f t e r i n c u b a t i o n f o r 30 min., absorbance of each w e l l a t 405 nm was r e a d . C o n t r o l v a l u e s were s u b t r a c t e d from sample v a l u e s . 3. Double-Sandwich ELISA A double-sandwich ELISA f o r the d e t e c t i o n of NAGase i n f i s h samples was used. A m i c r o t i t r e p l a t e was coated with 100 jiq/ml prepared r a b b i t anti-NAGase IgG and the p l a t e s incubated. Following removal of the c o a t i n g s o l u t i o n , 200yul of PBS B l o t t o was added and the p l a t e s incubated. Blocking s o l u t i o n was removed and PJ and FE samples or NAGase standards added. Control w e l l s contained no f i s h samples or NAGase. A f t e r incubation and washing, prepared chicken a n t i -NAGase (20 yAjg/ml i n PBS B l o t t o ) was added. Incubation and washing steps were repeated followed by a d d i t i o n of a n t i -chicken IgY ALP conjugate. Incubation and washing steps were repeated again and the enzyme subs t r a t e added. Twenty minutes l a t e r , absorbance at 405 nm was read. C o n t r o l values were subtracted from sample values. Linear r e g r e s s i o n was performed on the data obtained from the standards. Concentration of NAGase i n the f i s h samples was c a l c u l a t e d using the standard curve. F. R a d i a l Immunodiffusion Immunodiffusion p l a t e s were prepared using the method described by Mi 1 ford-Ward (1981) with m o d i f i c a t i o n s . In one t e s t - t u b e , 0.35 ml of r a b b i t a n t i - c h i c k e n IgG (Sigma, St. L o u i s , MO) was mixed with 1.65 ml of 0.2 M PBS, pH 7, and placed i n a 55°C water bath. In a second test-tube 0.07 g agarose (Sigma, St. L o u i s , MO) was mixed with 4.6 ml of PBS and 0.4 ml of a 0.35% NaN3 s o l u t i o n and placed i n a b o i l i n g water bath u n t i l the agarose d i s s o l v e d . The contents of both test-tubes were mixed and poured into r a d i a l immunodiffusion plates (ICN, Cleveland, OH). Following s o l i d i f i c a t i o n , 3 mm diameter holes were cut in the agarose using a template (ICN, Cleveland, OH). A standard curve was derived for each plate using chicken IgG (Sigma, St . Lou i s , MO) ranging in concentration from 0.2 to 1.2 mg/ml. Five m i c r o l i t r e s of samples and standards were appl ied to the wel l s . D i f fus ion was allowed to proceed for 3 days in a closed P e t r i plate containing moistened f i l t e r paper. IgG concentrations were determined by comparing the square of the diameter of the p r e c i p i t i n r ing (measured with a micrometre) versus the IgG concentration on the standard curve. G. SDS-Polyacrylamide Gel Electrophores i s SDS-PAGE was performed using method B already described in PART I , with modi f icat ions . Samples were d i l u t e d in sample buffer to give a prote in concentration of 2 mg/ml. Ten m i c r o l i t r e samples were appl ied to the ge l s . For analys i s of chicken anti-NAGase IgY, chicken IgG (Sigma, St . Lou i s , MO) was used as a standard. H. S t a t i s t i c a l Ana1ysis A one-way analys i s of variance was performed to determine i f there was a s i g n i f i c a n t d i f ference in NAGase concentrations between fresh and frozen PJ samples. A two-way analys i s of variance was performed to determine i f there was a s i g n i f i c a n t d i f ference in NAGase 58 c o n c e n t r a t i o n r a t i o s between sample d i l u t i o n s and between fre s h and frozen salmon samples. In the development of a standard curve f o r the double-sandwich ELISA for NAGase, l i n e a r r e g r e s s i o n was performed on the data. To determine i f the l i n e was s i g n i f i c a n t l y l i n e a r , the c o r r e l a t i o n c o e f f i c i e n t (r) was c a l c u l a t e d and compared to c r i t i c a l r values (Crow et a l . , 1978). The c o e f f i c i e n t of determination ( r 2 ) was a l s o c a l c u l a t e d . To determine the v a r i a t i o n about the r e g r e s s i o n l i n e , the standard e r r o r of estimate (SEE) was a l s o c a l c u l a t e d . 59 RESULTS AND DISCUSSION PART I: PRODUCTION OF MONOCLONAL ANTIBODIES (Mabs) SPECIFIC FOR AN ENTEROPATHOGENIC E.COLI (EPEC) A. Production of S p e c i f i c Mab Se c r e t i n g Hybridomas 1. Hybridoma Production The f i r s t step i n the production of monoclonal a n t i b o d i e s (Mabs) i s the establishment of hybridomas which secrete the s p e c i f i c antibody d e s i r e d . Hybridomas were produced using the f u s i o n procedure described. Two fusions were performed on two separate occasions. Fox-NY myeloma c e l l s were fused with spleen c e l l s from mice immunized with the outer membrane of an enteropathogenic E . c o l l (EPEC). Fox-NY c e l l s are d e f i c i e n t not only i n HGPRT, but a l s o adenosine phosphoribosyl t r a n s f e r a s e (APRT) (Taggart and Samloff, 1983). This allows the use of an a l t e r n a t e s e l e c t i o n system using adenine, aminopterin and thymidine, thus a l l o w i n g the s u r v i v a l of hybridomas that lose the X chromosome which codes f o r HGPRT during HAT s e l e c t i o n (Eshhar, 1985). Under t h i s s e l e c t i o n system, hybridomas formed between a spleen c e l l and a myeloma c e l l t hat d i d not lose t h i s X chromosome can u t i l i z e e i t h e r exogenous adenine and thymidine or hypoxanthine and thymidine i n a salvage pathway for nucleotide s y n t h e s i s . Therefore, the s e l e c t i o n media used i n t h i s experiment contained both aminopterin, hypoxanthine, adenine and thymidine. A f t e r approximately 10 days of growth, the hybridoma clones were v i s i b l e under a l i g h t microscope. Figure 4 60 d e p i c t s the hybridoma clones i n a s i n g l e w e l l of a microwell p l a t e . At t h i s stage of growth the w e l l s were t e s t e d f o r s p e c i f i c antibody production by ELISA. The E . c o l i OM p r e p a r a t i o n was used as the screening antigen. Table I shows the number and percentage of p o s i t i v e w e l l s obtained i n each f u s i o n . An absorbance value of 0.1 was a r b i t r a r i l y s e l e c t e d as the c u t - o f f point f o r p o s i t i v e clones. In f u s i o n 1, of the 480 w e l l s t e s t e d f o r antibody production, 11 (2.3%) t e s t e d p o s i t i v e . In f u s i o n 2, of the same number of w e l l s t e s t e d , 51 (10.6%) were p o s i t i v e for s p e c i f i c antibody production. Using t h i s f u s i o n p r o t o c o l , a l b e i t with a d i f f e r e n t antigen, as high as 80% of the w e l l s screened have been reported to be p o s i t i v e (Wieczorek,1989). Lane et a l . (1986) reported that while h i g h l y immunized spleens c o n t a i n thousands of s t i m u l a t e d B - c e l l s , l e s s that 1% of these w i l l fuse to become a n t i b o d y - s e c r e t i n g hybridomas. This number has been shown to depend on the antigen dose and the immunogenicity of the antigen. Increases i n immunizing antigen c o n c e n t r a t i o n tends to increase the y i e l d of hybridoma clones producing s p e c i f i c antibody ( S h i b i e r et a l . , 1988). I f the antigen i s a poor immunogen, antibody response w i l l be poor. The immunization p r o t o c o l a l s o seems to a f f e c t the y i e l d . S t a h l i et a l . (1983) showed that the s p e c i f i c e f f i c i e n c y , or y i e l d of p o s i t i v e hybridomas increased up to 3 days f o l l o w i n g immunization, then decreased. Olsson et a l . (1983) sta t e d that B - c e l l s should be i n a c e r t a i n stage of d i f f e r e n t i a t i o n f o r s u c c e s s f u l 61 Figure 4: Hybridoma clones as seen under a l i g h t microscope ( b r i g h t f i e l d ) i n a s i n g l e w e l l of a microwell p l a t e a f t e r approximately 10 days of growth i n AHAT media. (400X m a g n i f i c a t i o n ) 62 Table I : Number and P e r c e n t a g e of W e l l s C o n t a i n i n g s p e c i f i c A n t i b o d y P r o d u c i n g Hybridomas F u s i o n 1 2 a # of w e l l s t e s t e d 8 of s p e c i f i c w e l l s % of s p e c i f i c w e l l s 480 480 11 51 2.3% 10.6% a. F i v e m i c r o w e l l p l a t e s were used t o grow the c u l t u r e from each f u s i o n (96 w e l l s / p l a t e ) . b. Those w e l l s h a v i n g an A ^ Q ^ > 0.1 when t e s t e d by E L I S A c. The pe r c e n t a g e of w e l l s c o n t a i n i n g c l o n e s s p e c i f i c f o r the a n t i g e n . 63 h y b r i d i z a t i o n . There are a l s o some re p o r t s t h a t In v i t r o immunization i s more e f f i c i e n t i n terms of s p e c i f i c antibody production than immunization in_ v i v o (Eshhar, 1985) U l t i m a t e l y the success of a f u s i o n i s determined by the number of hybridomas s e c r e t i n g Mab with the d e s i r e d s p e c i f i c i t y and other p r o p e r t i e s . In t h i s experiment the f i n a l s e l e c t i o n of Mab s e c r e t i n g hybridomas was determined by c r o s s - r e a c t i v i t y patterns with other b a c t e r i a . Since i t was p r a c t i c a l l y impossible, due to l i m i t e d time and manpower, to proceed f u r t h e r with a l l of the p o s i t i v e clones, a working number was s e l e c t e d based on those w e l l s e x h i b i t i n g the highest t i t r e s (eg. highest absorbance values) since t h i s was a d e s i r a b l e property. From f u s i o n 1, hybridomas from 4 w e l l s were expanded and t h e i r r e a c t i v i t y confirmed by ELISA. These were then stored i n l i q u i d n i t r o g e n . From f u s i o n 2, hybridomas from 17 w e l l s were expanded and t h e i r r e a c t i v i t y confirmed by ELISA. These were a l s o stored i n l i q u i d n i t r o g e n . 2. Recloning A f t e r the i d e n t i f i c a t i o n and s e l e c t i o n of p o s i t i v e s , the next step was to r e d o n e the hybridomas i n the w e l l s . Since the o r i g i n a l p o s i t i v e w e l l s often contained more than one clone of hybridoma c e l l s (Figure 4), the d e s i r e d c e l l s could be outgrown by c e l l s that were not producing the antibody of i n t e r e s t . Recloning ensures monoclonality and e l i m i n a t e s undesirable hybridomas. In t h i s experiment r e c l o n i n g was performed by d i l u t i n g the c e l l s i n a s i n g l e p o s i t i v e w e l l i n a microwell p l a t e such that only 1 c e l l / w e l l was present. Subsequent growth l e d to a s i n g l e clone per w e l l as determined by microscopic examination Table I I shows the r e s u l t s of r e c l o n i n g . A l l 4 c u l t u r e s preserved from f u s i o n 1 were recloned, while only 5 of the 1 7 from f u s i o n 2 were recloned. On f u r t h e r expansion, only 4 of the 5 s e l e c t e d from f u s i o n 2 grew i n c u l t u r e . A t o t a l of 4 9 s i n g l e hybridoma clones were found to be s p e c i f i c for the E . c o l i OM by ELISA from f u s i o n 1 . From fu s i o n 2 , only 3 4 s i n g l e clones were found to be s p e c i f i c f o r the antigen. Again, since i t was impossible to t e s t a l l of the p o s i t i v e clones, only 8 were s e l e c t e d from each f u s i o n based on high t i t r e s i n ELISA. Since the absorbance readings for p o s i t i v e clones were g e n e r a l l y higher than the readings obtained p r i o r to r e c l o n i n g , a higher value ( A ^ Q ^ ) was chosen as the c u t - o f f point for s e l e c t i o n . The t o t a l of 1 6 hybridomas s e l e c t e d c o n s i s t e d of two from each o r i g i n a l p o s i t i v e w e l l from each f u s i o n . These hybridoma c u l t u r e s were recorded and stored i n l i q u i d n i t r o g e n . 3 . Immunofluorescence Screening S p e c i f i c i t y for the antigen (OM) was not the only property for which the Mab were s e l e c t e d . In t h i s experiment, patterns of c r o s s - r e a c t i v i t y with other b a c t e r i a was an important s e l e c t i o n c r i t e r i o n . To t e s t for c r o s s -r e a c t i v i t y , Mabs from the recloned hybridomas were screened against a panel of Enterobacteriaceae i n c l u d i n g both EPEC and non-EPEC s t r a i n s . A l l of the b a c t e r i a l s t r a i n s were obtained 6 5 Table II: Numbers of Positive Wells Selected for Recloning and Numbers of Resulting Positive Single Clones Fusion 1 2 a # of selected positive wells 4 17 # selected for recloning 4 5 # of single positive clones'3 49 34 after recloning a. Those positives exhibiting high t i tres prior to recloning b. Wells containing a single clone by microscopic examination and having an A 4 Q 5 > 0 . 5 in ELISA 66 from the B r i t i s h Columbia P r o v i n c i a l Health Laboratory and were i s o l a t e d from s t o o l specimens of both a d u l t s and i n f a n t s . EPEC 018:K77 d i d not grow when t r a n s f e r e d to a TSA s l a n t and therefore was excluded from the panel. Testing was done by i n d i r e c t immunofluorescence assay using undiluted supernatant f l u i d from hybridoma c u l t u r e s as the antibody source. Whole c e l l s were used as the antigens. Six hybridoma c u l t u r e s from the 16 s e l e c t e d reclones were randomly chosen f o r screening. The r e s u l t s are q u a l i t a t i v e s ince n e i t h e r the antibody nor antigen c o n c e n t r a t i o n were standardized. Table I I I shows the r e s u l t s of the immunofluorescence screening. For t h i s experiment a Mab which was s p e c i f i c to a l l EPEC yet d i d not react with other E n t e r o b a c t e r i a c i a e , i n c l u d i n g non-EPEC, was d e s i r a b l e . The data shows that the most promising antibody i n t h i s respect was 2F9 B3. I t was p o s i t i v e f o r a l l the EPEC s t r a i n s , showed minimal c r o s s -r e a c t i v i t y with other Enterobacteriaceae, and except f o r #3, d i d not c r o s s - r e a c t with the non-EPEC s t r a i n s on the panel. However, under the c o n d i t i o n s used i n t h i s assay the fluorescence observed was r e l a t i v e l y weak, i n c l u d i n g that for the p o s i t i v e c o n t r o l . Some c e l l s appeared to s t a i n and some d i d not, ^ w h i l e others were only p a r t i a l l y s t a i n e d . P o s i t i v e r e a c t i o n s with Mabs 3A5 CIO, 2E1 H6 and 5E10 B6 had a s i m i l a r f l u o r e s c e n t image. This may be due to low concentrations of antibody present i n the supernatant f l u i d of the hybridomas. I t i s a l s o p o s s i b l e that these Mabs are 67 Table I I I : R e s u l t s of the P r e l i m i n a r y S c r e e n i n g of S e l e c t e d Mabs A g a i n s t a Pane l of E n t e r o b a c t e r i a c e a e by Immunofluorescence Assay u s i n g Supernatant F l u i d From Hybridoma C u l t u r e s . Mabs B a c t e r i a 3A5C10 2H4H12 4D10C1 2E1H6 5E10B6 2F9B3 C * b c EPEC 0142:K86:H6 + + + + + + + + + + -EPEC 0128:K67 + - - 4 + + + EPEC 055:K59 +• - + + + + -EPEC 044:K74 + - - + + + + EPEC 0112:K68 + - +• - + -E . c o l i ( # l ) d — — — + _ _ E . c o l i 0157:H7 + - - + - - -E . c o l i 0157:K88:H19 - - - + - - -E . c o l i (#2) + - - + - - -E . c o l i (#3) + - - + + -S.marcescens + + — — _ + + C . f r e u n d i i + - - + + - -E . c l o a c a e + - + - + - -E . t a r d a - - - - - - -K.pneumoniae + - - + + - -P . m i r a b i l i s + - - - - - -P.morgani i - - + - + - -H . a l v e i + - - - + - -E . f e r q o s o n i l - - - - - - -E.hermani i + - - - + - -P . r e t t g e r i + - - + + - -K . a s c o r b i t a + - - - + - -A l k a l e s c e n s - + - - - + - -d i s p a r - I a . n e g a t i v e c o n t r o l b . p o s i t i v e c o n t r o l c . f l u o r e s c e n c e i n t e n s i t y i s on a s c a l e of - to +++ d . no s e r o t y p e i n f o r m a t i o n was a v a i l a b l e for numbered E . c o l i 68 s p e c i f i c f or only a minor antigen on the surface of the b a c t e r i a or i t s a n t i g e n i c determinant i s not f u l l y exposed. The weak fluorescence observed i n these r e a c t i o n s may make d e t e c t i o n of these b a c t e r i a d i f f i c u l t . Mab 2E1 H6 appears to be s p e c i f i c f o r a determinant common to E . c o l l since i t reacted with a l l of the E . c o l l on the panel, yet reacted with only two non-E.coli s t r a i n s . This antibody may ther e f o r e have p o t e n t i a l i n d e t e c t i n g E . c o l i as a group. Both Mabs 3A5 CIO and 5E10 B6 e x h i b i t e d extensive c r o s s - r e a c t i v i t y with the b a c t e r i a t e s t e d and t h e r e f o r e , were omitted from f u r t h e r s t u d i e s . Mabs 2H4 H12 and 4D10 CI were s e l e c t e d for f u r t h e r study since both were r e s t r i c t i v e i n t h e i r c r o s s - r e a c t i v i t i e s . In a d d i t i o n , the fluorescence observed when they reacted with the p o s i t i v e c o n t r o l was strong. This i n d i c a t e s that these a n t i b o d i e s are almost s o l e l y s p e c i f i c f or the E . c o l i s t r a i n to which they were r a i s e d and that the a n t i g e n i c determinant to which the an t i b o d i e s bind was not common to most of the b a c t e r i a on t h i s panel. The r e s u l t s obtained from screening the a n t i b o d i e s against EPEC 0128 :K67, EPEC 044 :K74 and S_;_ marcescens were in c o n c l u s i v e since the negative c o n t r o l s e x h i b i t e d some fluorescence. This may be due to n o n s p e c i f i c capture or attachment of a n t i b o d i e s to the b a c t e r i a s u r f a c e . 69 B. Batch Production and P u r i f i c a t i o n of Mabs Large amounts of the s e l e c t e d a n t i b o d i e s were obtained by growing the hybridomas in_ v i v o as a s c i t i c tumours i n mice. Eight p r i s t a n e - t r e a t e d mice were i n j e c t e d i n t r a p e r i t o n e a l l y with the s e l e c t e d hybridoma c u l t u r e s (2 mice for each c u l t u r e ) . A f t e r 7 days, the abdomens of the mice had swollen s u f f i c i e n t l y to be "tapped" f o r a s c i t e s f l u i d . A s c i t e s production y i e l d e d 30 ml of f l u i d c o n t a i n i n g Mab 2H4 H12, 14 ml of 2E1 H6, 7 ml of 4D10 C l and 9 ml of 2F9 B3 before the mice e x p i r e d . Mabs u s u a l l y only represent 10% of the t o t a l p r o t e i n content of the a s c i t e s f l u i d (Bruck et a l . , 1986). Other p r o t e i n s present include t r a n s f e r r i n , albumin and proteases (Kohler and M i l s t e i n , 1975) as w e l l as endogenous immunoglobulins ( B u r c h i e l , 1986). Therefore attempts were made to p u r i f y the Mab i n the a s c i t e s f l u i d . Both g e l f i l t r a t i o n and anion-exchange chromatography were t r i e d . Mouse a s c i t e s f l u i d c o n t a i n i n g Mab 2E1 H6 showed a s i n g l e major p r o t e i n peak when separated on a Sephacryl S-300 SF column (Figure 5). A small shoulder appeared at the l e a d i n g edge of t h i s peak suggesting the presence of another unresolved peak. P u r i f i c a t i o n of a s c i t e s f l u i d c o n t a i n i n g Mab 2F9 B3 showed a s i m i l a r p r o f i l e (Figure 6 ) , although a peak near the void volume of the column ( f r a c t i o n 14 or 28 ml) was more apparant i n t h i s case. The e l u t i o n p r o f i l e of a s c i t e s f l u i d c o n t a i n i n g Mab 4D10 C l shows a r e l a t i v e l y c l e a r separation of two peaks (Figure 7). Separation of a s c i t e s 70 2.0 0 10 20 30 40 50 Fraction no. F i g u r e 5: E l u t i o n p r o f i l e of Mab 2E1 H6 s e p a r a t e d by g e l f i l t r a t i o n chromatography. I m m u n o r e a c t i v i t y as determined by an i n d i r e c t ELISA i s a l s o shown. Column: S e p h a c r y l S-300 (1.8 X 34 cm); E l u t i n g b u f f e r : 0.1 M T r i s - H C l , pH 8, w i t h 0.5 M N a C l ; Flow r a t e : 2 ml h ; F r a c t i o n s : 2 m l . 71 2.0 Fraction no. F i g u r e 6: E l u t i o n p r o f i l e of Mab 2F9 B3 s e p a r a t e d by g e l f i l t r a t i o n chromatography. I m m u n o r e a c t i v i t y as determined by an i n d i r e c t ELISA i s a l s o shown. Column: S e p h a c r y l S-300 (1.8 X 34 cm); E l u t i n g b u f f e r : 0.1 M T r i s - H C l , pH 8, w i t h 0.5 M N a C l ; Flow r a t e : 2 ml h""* ; F r a c t i o n s : 2 m l . 72 Figure 7: E l u t i o n p r o f i l e of Mab 4D10 C l separated by gel f i l t r a t i o n chromatography. Immunoreactivity as determined by an i n d i r e c t ELISA is a lso shown. Column: Sephacryl S-300 (1.8 X 34 cm); E l u t i n g buffer: 0.1 M T r i s - H C l , pH 8, with 0.5 M NaCl; Flow rate: 2 ml h"^-; Frac t ions : 2 ml. 73 f l u i d c o n t a i n i n g Mab 2H4 H12 shows the same two peaks, with the f i r s t peak being much l a r g e r than l n the other p u r i f i e d a s c i t e s f l u i d s (Figure 8). In the e l u t i o n p r o f i l e s of 2E1 H6, 2F9 B3 and 4D10 C l the main p r o t e i n peak appears to be doubly peaked, suggesting that two unresolved peaks were present. In the e l u t i o n p r o f i l e of 2H4 H12 t h i s could a l s o be seen i n the presence of a small shoulder i n the second peak e l u t e d . The presence of the antibody peak was determined by r e a c t i n g the various f r a c t i o n s with the E . c o l i OM p r e p a r a t i o n i n an ELISA. Figures 5 and 7, show strong immunoreactivity i n the main peak, i n d i c a t i n g the presence of the Mab i n t h i s peak. Results from Figure 6 are i n c o n c l u s i v e since immunoreactivity was poor i n a l l f r a c t i o n s . This i s p o s s i b l y due to the presence of low concentrations of Mab or the Mab present had a poor a f f i n i t y f o r the antigen. Assay of f r a c t i o n s from p u r i f i e d Mab 2H4 H12 showed strong immunoreactivity i n the f i r s t e l u t e d peak (Figure 8). Immunoreactivity was a l s o observed i n the second peak. This suggests the presence of two antibody c l a s s e s with s i g n i f i c a n t l y d i f f e r e n t molecular weights. Since the f i r s t antibody eluted near the v o i d volume, i t was presumed to be of the IgM c l a s s which has an approximate molecular weight of 900 k i l o d a l t o n s (kD) ( C l e z a r d i n et a l . , 1986). Eluted peak f r a c t i o n s were subjected to e l e c t r o p h o r e s i s under reducing c o n d i t i o n s on a 10% SDS-polyacrylamide g e l and the separated p r o t e i n s i d e n t i f i e d by Coomassi s t a i n i n g . Figures 9 and 10 show the SDS-PAGE p r o f i l e s of s e l e c t e d column 74 ) F i g u r e 8: E l u t i o n p r o f i l e of Mab 2H4 H12 s e p a r a t e d by g e l f i l t r a t i o n chromatography. I m m u n o r e a c t i v i t y as determined by an i n d i r e c t ELISA i s a l s o shown. Column: S e p h a c r y l S-300 (1.8 X 34 cm); E l u t i n g b u f f e r : 0.1 M T r i s - H C l , pH 8, w i th 0.5 M N a C l ; Flow r a t e : 2 ml h"L ; F r a c t i o n s : 2 m l . 75 f r a c t i o n s . In Figure 9, lanes 2-5 show the p r o f i l e s of f r a c t i o n s from the p u r i f i e d a s c i t e s f l u i d c o n t a i n i n g Mab 2E1 H6. No major p r o t e i n bands were observed i n f r a c t i o n s 16 and 20 which correspond with the shoulder observed near the void volume. F r a c t i o n 22 showed 3 major p r o t e i n bands at approximately 60, 50 and 25 kD. The 50 and 25 kD bands correspond to the apparent molecular weights of the heavy chain and l i g h t chain of the IgG antibody (Brodeur and Tsang, 1986). The 60 kD band was presumably albumin which represents the l a r g e s t f r a c t i o n of a s c i t i c p r o t e i n s (Franek, 1986) and t y p i c a l l y migrates to t h i s p o s i t i o n i n SDS-PAGE ( C l e z a r d i n , 1986). F r a c t i o n 24 contained s i g n i f i c a n t l y more albumin i n a d d i t i o n to some IgG. S i m i l a r p r o f i l e s were observed for p u r i f i e d f r a c t i o n s of a s c i t e s f l u i d c o n t a i n i n g Mab 2F9 B3 (Figure 9 ). The f a c t that albumin co-eluted with the a n t i b o d i e s confirms the presence of two unresolved peaks. The f i r s t peaks, which eluted near the v o i d volume of the column, may be 2 macroglobulin which i s a common contaminant of a s c i t e s f l u i d and has a molecular weight of approximately 900 kD (Fahey and Terry, 1969). Because of i t s high molecular weight i t would not be resolved on the polyacrylamide gel used i n t h i s study. Figure 10 shows the e l e c t r o p h o r e t i c p r o f i l e s of f r a c t i o n s from the p u r i f i e d a s c i t e s f l u i d c o n t a i n i n g Mab 2H4 H12. F r a c t i o n 16 showed 3 major p r o t e i n bands. Due to the e a r l y e l u t i o n of the p r o t e i n peak, the antibody present was 76 MW (X1000) 97.4 66.2 45 31 21 14 .4 II 5 6 7 8 9 10 F i g u r e 9: SDS-PAGE p r o f i l e s of 2-ME-reduced samples from g e l f i l t r a t i o n p u r i f i c a t i o n . Lanes 1 and 6: m o l e c u l a r weight s t a n d a r d s ; Lanes 2-5: 2E1 H6 f r a c t i o n s 16, 20, 22, 24; Lanes 7-10: 2F9 B3 f r a c t i o n s 16, 20, 22, 24. 77 MW (X1000) 97.4 tap 56.2 * 45 31 2 i 14 .4 II 1 2 3 4 5 6 7 8 9 10 F i g u r e 10: SDS-PAGE p r o f i l e s of 2 -ME-reduced samples from g e l f i l t r a t i o n p u r i f i c a t i o n . Lanes 1 and 6: mo lecu lar weight s t a n d a r d s ; Lanes 2-5: 4D10 CI f r a c t i o n s 16, 20, 22, 24; Lanes 7-10: 2H4 H12 f r a c t i o n s 16, 20, 22, 24 . 78 presumed to be of the IgM class. The f i r s t band was expected to be IgM heavy chain since i t has been shown to have a higher molecular weight and therefore, a slower mobility than IgG heavy chain (Ghebrehiwet, 1986). The second band, with a molecular weight similar to IgG heavy chain may indicate the presence of this Ig class. Some IgG molecules present may have interacted with the IgM molecules so as to cause them to co-elute. If this were the case, the third protein band would consist of both IgM and IgG light chains. Although i t was not shown in this gel, the antibody may have been contaminated with a2-macroglobulin which has been shown to elute with IgM in gel f i l t r a t i o n because of their similar size (Fahey and Terry, 1969). Fraction 22, which corresponded to the shoulder observed in the second peak, showed the presence of albumin in addition to IgG heavy and l ight chain. Fraction 24, which corresponded to the second peak, appeared to contain primarily albumin. Given that each hybridoma produces an antibody of a single class (Burnet, 1957) i t would appear that two different hybridomas were injected into the mice during ascites production of Mab. In other words, recloning was not successful in isolating a single clone in this case. Either the number of clones was miscalculated or at the time of counting the clones the second clone was not v i s ib le due to slow growth. However, under conditions of ascites production, i t was given f u l l advantage to grow. This may account for the presence of two different antibody classes in 79 the a s c i t e s f l u i d . I t i s a l s o p o s s i b l e that the second antibody was endogenous. Gooi and F e i z i (1982) found that i n some instances, normal serum from untreated mice showed s u b s t a n t i a l l y higher binding to determinants on the antigen ( f e t a l g l y c o p r o t e i n s ) than d i d the a s c i t e s f l u i d sample c o n t a i n i n g Mab s p e c i f i c f o r the antigen. G e n e r a l l y , however, the amount of p o l y c l o n a l mouse Ig i s very low i n a s c i t e s f l u i d (Franek, 1986). In t h i s study, SDS-PAGE r e s u l t s i n d i c a t e d that the concentrations of the two a n t i b o d i e s were s i m i l a r (Figure 10). Another explanation f o r the presence of the two c l a s s e s may be that of c l a s s s w i t c h i n g . A small percentage of switch v a r i a n t hybridomas have been shown to spontaneously switch from producing one c l a s s or subclass of antibody to another while i n c u l t u r e . For example, S p i r a et a l . (1984) r e p o r t e d l y r a i s e d a hybridoma which produced IgM that bound to phosphorylcholine, but a l s o found IgG i n the c u l t u r e . This IgG was a l s o able to bind the antigen. Regardless of the expl a n a t i o n f o r the presence of the two c l a s s e s , f r a c t i o n s 14-18, corresponding to the f i r s t peak (Figure 8) were pooled, concentrated to 1 ml and termed Mab 2H4 H12. This antibody f r a c t i o n was s e l e c t e d since i t was the purer of the two and showed a higher immunoreactivity with the antigen. While IgG has been s u c c e s s f u l l y p u r i f i e d by g e l f i l t r a t i o n (Wieczorek, 1989), r e s o l u t i o n was poor under the co n d i t i o n s of t h i s study. Rather than attempting to modify 80 the c o n d i t i o n s of g e l f i l t r a t i o n , the use of anion-exchange for the p u r i f i c a t i o n of Mabs was i n v e s t i g a t e d using a s c i t e s f l u i d s c o n t a i n i n g Mabs 2F9 B3, 2E1 H6 and 4D10 C l . Mouse a s c i t e s f l u i d c o n t a i n i n g Mab 4D10 C l showed 3 major p r o t e i n peaks when separated on a DEAE-Sephacel column with a gradient of 0 - 1 M NaCl (Figure 11). A f o u r t h peak appeared at f r a c t i o n 7 i n d i c a t i n g a p r o t e i n contaminant which d i d not bind to the column. This peak was present i n a l l 3 samples p u r i f i e d . A n a l y s i s of the f r a c t i o n s e l u t e d under the gradient by ELISA showed the antibody f r a c t i o n to be located i n the f i r s t peak. This peak was el u t e d at a s a l t c o n c e n t r a t i o n of approximately 0.5 M NaCl. The second and t h i r d peaks e l u t e d at a s a l t c o n c e n t r a t i o n of 0.65 M and 0.8 M NaCl r e s p e c t i v e l y . Figure 12 shows the e l u t i o n p r o f i l e of a s c i t e s f l u i d c o n t a i n i n g Mab 2F9 B3. Again, 3 major p r o t e i n peaks appeared under the e l u t i o n c o n d i t i o n s . However, the f i r s t two peaks were unresolved. The f i r s t peak appeared to e l u t e at approximately 0.55 M NaCl while the second peak e l u t e d at 0.65 M NaCl. The t h i r d peak was much l a r g e r i n t h i s sample and e l u t e d at 0.8 M NaCl. An examination of the f r a c t i o n s by ELISA i n d i c a t e d a low immunoreactivity, although i t was predominantly i n the f i r s t of the two unresolved peaks. In the p u r i f i c a t i o n of a s c i t e s f l u i d c o n t a i n i n g Mab 2E1 H6 only 2 major peaks were e l u t e d (Figure 13). The f i r s t peak eluted at 0.65 M NaCl, c o n s i s t e n t with the second peak observed i n the two previous a s c i t e s samples. The second 81 Figure 11: E lu t ion p r o f i l e of Mab 4D10 C l separated by anion-exchange chromatography. Immuno-r e a c t i v i t y as determined by an ind irec t ELISA is also shown. Column: DEAE-Sephacel (1.8 X 7 cm); E l u t i n g buffer: 10 mM T r i s - H C l , pH 8, NaCl concentration gradient; Flow rate : 5 ml h"-*- ; Fract ions : 1 ml. 82 Figure 12: E l u t i o n p r o f i l e of Mab 2F9 B3 separated by anion-exchange chromatography. Immuno-r e a c t i v i t y as determined by an i n d i r e c t ELISA is a lso shown. Column: DEAE-Sephacel (1.8 X 7 cm); E l u t i n g buffer: 10 mM T r i s - H C l , pH 8, NaCl concentration gradient; Flow rate : 5 ml n - l . Frac t ions : 1 ml. 8 3 F i g u r e 13: E l u t i o n p r o f i l e of Mab 2E1 H6 s e p a r a t e d by anion-exchange chromatography. Immuno-r e a c t i v i t y as determined by an i n d i r e c t ELISA i s a l s o shown. Column: DEAE-Sephace l (1.8 X 7 cm); E l u t i n g b u f f e r : 10 mM T r i s - H C l , pH 8, NaCl c o n c e n t r a t i o n g r a d i e n t ; Flow r a t e : 5 ml n - l . F r a c t i o n s : 1 m l . 84 peak eluted close to 0.9 M NaCl. ELISA showed the antibody fraction to be located in the second peak. The peak fractions were subjected to electrophoresis under reducing conditions. Figures 14 and 15 show the SDS-PAGE profiles of selected fractions of purified mouse ascites f luids . In Figure 14, lanes 2-4 show the profiles of fractions from the purified ascites f luid containing Mab 2F9 B3. The antibody was located in fraction 40 which corresponded to the f i r s t peak in the elution profi le (Figure 12), and was evident by the heavy and light chains of IgG at approximately 50 and 25 kD respectively. Another protein band appeared at 66 kD. This corresponded to the apparent molecular weight of transferrin which commonly co-elutes with IgG in anion-exchange chromatography (Burchiel et a l . , 1984). Fraction 45, which corresponded to the second peak, showed a large protein band between 50 and 65 kD. This was presumably comprised predominantly of albumin. No bands appeared in fraction 51, probably due to a low protein concentration or the inab i l i ty of the gel to resolve the protein. Lanes 6-8 show the profiles of fractions from the purified ascites f luid containing Mab 2E1 H6. Fraction 41, which corresponded to the shoulder observed at the leading edge of the main peak (Figure 13), showed a single protein band which was probably transferrin. Fraction 46, which corresponded to the main peak, showed bands corresponding to the heavy and light chains of Ig in addition to albumin. Fraction 56, from the third peak, contained no bands. 85 MW (X1000) F i g u r e 14: SDS-PAGE p r o f i l e s of 2 -ME-reduced samples from ion-exchange p u r i f i c a t i o n . Lane 1: m o l e c u l a r weight s t a n d a r d s ; Lanes 2-4: 2F9 B3 f r a c t i o n s 40, 45, 51; Lanes 6-8: 2E1 H6 f r a c t i o n s 41, 46, 56. 86 Figure 15 shows the profiles of fractions from the purified ascites f luid containing Mab 4D10 C l . Fraction 40, corresponding to the f i r s t peak, c learly showed the presence of the heavy and light chains of Ig in addition to transferrin. The second peak consisted primarily of albumin which was shown by the heavy protein band in fraction 46. Fractions 50 and 53 showed no major protein bands. Mabs 4D10 Cl and 2F9 B3 appeared to have a similar charge density since they eluted at similar salt concentrations. On the other hand, Mab 2E1 H6 appeared to have a similar charge density to albumin which explains their co-elution. In fact, Burchiel et a l . (1984) already demonstrated that different monoclonal mouse igGs elute at different times under identical conditions. In addition, Horejsi and Hilgert (1986) mentioned that IgG Mabs can dif fer markedly in their isoelectric points and so would have different charge profiles at similar pHs. The elution conditions for the optimal separation of monoclonal IgG molecules from ascites f luid are different for each antibody. In this situation, i t may be advantageous to use purif ication procedures that separate on the basis of properties such as size, not change greatly between different antibodies. Optimization of gel f i l t r a t i o n conditions for separation of the Mabs may prove to be a better procedure in this respect. The transferrin that co-eluted with the Mab in anion-exchange is often removed in a second purification step, usually gel f i l t ra t ion (Burchiel et a l . , 1984). This was not 87 MW (X1000) 45 M ^ 31 mm 21 M l 14.4 1 2 3 4 5 6 Figure 15: SDS-PAGE p r o f i l e s of 2-ME-reduced samples from ion-exchange p u r i f i c a t i o n . Lane 1: molecular weight standards; Lanes 3-6: 4D10 Cl fractions 40, 46, 50, 53. 88 done i n t h i s experiment s i n c e p u r i t y was not a c r i t i c a l f a c t o r . For the same reason, f u r t h e r p u r i f i c a t i o n of Mab 2E1 H6 was not attempted despite i t s ' c o - e l u t i o n with albumin. F r a c t i o n s from the peaks c o n t a i n i n g immunoglobulin were pooled and concentrated to 1 ml. C. C h a r a c t e r i z a t i o n of the Mabs and t h e i r Antigens The p u r i f i e d Mabs were c h a r a c t e r i z e d on the basis of t h e i r immunoreactivity with the antigen, isotype and s p e c i f i c i t y . Information on t h e i r r e s p e c t i v e antigens was obtained by t h e i r r e a c t i o n i n an immunoblot assay. 1. Immunoreactivity The immunoreactivity or antigen-binding c a p a c i t y of the Mabs was determined using an i n d i r e c t ELISA. OM preparation of E . c o l i was used as the antigen. Since the s p e c i f i c c o n c e n t r a t i o n of immunoglobulin was not known, r e a c t i v i t y was determined on a t o t a l p r o t e i n b a s i s . Figure 16 shows the r e s u l t s . From t h i s graph the t i t r e s (the minimum p r o t e i n c o n c e n t r a t i o n at which s i g n i f i c a n t p r o t e i n r e a c t i v i t y was observed) can be compared. Neither Mab 2E1 H6 nor 2F9 B3 showed very high r e a c t i v i t y even at the highest p r o t e i n c o n c e n t r a t i o n tested (0.01%). Mabs 4D10 CI and 2H4 H12 e x h i b i t e d a markedly higher r e a c t i v i t y even at concentrations as low as 0.0001% i n the case of 4D10 CI. Concentrations above 0.001% were not tes t e d for Mabs 4D10 C l and 2H4 H12 due to the low p r o t e i n concentrations of these Mab preparations as w e l l as t h e i r l i m i t e d supply. 89 10 " 7 10 " 8 10 " 5 1 o - 4 10 " 3 1 o - 2 10 % Protein F i g u r e 16: A n t i g e n b i n d i n g a c t i v i t y of Mabs as determined by an i n d i r e c t E L I S A . Mab 2E1 H6 ( • ) ; 2F9 B3 ( X ) ; 4D10 C l ( A ) ; 2H4 H12 ( Q ) • 90 The low r e a c t i v i t y observed f o r Mabs 2E1 H6 and 2F9 B3 may be due to a low concent r a t i o n or a low a f f i n i t y of these Mabs f o r the antigen. Another ex p l a n a t i o n may be that the a n t i g e n i c determinants they are s p e c i f i c f o r represent only a small p o r t i o n of the OM. This could r e s u l t i n l e s s antibody binding and t h e r e f o r e , a lower s i g n a l than i f more determinants were present. 2. Isotype A n a l y s i s To determine the c l a s s or subclass of the Mabs, they were reacted with anti-mouse c l a s s s p e c i f i c a n t i b o d i e s i n an i n d i r e c t ELISA. The r e s u l t s are shown i n Table IV. Both Mab 2E1 H6 and Mab 2F9 B3 reacted p o s i t i v e l y with the a n t i - I g G l antibody. Mab 4D10 C l reacted p o s i t i v e l y w i t h both a n t i -IgG2a and anti-IgG2b, suggesting that both subclasses were present i n the p u r i f i e d antibody p r e p a r a t i o n . This would imply that an e r r o r was made i n r e c l o n i n g and monoclonality was not achieved. However, as was mentioned p r e v i o u s l y , the p o s s i b i l i t y e x i s t s that the hybridoma producing these a n t i b o d i e s was a subclass switch v a r i a n t . Preud'homme et a l . (1975) observed that IgG2b producing myeloma c e l l s often spontaneously switch to expressing IgG2a molecules. This may account for the presence of both antibody subclasses. Mab 2H4 H23 reacted p o s i t i v e l y with anti-IgM and a n t i -IgG3. This confirms the presence of these two c l a s s e s which was already e l u c i d a t e d from the O D 2 8 O p r o f i l e of t h i s Mab when p u r i f i e d by g e l f i l t r a t i o n (Figure 8 ) . 91 Table IV: Results of isotyplng of Mabs Mab 2F9 B3 2E1 H6 4D10 CI 2H4 H12 Ig Isotypec IgGl IgGl IgG2a, IgG2b IgM, IgG3 a. Anti-lsotype specific antibodies with which the Mabs reacted posit ively in an indirect ELISA. 92 3. Immunofluorescence Screening Mabs were screened against a panel of b a c t e r i a to t e s t t h e i r s p e c i f i c i t y . An i n d i r e c t immunofluorescence assay, as was done for the hybridoma c u l t u r e f l u i d s p r e v i o u s l y , was used. Table V shows the r e s u l t s of the screening. Both 2H4 H12 and 4D10 CI showed very l i t t l e cross r e a c t i o n with the b a c t e r i a on the panel. Mab 2H4 H12 c r o s s -reacted weakly with EPEC 0112:K68 and K.pneumoniae. Mab 4D10 CI cross reacted weakly with EPEC 055:K59, E.cloacae and P.morganii. This would suggest that these b a c t e r i a possess a n t i g e n i c determinants which are close enough i n s t r u c t u r e to the true determinant so as to bind weakly to the antibody. The weak fluorescence may a l s o be due to n o n - s p e c i f i c s t a i n i n g . N o n - s p e c i f i c s t a i n i n g i s a r e s u l t of non-immunologic attachment of a n t i b o d i e s to the b a c t e r i a or to the presence of n a t u r a l a n t i b o d i e s i n the Mab preparation which c r o s s - r e a c t with determinants on the b a c t e r i a ( B i g a z z i and T i l t o n , 1980). A l s o , since optimum d i l u t i o n s of Mab were not determined, i t i s p o s s i b l e that too high concentrations were present. High concentrations of antibody may lead to an increase i n c r o s s - r e a c t i o n s (Borrebaeck and Glad, 1989). C r o s s - r e a c t i o n of Mabs 2E1 H6 and 2F9 B3 with the b a c t e r i a was ext e n s i v e . Both reacted w i t h a l l the E . c o l i serotypes i n a d d i t i o n to many of the Enterobacteriaceae on the panel. This would suggest the presence of a common or s i m i l a r a n t i g e n i c determinant i n the outer membrane of these b a c t e r i a . The pa t t e r n of c r o s s - r e a c t i v i t y was much d i f f e r e n t 93 Table V: R e s u l t s of the Second s c r e e n i n g of s e l e c t e d Mabs A g a i n s t a Pane l of E n t e r o b a c t e r i a c e a e by Immuno-f l u o r e s c e n c e as say u s i n g P u r i f i e d Mabs. Mabs B a c t e r i a 2H4 H12 4D10 C l 2E1 H6 2F9 B3 C b c EPEC 0142:K86:H6 4-4-4- + + + + + + + -EPEC 0128:K67 - - 4-4- 4- + -EPEC 055:K59 - + + + + + -EPEC 044:K74 - - f f + 4- -EPEC 0112:K68 + - 4-4- 4-4- -d E . c o l i (#1) — — 4-4- 4-4- _ E . c o l i 0157:H7 - - 4-4- 4-4- -E . c o l i 0157:K88:H19 - - + + + + -E . c o l i (#2) - - + + + + 4 E . c o l i (#3) - - + + + + -S . m a r c e s c e n s - — + + + + _ C . f r e u n d i i - - + 4- + + -E . c l o a c a e - + + + + + -E . t a r d a - - - - -K.pneumoniae + - + + 4 + -P . m i r a b i l i s - - 4- - -P.morgani i - + + + 4-4- -H . a l v e i - - - 4-4- -E . f u r g o s o n i i - - - - -E.hermani i - - + - -P. r e t t q e r i - - - - -K . a s c o r b i - - - - -A l k a l e s c e n s - d i s p a r - I - - + - -a . n e g a t i v e c o n t r o l b . p o s i t i v e c o n t r o l c . f l u o r e s c e n c e i n t e n s i t y i s on a s c a l e of - to 4-4-4-d . no s e r o t y p e i n f o r m a t i o n was a v a i l a b l e for numbered E . c o l i 94 than that observed when hybridoma culture f luid was used as the antibody source (Table III) . The concentration of Mab may have been too low in the hybridoma culture f luid to stain some ce l l s . This would also explain the weaker fluorescence seen in those positive samples as compared to the same samples in the second screening. It is also possible that the spec i f ic i ty of the Mab changed during growth in ascites. Cross-reactivit ies in some Mabs have been shown to differ markedly when raised in ascites as compared to the same Mab raised in culture (Bosch et a l . , 1982). Mutations have been shown to occur in the immunoglobulin gene of some cultured antibody-producing ce l l s (Cotton et a l . , 1973; Bruggeman et a l . , 1982). If the growth environment of the cel ls is altered s ignif icant ly , such as in ascites growth, the mutant may develop a growth advantage and outgrow the original c e l l population. Immunoglobulins produced by the mutant could show different spec i f ic i t ies (Bosch et a l . , 1982). Another explanation may be that monoclonality was not achieved during recloning and a second hybridoma producing antibody with a different spec i f ic i ty was present during growth in ascites. Due to slow growth this hybridoma may not have been producing sufficient antibody in hybridoma culture to s ignif icantly influence immunofluoresence results. Growth of this hybridoma may have been stimulated in ascites and therefore, more of this antibody was produced. This may have contributed to the change in the immunofluorescence results. 95 4. Immunoblot A n a l y s i s Immunoblotting was c a r r i e d out t o . g a i n information on the antigen i t s e l f . Three EPEC, 3 non-EPEC and 6 non-E.coli s t r a i n s were randomly s e l e c t e d from the b a c t e r i a screening panel f o r a n a l y s i s . SDS-PAGE p r o f i l e s of c e l l l y s a t e s were obtained then t r a n s f e r e d e l e c t r o p h o r e t i c a l l y to a p o l y v i n y l i d e n e d i f l u o r i d e membrane. R e p l i c a t e s were prepared and each allowed to react with a d i f f e r e n t Mab i n an ELISA. Figure 17 d e p i c t s the e l e c t r o p h o r e t i c p r o f i l e s of the c e l l l y s a t e s under reducing c o n d i t i o n s . Figures 18 to 21 d e p i c t the immunoblots of the b a c t e r i a samples with Mabs 2E1 H6 and 2F9 B3. Both were s t r o n g l y r e a c t i v e with a p r o t e i n band i n the c o n t r o l EPEC with an apparant molecular weight of approximately 35 kD (Figure 18). This band was common to a l l the EPEC t e s t e d . A second band appeared at approximately 30 kD i n the 2E1 H6 and 2F9 B3 b l o t s of the c o n t r o l EPEC, but not i n the other EPEC b l o t s . Figure 19 shows the immunoblots of non-EPEC with the same Mabs. Again, the Mabs reacted with a 35 kD p r o t e i n band. A 30 kD band was a l s o v i s i b l e i n the non-EPEC b l o t s . In Figure 20 i t can be seen that C . f r e u n d i i , E.cloacae, and K.pneumoniae contained a c r o s s - r e a c t i v e p r o t e i n of approximately the same molecular weight as the 35 kD p r o t e i n i n the c o n t r o l i n both Mab b l o t s . In a d d i t i o n a 30 kD c r o s s -r e a c t i v e p r o t e i n was a l s o present. In the 2F9 B3 b l o t of the c o n t r o l two bands appeared, one at 30 kD and another at 28 kD. Since the 28 kD band d i d not appear i n previous b l o t s of 96 MW (X1000) 9 10 11 12 13 14 15 16 F i g u r e 17: SDS-PAGE p r o f i l e of 2 -ME-reduced b a c t e r i a samples . Lanes 1 and 9: m o l e c u l a r weight s t a n d a r d s ; Lanes 2 and 10: EPEC 0142:K86:H6; Lane 3: EPEC 0128:K67; Lane 4: EPEC 055:K59; Lane 5: EPEC 044:K74; Lane 6: E . c o l i 0157: H7; Lane 7: E . c o l i 0157:K88:H19; Lane 8: E . c o l i # non-EPEC; Lane 11: C . f r e u n d i i ; Lane 12: E . c l o a c a e ; Lane 13: K.pneumoniae; Lane 14: S .marcescens: Lane 15: E . h e r m a n i i ; Lane 16: P . m i r a b i l i s . 97 MW (X1000) 97 . 4 66 . 2 45 31 21 2E1 H6 2F9 B3 1 2 3 4 1 2 3 4 Figure 18: Immunoblots of EPEC with Mabs 2E1 H6 and 2F9 B3. Lane 1: EPEC 0142:K86:H6 ( c o n t r o l ) ; Lane 2: EPEC 0128:K67; Lane 3: EPEC 055:K59; Lane 4: EPEC 044 K74. 98 MW (X1000) 2E1 H6 2F9 B3 97.4-66.2 45 31-21-14.4-1 2 3 1 2 3 4 Figure 19: Immunoblots of non-EPEC with Mabs 2E1 H6 and 2F9 B3. Lane 1: EPEC 0142:K86:H6 (control); Lane 2: E . c o l i 0157:H7; Lane 3: E . c o l i 0157:K88: H19; Lane 4: E . c o l i , non-EPEC. 99 MW (X1000) 2E1 H6 2F9 B3 97.4 66 .2 45 31-21-14.4-tmm 2 3 4 i 2 3 Figure 20: Immunoblots of Enterobacteriaceae with Mabs 2E1 H6 and 2F9 B3. Lane 1: EPEC 0142:K86:H6 (control); Lane 2: C.freundii; Lane 3: E.  cloacae; Lane 4: K.pneumoniae. 100 the control i t was not considered to be a major cross-reaction. The 30 kD band did not appear in the 2E1 blot of the control or the control blots of Figure 21. This may be due to i n e f f i c i e n t transfer of thi s protein during b l o t t i n g . A l t e r n a t i v e l y , i t may r e f l e c t the i n s t a b i l i t y of t h i s protein under SDS-denaturing conditions. A fai n t band of approximately 20 kD also appeared in the 2F9 B3 blot of C.  freundii and the 2E1 H6 blot of C.freundii and K.pneumoniae. This probably represents a very minor cross-reaction. In the 2E1 H6 blot of S.marcescens (Figure 21), only a 30 kD cross-reactive protein band appeared. E.hermanni contained both a 35 kD and a 30 kD protein which were cross-reactive with 2E1 H6. Mab 2E1 H6 was not reactive with P.mirabllis. Mab 2E1 H6 did not cross react with S.marcescens, E.hermani1 or P.mirabilis, suggesting that these bacteria do not share common or similar determinants with the control E . c o l i . It cannot be stated whether the cross-reacting proteins observed in the bacteria were the same as those found in the control EPEC. In spite of their similar electrophoretic m o b i l i t i e s , they may be d i f f e r e n t proteins with shared or simi l a r antigenic determinants. The extensive c r o s s - r e a c t i v i t y pattern observed with Mabs 2E1 H6 and 2F9 B3 among the bacteria panel was not unusual since antigenic c r o s s - r e a c t i v i t y among the outer membrane proteins is a common phenomenon in the Enterobacteriaceae family (Hofstra and Dankert, 1980). The 101 MW (X1000) 2E1 H6 2F9 B3 F i g u r e 21: Immunoblots of E n t e r o b a c t e r i a c e a e wi th Mabs 2E1 H6 and 2F9 B3. Lane 1: EPEC 0142:K86:H6; Lane 2: S .marcescens ; Lane 3: E . h e r m a n i i ; Lane 4: P . m i r a b i l i s . 102 cross-reactive 35 kD protein observed in nearly a l l the blots corresponded c l o s e l y to the electrophoretic mobility of the heat-modified form of the outer membrane protein (Omp) A which has been well characterized in E . c o l i K12 (Lambert, 1988). Proteins cross-reactive with Omp A have previously been detected in a l l strains of E . c o l i in addition to many other Enterobacteriaceae (Hofstra and Dankert, 1980). It i s possible that protein bands that appeared at 28 to 30 kD were unmodified portions of the same protein present due to i n s u f f i c i e n t heating. Hofstra and Dankert (1980) reported that the unmodified form of the proteins cross-reactive with Omp A protein in the family Enterobacteriaceae were in the molecular weight range of 26.5 to 31 kD. Table VI compares the results of the immunoblots with those of the immunofluorescence assay. Aggreement between results was observed in a l l cases except with S.marcescens and P.mirabilis. Mab 2F9 B3 reacted p o s i t i v e l y with S.marcescens in the immunofluorescence assay, but reacted negatively in the immunoblot. S i m i l a r l y , Mab 2E1 H6 reacted p o s i t i v e l y with P.mirabilis in the immunofluorescence assay, but negatively in the immunoblot. While these antibodies were capable of detecting the cross-reacting determinants in the whole c e l l , the determinants were seemingly not recognizable when denatured as on blots . Neither Mab 4D10 CI nor Mab 2H4 H12 reacted in the immunoblots ( i . e . there were no v i s i b l e reacting protein bands) either under reducing or non-reducing conditions. 103 Table VI: Results of Assay Immunoblotting vs. Immunofluorescence B a c t e r i a Immunoblot Immunofluoresence 2F9 B3 2E1 H6 2F9 B3 2E1 H6 EPEC 0142:K86:H6a b + 4- + + EPEC 0128:K67 + + + + EPEC 055:K59 + 4- + 4-EPEC 044:K74 + + + 4-NON-EPEC 0157:H7 + + + 4-NON-EPEC 0157:K88:H19 + + + 4-NON-EPEC, not 0157 + + + 4-C . f r e u n d i i + + + 4-E.cloacae + + + + K.pneumoniae + + + 4-S.marcescens - + + 4-E.hermani i - + - 4-P . m i r a b i l i s - - - 4-a. p o s i t i v e c o n t r o l b. + = p o s i t i v e r e a c t i o n - = no r e a c t i o n 104 This i s thought to be due to t h e i r i n a b i l i t y to detect the a n t i g e n i c determinant under SDS-denaturing c o n d i t i o n s , since not a l l a n t i g e n i c s i t e s r e t a i n t h e i r n a t i v e c o n f i g u r a t i o n a f t e r SDS treatment (Towbin and Gorden, 1984). The increased s p e c i f i c i t y of Mabs over p o l y c l o n a l a n t i b o d i e s can r e a d i l y be seen by comparing immunoblots prepared with both monoclonals and p o l y c l o n a l s . Figure 22 d e p i c t s b l o t s of various b a c t e r i a c e l l l y s a t e s reacted with p o l y c l o n a l antiserum obtained from the egg yolk s of chickens immunized with E . c o l i 0142:K86:H6 (preparation of s p e c i f i c a n t i b o d i e s from egg yolk i s discussed i n Part I I of t h i s t h e s i s ) . The m u l t i p l e banding represents the presence of an t i b o d i e s with v a r y i n g s p e c i f i c i t i e s , which i s t y p i c a l of p o l y c l o n a l a n t i s e r a . By comparison, the Mabs e x h i b i t e d a narrow range of s p e c i f i c i t y as shown by the l i m i t e d number of r e a c t i n g p r o t e i n bands (Figures 18 to 21). Although the p o l y c l o n a l antiserum was not t e s t e d f o r c r o s s - r e a c t i v i t y with a l l of the Enterobacteriaceae on the panel, i t i s reasonable to expect the degree of c r o s s - r e a c t i v i t y with these b a c t e r i a to be high since the presence of c r o s s - r e a c t i n g antigens among the f a m i l y Enterobacteriaceae i s a l s o high (Hofstra and Dankert, 1980). D. Conclusion Under the c o n d i t i o n s of t h i s experiment there was no success i n o b t a i n i n g a Mab uniquely s p e c i f i c to EPEC. This does not imply that such a Mab does not e x i s t among the antibody r e p e r t o i r e of the many hybridomas r a i s e d , only that 105 2 3 4 5 6 7 8 Figure 22: Immunoblot of EPEC and other Enterobacteriaceae with polyclonal antiserum. Lanes 1 and 5: EPEC 0142:K86:H6 (control); Lane 2: EPEC 0128:K67; Lane 3: EPEC 055:K59; Lane 4: EPEC 044:K74; Lane 6: C.freundii; Lane 7: E.cloacae; Lane 8: K.pneumoniae. 106 t o the e x t e n t of s c r e e n i n g performed i n t h i s s t u d y such an a n t i b o d y was not found. Given s u f f i c i e n t time and manpower i t i s f e a s i b l e t h a t , i f t h e r e i s a u n i q u e l y s p e c i f i c s u r f a c e a n t i g e n common o n l y t o EPEC, a c o m p l i m e n t a r y Mab c o u l d be found. I t i s p o s s i b l e t h a t the 94 kD OM p r o t e i n e x p r e s s e d i n some C l a s s I EPEC s t r a i n s , r e p o r t e d i n the l i t e r a t u r e r e v i e w , i s such an a n t i g e n . I s o l a t i o n of t h i s p r o t e i n and i t s use as the immunogen may v a s t l y d e c r e a s e s c r e e n i n g t i m e . A Mab s p e c i f i c f o r t h i s p r o t e i n may have p o t e n t i a l use i n an a s s a y t o d i s t i n g u i s h t h i s E . c o l i type from o t h e r s . N e v e r t h e l e s s , i n t h i s s t u d y two Mabs (4D10 CI and 2H4 H12) were produced which were s t r o n g l y r e a c t i v e w i t h a s i n g l e EPEC s e r o t y p e i n an immunofluorescence a s s a y , w h i l e showing m i n i m a l c r o s s - r e a c t i v i t y w i t h o t h e r b a c t e r i a . T h i s Mab may have p o t e n t i a l use i n the d e t e c t i o n of t h i s E . c o l i s e r o t y p e i n the f e c e s of i n f a n t s or i n food systems. Because of the s t r o n g f l u o r e s c e n c e o b s e r v e d , t h i s EPEC c o u l d e a s i l y be d e t e c t e d . A second p a i r of a n t i b o d i e s were a l s o produced which were weakly r e a c t i v e w i t h a l l the E . c o l i s e r o t y p e s t e s t e d i n a d d i t i o n t o most of the o t h e r E n t e r o b a c t e r i a c e a e on the p a n e l . Immunoblotting showed the s e Mabs t o be r e a c t i v e i n most cases w i t h b o t h a 35 kD and a 30 kD p r o t e i n . I t i s p o s s i b l e t h a t the h e a v i e r p r o t e i n i s s i m i l a r t o the 35 kD Omp A p r o t e i n d e s c r i b e d i n the l i t e r a t u r e (Lambert, 1988). These Mabs may have p o t e n t i a l use i n d e t e c t i n g b a c t e r i a b e l o n g i n g t o the E n t e r o b a c t e r i a c e a e f a m i l y . 107 Obviously further experiments testing for antigenic c r o s s - r e a c t i v i t y among a larger panel of bacteria are required. In addition non-denaturing immunoblots with Mabs 4D10 CI and 2H4 H12 should be performed in order to gain information on the reactive antigen. While Mabs 4D10 Cl and 2H4 H12 showed a strong a f f i n i t y and 2E1 H6 and 2F9 B3 a weak a f f i n i t y for the OM preparation in ELISA, i t remains to be shown i f they are reactive with the whole c e l l in t h i s type of assay. In using Mabs, one must be aware of the potential problems associated with them. Very often t h e i r s t a b i l i t y to chemical and physical treatments, such as changes in pH and temperature, are less than that observed with polyclonal antibodies (Kurstak, 1986). As was shown in t h i s thesis and has been reported previously (Bosch et a l . , 1982), the cross-r e a c t i v i t y p r o f i l e s of the Mabs may change due to changes in the growth conditions of the hybridomas. In addition, some hybridoma clones that produce desired antibodies can spontaneously become non-producers (Young, 1984). Unless the antigen contains multiple i d e n t i c a l determinants, p r e c i p i t a t i o n reactions between Mabs and antigens generally do not occur (Galfre and M i l s t e i n , 1981). Because the Mab is s p e c i f i c for a single antigenic determinant, negative results do not necessarily imply absence of antigen since changes in the environment of the determinant or the way the antigen is presented could s i g n i f i c a n t l y a l t e r r e s u l t s . 108 D e s p i t e t h e s e problems, however, monoclonal a n t i b o d i e s have s i g n i f i c a n t advantages over p o l y c l o n a l a n t i b o d i e s and w i l l remain a p o w e r f u l t o o l f o r many s c i e n t i f i c d i s c i p l i n e s , i n c l u d i n g food s c i e n c e . 109 PART I I : USE OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) TO DETECT B-N-ACETYLGLUCOSAMINIDASE (NAGase) A. Production of Antibodies S p e c i f i c f o r NAGase Both White Leghorn hens and New Zealand white r a b b i t s were i n j e c t e d with a commercial bovine 6-N-acetylglucosaminidase (NAGase) pr e p a r a t i o n . Immunoglobulin Y (IgY) was separated from the egg yolk of immunized chickens by polyethylene g l y c o l p r e c i p i t a t i o n followed by DEAE-Sephacel chromatography. This experiment was performed i n t r i p l i c a t e . The Ig f r a c t i o n of the blood serum from immunized r a b b i t s was i s o l a t e d by ammonium sulphate p r e c i p i t a t i o n . P u r i t y of the i s o l a t e d IgY was examined by SDS-PAGE and r a d i a l immunodiffusion. Figure 23 shows the SDS-PAGE p r o f i l e s of the three f r a c t i o n s compared to standard IgY. A l l three f r a c t i o n s e x h i b i t e d p r o t e i n bands t y p i c a l of the heavy and l i g h t chains of IgY. R a d i a l immunodiffusion showed the three f r a c t i o n s of IgY to have a p u r i t y of between 80 and 82%. Ammonium sulphate p r e c i p i t a t i o n was the only p u r i f i c a t i o n step performed on the r a b b i t blood serum si n c e i t i s reported that t h i s method i s s u f f i c i e n t to y i e l d an IgG f r a c t i o n of approximately 95% p u r i t y (Garvey et a l . 1977). This method was performed on blood serum samples obtained from each of the two r a b b i t s used i n t h i s experiment. Figure 24 shows the reduced SDS-PAGE p r o f i l e s of the two f r a c t i o n s compared to molecular weight standards. A large p r o t e i n band 110 - • f • • • a § HC LC F i g u r e 23: SDS-PAGE p r o f i l e s of 2 -ME-reduced p u r i f i e d IgY f r a c t i o n s . Lane 1: IgY s t a n d a r d ; Lanes 3, 5 and 7: IgY f r a c t i o n s 1, 2 and 3. Heavy c h a i n s (HC) and l i g h t c h a i n s (LC) of Ig are shown. I l l MW (X1000) 97 .4 66 . 2 45 31 21 14 . 4 HC LC F i g u r e 24: SDS-PAGE p r o f i l e s of 2 -ME-reduced p u r i f i e d r a b b i t IgG f r a c t i o n s . Lane 1: m o l e c u l a r weight s t a n d a r d s ; Lanes 3 and 5: IgG f r a c t i o n s from r a b b i t s 1 and 2. Heavy c h a i n s (HC) and l i g h t c h a i n s (LC) of Ig are shown. 112 appears at approximately 45 kD. This value is close to the molecular weight of the heavy chain of rabbit IgG (Marler et a l . , 1964). A diffuse band also appeared at approximately 25 kD which corresponded to the molecular weight of the l ight chain of rabbit IgG (Marler et a l . , 1964). Other minor bands reflected the presence of some contaminating proteins. Radial immunodiffusion was not performed on the rabbit IgG fractions since anti-rabbit antibody and standard rabbit IgG were not available. It has already been established by McCannel (1988) that chickens immunized with NAGase are capable of generating an antibody response to this immunogen. To verify this , the immunoreactivity of the IgY fractions against NAGase was tested. This was done using an indirect ELISA. IgY fractions at various protein concentrations were tested. Figure 25 shows the results . As the concentration of IgY was increased there was a subsequent increase in absorbance at 405 nm indicating an increase in reaction with NAGase. Above a concentration of 0.01% protein, the ac t iv i ty decreased. This is unusual since one would expect the absorbance values (and thus immunoreactivity) to reach a plateau once the binding sites on the NAGase bound to the microwell plate were saturated. Instead, higher concentrations seemed to promote a decrease in the antigen-antibody interaction. This phenomenon is not uncommon to ELISAs (de Savigny and Vol ler , 1980) and is considered to be a type of "prozone" effect. Prozone effects are generally observed in precipitation and 113 F i g u r e 25: A n t i g e n b i n d i n g a c t i v i t y of IgY f r a c t i o n s as determined by an i n d i r e c t E L I S A . F r a c t i o n 1 ( X ) ; F r a c t i o n 2 ( O ); F r a c t i o n 3 ( Q ) . 1 1 4 agglutination reactions whereby in the presence of antibody excess no reaction between antigen and antibody is v is ible (Weir, 1988). The exact mechanism for this phenomenon is not known. The a b i l i t y of the rabbit IgG to bind to NAGase was also examined using an indirect ELISA. Figure 26 indicates that, l ike the chickens, rabbits immunized with NAGase produced antibodies which were capable of reacting with NAGase. Again, antibody act iv i ty peaked and then declined beyond a certain concentration. In spite of this prozone effect, i t has been shown that the antibodies produced in the rabbits and chickens were capable of reacting with NAGase and therefore have potential use in an ELISA for this enzyme. B. Preparation of Anti-NAGase - Alkaline Phosphatase  (ALP) Conjugates Anti-NAGase antibody bound to an enzyme label was required to conduct the sandwich ELISA shown (Figure 27). Anti-NAGase chicken IgY was bound to alkaline phosphatase (ALP) using two methods. The f i r s t method involved linking free amino groups of the antibody and the enzyme by reacting with glutaraldehyde. The reaction mixture was then purified by gel f i l t r a t i o n to remove unreacted antibody and enzyme. To determine the working di lut ion of the conjugate, an ELISA was performed on various dilutions of conjugate with NAGase. The conjugate had v ir tua l ly no act iv i ty below a 1/10 di lut ion (Figure 28). The most noticable problem with the use of this 115 2.5 E C 2.0 -— m o t^- -1.5 — Q) O C o 1.0 P . V-O CO --Q 0.5 — < 0.0 -10 10 55 Protein F i g u r e 26: Antigen b i n d i n g a c t i v i t y of i s o l a t e d r a b b i t IgG f r a c t i o n s as determined by an i n d i r e c t ELISA. Rabbit 1 ( X ) ; Rabbit 2 ( O ) . 116 Sandwich ELISA A n t i - N A G a s e - A L P c o n j u g a t e N A G a s e A n t i - N A G a s e A b ( c h i c k e n ) Figure 27: Diagram of the sandwich ELISA 117 2 . 5 F i g u r e 28: D e t e r m i n a t i o n of the working d i l u t i o n of the g l u t a r a l d e h y d e prepared a n t i - N A G a s e - ALP c o n j u g a t e . ( X ) w i th NAGase c o a t i n g ; ( • ) wi thout NAGase c o a t i n g . 118 conjugate was that even in the absence of NAGase (plate was s t i l l blocked), a very high absorbance was recorded when the conjugate was added. The absorbance was even greater than when NAGase was present. This indicates that the conjugate was binding non-specifically to the plate. When compared with results from a similar ELISA using the same antibody (unconjugated), followed by addition of commercial anti-chicken IgY - ALP conjugate, non-specific binding did not occur ( i . e . there was no significant absorbance reading in the absence of NAGase). This can be seen in Figure 29. This suggests that perhaps some change in the antigen binding site on the antibody molecule had taken place as a result of conjugation with ALP, resulting in an increase in non-specific binding. The non-specific binding may also be attributed to the low dilutions required for an observed antibody-antigen interaction. Masseyeff and Ferrua (1979) observed that in some instances at a high concentration of conjugate the maximal binding was the same whether antigen was present or not. Since the low react ivity and non-specific binding of the conjugate may be due to the method used to link the antibody and enzyme, a second conjugation method was chosen. The second method employed sodium periodate as the active reagent. Sodium periodate oxidized carbohydrates to aldehydes on the enzyme which were then allowed to react with free amino groups on the antibody. The reaction mixture was purified by gel f i l t r a t i o n . The working di lut ion of the 119 F i g u r e 29: D e t e r m i n a t i o n of the working d i l u t i o n of the IgY f r a c t i o n used i n the p r e p a r a t i o n of a n t i - N A G a s e -ALP c o n j u g a t e s . ( X ) wi th NAGase c o a t i n g ; ( Q ) w i thout NAGase c o a t i n g . 120 conjugate was determined by ELISA. Results similar to those for the glutaraldehyde prepared conjugate were observed (Figure 30). While the conjugate retained some act iv i ty at as high as a 1/20 d i lu t ion , there was also a high degree of non-specific binding in the absence of NAGase. Unti l the problems associated with these conjugates are solved, a sandwich ELISA for NAGase cannot be developed C. Application of an ELISAs to detect NAGase An attempt was made to derive standard curves for the detection of NAGase using a competitive and a double-sandwich ELISA. Application of these assays to detect NAGase in fish samples was also examined. 1. Competitive ELISA A competitive ELISA (Figure 31) was performed on three separate days using NAGase concentrations ranging from 0 jug/ml to 20/ig/ml. Microwell plates were coated with a fixed concentration of NAGase. A fixed concentration of ant i -NAGase Ab was added at the same time as the NAGase standards. A competition for binding sites occured. Anti-NAGase not bound to free NAGase was able to react with NAGase coating the plate. Anti-chicken - ALP conjugate was then added. Figure 32 depicts an average standard curve. For a l l three days, absorbance values increased with increasing NAGase concentration to 0.5 /ig/ml then decreased with further increases in the concentration of NAGase. This relationship is not typical of a competitive ELISA. A continual decrease 121 F i g u r e 30: D e t e r m i n a t i o n of the working d i l u t i o n of the p e r i o d a t e - o x i d a t i o n prepared ant i -NAGase - ALP c o n j u g a t e . ( X ) w i th NAGase c o a t i n g ; ( • ) w i thout NAGase c o a t i n g . 122 Competitive ELISA NAGase + Anti -NAGase Ab (chicken) NAGase Anti-chicken - ALP conjugate Anti -NAGase Ab (chicken) NAGase F i g u r e 31: Diagram of the c o m p e t i t i v e E L I S A . 123 E l O i.i -I o cu 0.9 H o c D J Q 0 . 7 - H o < 0.5 o ID • 0.3 | 1 1 1 l | 1 1 1 i | 1 1 1 1 | i 1 i 1 1 1 I 1 I 0.0 5.0 10.0 15.0 20.0 25.0 NAGase Cone, (ug/ml) F i g u r e 32: R e l a t i o n s h i p between NAGase c o n c e n t r a t i o n and absorbance a t 405 nm i n a c o m p e t i t i v e E L I S A . 124 i n absorbance values as the antigen c o n c e n t r a t i o n increases was expected (Huang et a l . , 1987). The data presented here suggests that below a c r i t i c a l NAGase co n c e n t r a t i o n , increases i n NAGase con c e n t r a t i o n cause an increase i n binding of anti-NAGase to NAGase already c o a t i n g the p l a t e . The reason why t h i s occured i s unknown. No previous repo r t s of such a phenomenon could be found i n the l i t e r a t u r e . Due to the nature of these r e s u l t s , a standard curve could not be f i t t e d to t h i s data. Therefore, t h i s ELISA was not s u i t a b l e for the determination of NAGase concentrations i n f i s h samples. 2. Double-sandwich ELISA A double-sandwich ELISA (Figure 33) was performed on three separate days using NAGase concentrations ranging from 0.031/jg/ml to 20/ig/ml. The r e l a t i o n s h i p between the l o g of the standard concentrations and the absorbance at 405 nm was s i g n i f i c a n t l y l i n e a r ( a=0.01) on each day. A t y p i c a l standard curve i s depicted i n Figure 34. In t h i s ELISA, prepared r a b b i t anti-NAGase was used to coat the microwell p l a t e and chicken anti-NAGase was used as the second antibody. The ELISA performed i n t h i s way had an average blank absorbance of 0.027 at 405 nm when no NAGase was added. When the p l a t e s were coated with chicken a n t i -NAGase and r a b b i t anti-NAGase was used as the second antibody, however, the absorbance of the blanks at 405 nm averaged 0.39. This information suggests the commercial a n t i - r a b b i t antibody - ALP conjugate c r o s s - reacted with the Double Sandwich ELISA A n t i - c h i c k e n - A L P c o n j u g a t e A n t i - N A G a s e a b ( c h i c k e n ) N A G a s e A n t i - N A G a s e A b ( r a b b i t ) Figure 3 3 : Diagram of the double sandwich ELISA. 1 2 6 1.0 -2.0 -1.0 0.0 1.0 2.0 Log NAGase (ug/ml) F i g u r e 34: A t y p i c a l s t a n d a r d curve f o r NAGase as determined by a d o u b l e - s a n d w i c h ELISA {zz = 0.96; SEE = 0.066) 127 chicken IgY. The f a c t t h a t the blank values were low when r a b b i t IgG was used as the c o a t i n g antibody i n d i c a t e s that there was no c r o s s - r e a c t i o n between a n t i - c h i c k e n IgY - ALP conjugate and r a b b i t IgG or the prepared chicken IgY and the r a b b i t IgG. A p o s s i b l e e x p l a n a t i o n f o r these r e s u l t s i s t h a t the same or s i m i l a r determinants to which the a n t i - r a b b i t Ig were r a i s e d a l s o e x i s t on the chicken IgY molecule. D. A p p l i c a t i o n of a Double-sandwich ELISA to Detect NAGase  in F i s h Muscle The double-sandwich ELISA was a p p l i e d to the examination of press j u i c e (PJ) and f i s h e x t r a c t (FE) samples from salmon muscle. The concentration of NAGase i n the samples was c a l c u l a t e d using a standard curve. F i v e d i l u t i o n s of the PJ and FE samples were examined (no d i l u t i o n , 1/2, 1 / 4 , 1/8 and 1/16). Table VII shows the r e s u l t s from the 3 salmon samples examined. Results of only the f i r s t three d i l u t i o n s are shown since absorbance values obtained from higher d i l u t i o n s were too low to enable c a l c u l a t i o n of NAGase concentrations from the standard curve. S i m i l a r NAGase concentrations would be expected f o r the d i f f e r e n t d i l u t i o n s once the d i l u t i o n f a c t o r had been taken i n t o c o n s i d e r a t i o n . This was not the case. Instead, i n a l l but three samples the c a l c u l a t e d NAGase concentration increased with an increase i n d i l u t i o n . McCannel (1988) reported a s i m i l a r occurence i n her examination of d i l u t e d PJ and FE samples by an i n d i r e c t ELISA. She suggested that i n the i n d i r e c t ELISA, other substances were co a t i n g the p l a t e 128 Table VII: Levels of (3-N-Acetylglucosaminldase (NAGase) In Fresh and Frozen Salmon Samples as Determined by a Double-sandwich ELISA. NAGase Cone, (ug/ml) Sample Dilution Sample Preparation 1 1/2 1/4 Salmon 1 Fresh Press Juice 0. 235 0.342 0.536 Salmon 2 0. 210 0.326 0.424 Salmon 3 0. 251 0.370 0.360 Salmon 1 Fresh Extract 0. 320 0.538 0.360 Salmon 2 0. 193 0.248 0.336 Salmon 3 0. 168 0.256 0.340 Salmon 1 Frozen Press Juice 0. 520 0.682 0.588 Salmon 2 0. 309 0.442 0.596 Salmon 3 0. 240 0.342 0.544 Salmon 1 Frozen Extract 0. 387 0.462 0.620 Salmon 2 0. 239 0.322 0. 496 Salmon 3 0. 221 0.286 0.432 a. Concentrations presented were after multiplication by di lut ion factors. 129 which interfered with NAGase binding. Dilution of the fish samples therefore diluted the interfering substances and thus an increased amount of NAGase bound to the plate. A similar explanation can be offered for the results observed in this experiment. Dilution of the samples diluted other substances which possibly interfered with antibody-antigen interaction. Therefore, more NAGase bound to the anti-NAGase antibody coating the plate. The result was an observed increase in absorbance at 405 nm and the calculated NAGase concentration. The data in Table VII was converted into ratios of NAGase concentration in the PJ fraction to the total NAGase concentration (NAGase concentration in the PJ + NAGase concentration in the FE). The ratios for the fresh and frozen samples is shown in Table VII. The expression of these data as the ratios of the NAGase concentration in the PJ to the total NAGase concentration appeared to eliminate the di lut ion effect (Table VIII). The ratios derived for each di lut ion were not s ignif icantly different as determined by analysis of variance ( a=0.05). Therefore, i t is reasonable to assume that any di lut ion of PJ or FE samples could be used to derive NAGase concentrations provided the absorbance values recorded are within the bounds of the standard curve. Rehbein et a l . (1978) used a similar ratio (NAGase enzyme act iv i ty in PJ compared to total enzyme activity) to decrease var iab i l i ty in the data due to species of f i sh . Use of ratios may also eliminate var iab i l i ty due to other factors such as age of f ish, duration of storage, etc. 130 Table VIII: concentration Ratios of 8-N-Acetylglucosaminldase (NAGase) In Fresh and Frozen Salmon Samples (calculated from Table VII). Concentration Ratio Sample Dilution Sample Preparation 1 1/2 1/4 Salmon 1 Fresh 0.423 0.388 0.598 Salmon 2 0.521 0.568 0.558 Salmon 3 0.599 0.591 0.514 Salmon 1 Frozen 0.573 0.596 0.487 Salmon 2 0.564 0.578 0.545 Salmon 3 0.520 0.545 0.538 ( PJ ) a. concentration ratio = ( PJ + FE ) 131 When McCannel (1988) performed an indirect ELISA on salmon samples, she reported that NAGase concentrations decreased in the frozen samples. It was suggested that this was possibly due to part ia l denaturation of the native NAGase structure by freezing, resulting in reduced recognition of the NAGase by its specific antibody. Using the double sandwich ELISA, NAGase concentrations in frozen PJ samples were s ignif icantly higher as determined by analysis of variance (a=0.05), than in the same samples when fresh (Table VII). This is an expected result since i t has already been established that some lysosomal enzymes including NAGase, are released during freeze/thawing of fish muscle (Rehbein et a l . , 1978). Therefore, the concentration of NAGase is expected to be higher in the frozen PJ samples. Analysis of variance was performed on the concentration ratios for the fresh samples versus samples frozen for one week then thawed. The analysis showed that there was no significant difference (a =0.05) between the two sets of ratios and therefore the two treatments. This indicates that while the levels of NAGase increased in the frozen PJ samples, so also did the total NAGase levels. Total NAGase levels would be expected to remain the same for both the fresh and the frozen treatments. One explanation for the difference in total NAGase levels may be that the conditions used for the preparation of fish extract were not severe enough for the disruption of lysosomes and release of NAGase in the fresh f i sh . Lysosomes in the frozen fish muscle, on 132 the other hand, may have s u f f e r e d freeze damage. As a r e s u l t they were more s u s c e p t i b l e to lysosome d i s r u p t i o n and NAGase r e l e a s e . The r e s u l t was that the NAGase l e v e l s i n the frozen FE were higher than i n the f r e s h FE. Therefore, the c a l c u l a t e d t o t a l NAGase l e v e l s were higher i n the frozen samples. In order to use t h i s ELISA to d i f f e r e n t i a t e between frozen/thawed and f r e s h f i s h , a method i s needed for f i s h e x t r a c t preparation which would r e s u l t i n c o n s i s t a n t t o t a l NAGase concentrations i n f r e s h and frozen/thawed samples. Rehbein et a l . (1978) used a f i s h e x t r a c t p r e p a r a t i o n method s i m i l a r to the one described i n t h i s experiment. However, T r i t o n X-100 was added p r i o r to homogenization. This detergent probably aided i n membrane d i s r u p t i o n and s o l u b i l i z a t i o n of membrane bound p r o t e i n s . The r e s u l t was a more r e l i a b l e e s t i m a t i o n of, i n t h i s case, t o t a l NAGase enzyme a c t i v i t y . By comparing the NAGase a c t i v i t y i n the press j u i c e as a % of the t o t a l a c t i v i t y , these researchers were able to detect s i g n i f i c a n t d i f f e r e n c e s between f r e s h o f i s h f i l l e t s and f i l l e t s frozen for 1 day at -26 to -29 C. T r i t o n X-100 was not added i n the present study because i t was thought that i t may a f f e c t antigen-antibody binding. Experiments to determine i f t h i s were t r u e , however, were not performed. To e f f e c t i v e l y use t h i s ELISA, one has to e s t a b l i s h that the d i f f e r e n c e s i n NAGase concentration r a t i o s are s o l e l y due to f r e e z i n g . Experiments to t e s t t h i s hyothesis were not 133 done i n t h i s t h e s i s . Rehbein et a l . (1978) have already reported that during a u t o l y s i s and b a c t e r i a l s p o i l a g e of f i s h muscle, enzymes had been released as i n d i c a t e d by a c t i v i t y r a t i o s for s p o i l e d f i s h which were n e a r l y as high as i n the frozen/thawed f i l l e t s . However, f o r NAGase, the a c t i v i t y r a t i o s increased a f t e r 10 days of storage of f i s h on i c e . The f i s h were not s e l l a b l e at that point based on or g a n o l e p t i c and chemical t e s t s (Rehbein et a l . , 1978). Therefore, the e s t i m a t i o n of NAGase l e v e l s i n f i s h muscle may s t i l l be a u s e f u l i n d i c a t o r of frozen/thawed f i s h muscle. E. Conclusion P o l y c l o n a l a n t i b o d i e s s p e c i f i c f o r NAGase were s u c c e s s f u l l y derived from both chickens and r a b b i t s . However, chicken IgY - ALP conjugates e x h i b i t e d low immunoreactivity with the antigen as w e l l as a high degree of n o n - s p e c i f i c binding when prepared by r e a c t i o n with glutaraldehyde or by periodate o x i d a t i o n . Therefore, these conjugates were considered unsuitable reagents f o r use i n a sandwich ELISA. Problems were a l s o encountered i n the development of a competitive ELISA for NAGase as seen by the a t y p i c a l standard curve produced by t h i s assay. Development of a double-sandwich ELISA f o r NAGase was s u c c e s s f u l . However, when t h i s ELISA was a p p l i e d to the d e t e c t i o n of t h i s enzyme i n f i s h muscle samples, the information obtained was i n c o n c l u s i v e . While increases i n the l e v e l s of t h i s enzyme were observed i n frozen PJ samples, con c e n t r a t i o n r a t i o s remained the same. 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