UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Development of a general ligand for immunoaffinity partitioning in two phase aqueous polymer systems Stocks, Susan Jill 1986

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1986_A6_7 S83.pdf [ 5.48MB ]
Metadata
JSON: 831-1.0059392.json
JSON-LD: 831-1.0059392-ld.json
RDF/XML (Pretty): 831-1.0059392-rdf.xml
RDF/JSON: 831-1.0059392-rdf.json
Turtle: 831-1.0059392-turtle.txt
N-Triples: 831-1.0059392-rdf-ntriples.txt
Original Record: 831-1.0059392-source.json
Full Text
831-1.0059392-fulltext.txt
Citation
831-1.0059392.ris

Full Text

DEVELOPMENT OF A GENERAL LIGAND FOR IMMUNOAFFINITY PARTITIONING IN TWO PHASE AQUEOUS POLYMER SYSTEMS by SUSAN J I L L STOCKS B . S c , Reading U n i v e r s i t y , 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Chemistry We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1986 © Susan J i l l S tocks, 1986 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e The U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my Department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D epartment o f C h e m i s t r y The U n i v e r s i t y of B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5 Date: A p r i l 1986 Abstract The p a r t i t i o n of erythrocytes in a two phase aqueous polymer system composed of dextran T500, poly(ethylene glycol)8000 (PEG 8000) and buffer was studied and the effect of a combination of a f f i n i t y ligands, namely rabbit IgG and PEG 1900 modified monoclonal IgG, was examined as a poten t i a l c e l l separation technique. Several hybridoma l i n e s secreting mouse monoclonal IgG s p e c i f i c for the F c receptor of rabbit IgG were produced by the fusion of immunised mice spleen c e l l s and mouse myeloma c e l l s . The monoclonal IgG was modified by cyanuric chloride attachment of PEG 1900. The modified monoclonal antibody partitioned predominantly into the PEG r i c h upper phase of a two phase aqueous polymer system containing PEG 8000 and dextran T500. The PEG-modified monoclonal IgG was used as an a f f i n i t y ligand in the two phase polymer system to s p e c i f i c a l l y increase the p a r t i t i o n of rabbit anti-NN glycophorin A IgG. The rabbit IgG was used together with the PEG-modified monoclonal IgG to increase the p a r t i t i o n of human erythrocytes. The same system had no ef f e c t on rabbit erythrocytes. In summary, i t was demonstrated that a monoclonal antibody can be modified and used to a l t e r c e l l p a r t i t i o n in two phase aqueous polymer systems in an immunologically s p e c i f i c manner. i i Table of Contents A. I n t r o d u c t i o n 1 1. The Theory of Phase S e p a r a t i o n 3 2. The Phase Diagram 7 3. The Theory of M o l e c u l a r P a r t i t i o n 9 4. The Theory of E l e c t r o l y t e P a r t i t i o n 10 5. The A p p l i c a t i o n s of A f f i n i t y P a r t i t i o n i n g 13 6. The Theory of A f f i n i t y P a r t i t i o n 15 7. The Theory of C e l l and P a r t i c l e P a r t i t i o n 17 8. The Theory of Monoclonal A n t i b o d i e s 21 9. A n a l y t i c a l Techniques used in the Study 36 B. M a t e r i a l s and Methods 41 1. Immunisation of the R a b b i t s 41 2. P u r i f i c a t i o n of the Rabbit anti-NN G l y c o p h o r i n A Immunoglobulin G 41 3. M i c r o t i t r e of the anti-NN g l y c o p h o r i n A Immunoglobulin G 42 4. Fragmentation of Rabbit Immunoglobulin G by Papain 44 5. Sodium Dodecyl Sulphate P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s 44 6. Immunisation of BALB/c Mice 50 7. C u l t u r e of NS 1 C e l l s .51 8. P r e p a r a t i o n of C e l l s f o r F u s i o n 52 9. F u s i o n P r o t o c o l 53 10. The Enzyme-linked Immunosorbent Assay(ELISA) ....54 11. Recloning by L i m i t i n g D i l u t i o n 56 12. Antibody P r o d u c t i o n and P u r i f i c a t i o n 56 13. F r e e z i n g and Thawing of Hybridoma C e l l s 58 i i i 1 4 . C h a r a c t e r i s a t i o n o f t h e P u r i f i e d M o n o c l o n a l A n t i b o d y by a W e s t e r n B l o t 5 8 1 5 . A f f i n i t y C h r o m a t o g r a p h y o f A n t i b o d i e s 6 0 1 6 . M o d i f i c a t i o n o f t h e M o n o c l o n a l A n t i b o d y w i t h PEG 1 9 0 0 61 1 7 . Degree o f PEG 1 9 0 0 M o d i f i c a t i o n o f t h e M o n o c l o n a l A n t i b o d y 6 4 1 8 . A s s a y of t h e B i n d i n g A c t i v i t y of t h e P E G - a n t i b o d y c o n j u g a t e 6 7 1 9 . S i n g l e Tube P a r t i t i o n E x p e r i m e n t s 6 8 C. R e s u l t s and D i s c u s s i o n 7 2 1 . R a b b i t a n t i - N N G l y c o p h o r i n A 7 2 2 . P a p a i n D i g e s t i o n o f R a b b i t I m m u n o g l o b u l i n G 7 7 3 . P r o d u c t i o n and S e l e c t i o n o f H y b r i d o m a s 7 8 4 . P u r i f i c a t i o n o f t h e M o n o c l o n a l A n t i b o d y 8 2 5 . A c t i v i t y o f t h e P u r i f i e d M o n o c l o n a l A n t i b o d y . . . . 8 5 6 . S y n t h e s i s and A n a l y s i s o f t h e P E G - M o n o c l o n a l A n t i b o d y 8 8 7 . R e s u l t s o f t h e TNBS and F l u o r e s c a m i n e A s s a y s . . . . 8 9 8 . The A c t i v i t y o f t h e P E G - M o d i f i e d A n t i b o d y 9 3 9 . P a r t i t i o n i n g o f N a t i v e and M o d i f i e d P r o t e i n s . . . . 9 5 1 0 . E f f e c t i v e A t t a c h e d PEG 1 9 0 0 9 7 1 1 . P a r t i t i o n o f Human E r y t h r o c y t e s i n t h e a b s e n c e of A n t i b o d y 1 0 0 1 2 . The e f f e c t o f t h e a n t i b o d i e s on e r y t h r o c y t e v p a r t i t i o n 1 0 4 1 3 . The e f f e c t o f a l t e r i n g t h e r a t i o s o f a n t i b o d i e s 1 0 9 1 4 . The e f f e c t of t h e a n t i b o d i e s on r a b b i t e r y t h r o c y t e p a r t i t i o n 1 1 1 1 5 . Summary 1 1 3 CONCLUSION 1 1 6 i v GLOSSARY OF TERMS 117 GLOSSARY OF SYMBOLS AND ABBREVIATIONS 120 BIBLIOGRAPHY 122 v L i s t of F i g u r e s F i g . 1 . G e n e r a l Phase D i a g r a m f o r a Two P o l y m e r / B u f f e r Phase System. 8 F i g . 2 . Phase D i a g r a m s f o r D e x t r a n T500/PEG 8 0 0 0 / B u f f e r S ystems 1 1 F i g . 3 . The C l o n a l S e l e c t i o n T h e o r y 23 F i g . 4 . S t r u c t u r e o f t h e IgG M o l e c u l e 24 F i g . 5 . P r o d u c t i o n o f H y b r i d o m a s 27 F i g . 6 . M e t a b o l i c Pathways r e l e v a n t t o Hybridoma S e l e c t i o n i n HAT Medium 29 F i g . 7 . S c h e m a t i c R e p r e s e n t a t i o n o f t h e Mechanism of I m m u n o a f f i n i t y P a r t i t i o n 35 F i g . 8 . The E n z y m e - l i n k e d Immunosorbent A s s a y ( E L I S A ) 39 F i g . 9 . FPLC P r o f i l e o f ' R a b b i t Serum P r o t e i n s 43 F i g . 1 0 . FPLC P r o f i l e o f t h e P a p a i n D i g e s t o f R a b b i t IgG 45 F i g . 1 1 . SDS-PAGE o f R a b b i t IgG and t h e P a p a i n D i g e s t o f R a b b i t IgG 46 F i g . 1 2 . FPLC P r o f i l e o f M o n o c l o n a l IgG 57 F i g . 1 3 . R e a c t i o n Scheme f o r P r o t e i n M o d i f i c a t i o n w i t h PEG 62 F i g . 1 4 a . R e a c t i o n o f F l u o r e s c a m i n e w i t h P r i m a r y Amines 66 F i g . 1 4 b . R e a c t i o n o f TNBS w i t h P r i m a r y Amines 66 F i g . 1 5 . SDS-PAGE o f N a t i v e and Reduced IgG 71 v i F i g . 1 6 . C o n c e n t r a t i o n Dependence of t h e B i n d i n g o f MN and NN G l y c o p h o r i n A by A n t i - N N G l y c o p h o r i n A R a b b i t IgG 75 F i g . 1 7 . C e l l C u l t u r e s 79 F i g . 1 8 . SDS-PAGE o f M o n o c l o n a l and P o l y c l o n a l A n t i b o d i e s 81 F I G . 1 9 . C o n c e n t r a t i o n Dependence o f t h e B i n d i n g o f R a b b i t F c Fragment o f IgG by U n p u r i f i e d M o n o c l o n a l A n t i - r a b b i t F c Fragment 83 F i g . 2 0 . C o n c e n t r a t i o n Dependence o f t h e B i n d i n g o f R a b b i t F c Fragment by P u r i f i e d M o n o c l o n a l A n t i b o d y 84 F i g . 2 1 a . W e s t e r n B l o t of M o n o c l o n a l A n t i - F c Fragment a g a i n s t P a p a i n D i g e s t F r a g m e n t s and Reduced R a b b i t IgG 87 F i g . 2 1 b . D i a g r a m o f P r o t e i n T r a n s f e r i n a W e s t e r n B l o t 87 F i g . 2 2 a . L i n e a r R e g r e s s i o n A n a l y s i s o f F l u o r e s c a m i n e S t a n d a r d C u r v e s f o r N a t i v e and P E G - m o d i f i e d M o n o c l o n a l A n t i b o d y 90 F i g . 2 2 b . L i n e a r R e g r e s s i o n A n a l y s i s o f TNBS S t a n d a r d C u r v e s f o r N a t i v e and P E G - m o d i f i e d M o n o c l o n a l A n t i b o d y 92 F i g . 2 3 . R e s u l t s of B i n d i n g A s s a y f o r P E G - m o d i f i e d M o n o c l o n a l IgG t o R a b b i t IgG 94 v i i 5 L i s t of T a b l e s T a b l e 1. R e s u l t s of M i c r o t i t r e o f R a b b i t A n t i - N N G l y c o p h o r i n A IgG a g a i n s t Human E r y t h r o c y t e s o f NN, MM o r MN S p e c i f i c i t i e s 73 T a b l e 2. R e s u l t s of F r e e C h l o r i d e T i t r a t i o n of PCC 1900 88 T a b l e 3. P a r t i t i o n o f N a t i v e and M o d i f i e d A n t i b o d i e s 96 T a b l e 4. E f f e c t i v e Amount of PEG 1900 a t t a c h e d t o t h e M o n o c l o n a l A n t i b o d y 98 T a b l e 5. E f f e c t of B u f f e r s on E r y t h r o c y t e P a r t i t i o n 101 T a b l e 6. E f f e c t of A n t i b o d i e s on E r y t h r o c y t e P a r t i t i o n 104 T a b l e 7. E f f e c t o f A l t e r i n g t h e R e l a t i v e Amounts o f A n t i b o d i e s on E r y t h r o c y t e P a r t i t i o n 109 T a b l e 8. E f f e c t of A n t i b o d i e s on Human and R a b b i t E r y t h r o c y t e P a r t i t i o n 111 v i i i Acknowledgements I would l i k e t o thank e v e r y b o d y i n t h e l a b f o r t h e i r i n v a l u a b l e a d v i c e and a s s i s t a n c e , i n p a r t i c u l a r , Nancy H a m i l t o n and E r i c Wong f o r t h e i r h e l p i n making t h e m o n o c l o n a l a n t i b o d y . I am a l s o i n d e b t e d t o Kim S h a r p , Raymond N o r r i s - J o n e s , J o hn Cavanagh and J o h a n n J a n z e n f o r t h e i r h e l p and p a t i e n c e . Most o f a l l I would l i k e t o thank Don B r o o k s f o r making t h i s t h e s i s p o s s i b l e w i t h h i s a d v i c e , s u g g e s t i o n s and p a r t i e s . i x 1 A. INTRODUCTION T h e r e a r e many e x i s t i n g p r o c e d u r e s t o i d e n t i f y and i s o l a t e p u r e m o l e c u l a r s p e c i e s f r o m complex s o l u t i o n s b u t methods t o s e p a r a t e s u b - p o p u l a t i o n s o f c e l l s f r o m m i x e d s u s p e n s i o n s a r e l e s s w e l l d e v e l o p e d . P r o c e d u r e s u s i n g c e l l s i z e , d e n s i t y o r shape a r e l e s s u s e f u l t h a n t h o s e b a s e d on c e l l s u r f a c e p r o p e r t i e s , a s t h e l a t t e r a r e most l i k e l y t o be a s s o c i a t e d w i t h t h e f u n c t i o n o f t h e c e l l . F o r example, l y m p h o c y t e s s e c r e t i n g d i f f e r e n t a n t i b o d i e s p r o b a b l y have s i m i l a r s i z e and g e o m e t r y but d i f f e r e n t s u r f a c e a n t i g e n s . T h e r e a r e c u r r e n t l y f i v e main c l a s s e s o f c e l l s e p a r a t i o n methods b a s e d on membrane c h a r a c t e r i s t i c s : 1 . C e l l e l e c t r o p h o r e s i s o r i s o e l e c t r i c f o c u s i n g ( 1 , 2 ) . 2 . I n d i v i d u a l c e l l s o r t i n g u s i n g f l u o r e s c e n t c e l l s u r f a c e l a b e l s ( 3 ) o r a l i g h t s c a t t e r i n g s i g n a t u r e ( 4 ) . 3 . I m m u n o a d s o r p t i o n on a f f i n i t y c o lumns ( 5 , 6 ) . 4 . A n t i b o d y - m e d i a t e d complement l y s i s o f c o n t a m i n a t i n g c e l l s u b - p o p u l a t i o n s ( 7 ) . 5 . P a r t i t i o n and c o u n t e r c u r r e n t d i s t r i b u t i o n i n two pha s e aqueous p o l y m e r s y s t e m s ( 8 , 9 ) . Of t h e s e f i v e c l a s s e s , p a r t i t i o n o f c e l l s i n two p h a s e a q u e o u s p o l y m e r s y s t e m s was e x a m i n e d i n t h i s s t u d y . The f a c t t h a t most m i x t u r e s o f p o l y m e r s show i n c o m p a t a b i l i t y under c e r t a i n w e l l d e f i n e d c o n d i t i o n s , i n b o t h o r g a n i c and aqueous s o l v e n t s , i s a w e l l e s t a b l i s h e d g e n e r a l phenomenon ( 1 0 ) . Two t y p e s o f phase s e p a r a t i o n can 2 o c c u r as a r e s u l t of t h i s i n c o m p a t a b i l i t y : one p o l y m e r c o l l e c t s p r e d o m i n a n t l y i n one p h a s e and t h e o t h e r p o l y m e r m o s t l y i n t h e o t h e r p h a s e ; o r b o t h p o l y m e r s c o l l e c t i n t h e same p h a s e , t h e o t h e r p h a s e b e i n g m o s t l y s o l v e n t ( 1 1 ) . The t e r m " c o a c e r v a t i o n " i s u s e d t o d e s c r i b e t h e g e n e r a l phenomenon o f p h a s e s e p a r a t i o n o f p o l y m e r m i x t u r e s and t h e c a s e where o p p o s i t e l y c h a r g e d p o l y m e r s c o l l e c t i n t h e same pha s e i s t e r m e d "complex c o a c e r v a t i o n " . I t i s a l s o p o s s i b l e t o f o r m a two phase s y s t e m by c o m b i n i n g a s i n g l e n e u t r a l p o l y m e r o r p o l y e l e c t r o l y t e w i t h a s a l t i n a common s o l v e n t . The most commonly u s e d phase s y s t e m s f o r p a r t i t i o n i n g b i o l o g i c a l m a t e r i a l s a r e t h o s e b a s e d on two p o l y m e r s s u c h as d e x t r a n / p o l y ( e t h y l e n e g l y c o l ) ( P E G ) o r d e x t r a n / f i c o l l . P hase s e p a r a t i o n r e s u l t s f r o m an u n f a v o u r a b l e i n t e r a c t i o n e n e r g y between t h e two p o l y m e r s . S o l u t e s w i l l p a r t i t i o n s e l e c t i v e l y between t h e two p h a s e s o r , i n t h e c a s e o f p a r t i c l e s , between t h e i n t e r f a c e and one o f t h e two p h a s e s , d e p e n d i n g on t h e c h e m i c a l p o t e n t i a l o f t h e s o l u t e o r s u r f a c e f r e e e n e r g y of t h e p a r t i c l e i n e a c h p h a s e o r a t t h e i n t e r f a c e . B e s i d e s t h e i n c o m p a t a b i l i t y o f t h e s o l u t e o r p a r t i c l e w i t h t h e p h a s e p o l y m e r s , o t h e r f a c t o r s s u c h as s a l t c o n c e n t r a t i o n o r t y p e , pH and t h e p r e s e n c e o f a f f i n i t y l i g a n d s ( m o l e c u l e s w h i c h a s s o c i a t e s p e c i f i c a l l y w i t h t h e m a t e r i a l b e i n g d i s t r i b u t e d ) w i l l a f f e c t t h e p a r t i t i o n c o e f f i c i e n t o f t h e p a r t i c l e o r s o l u t e . 3 1. THE THEORY OF PHASE SEPARATION The s t a t i s t i c a l t h e r m o d y n a m i c s d e s c r i b i n g p h a s e s e p a r a t i o n of p o l y m e r s was d e r i v e d by F l o r y ( l 2 ) and H u g g i n s ( 1 3 ) . The i n i t i a l a p p r o a c h was t o d e r i v e an e x p r e s s i o n d e s c r i b i n g t h e f r e e e n e r g y change on m i x i n g a s i n g l e p o l y m e r and a s o l v e n t . E x p r e s s i o n s f o r t h e e n t h a l p i c and e n t r o p i c c o n t r i b u t i o n s t o t h e f r e e e n e r g y c h a n g e were d e r i v e d , t h e n c ombined u s i n g t h e G i b b s - H e l m h o l t z r e l a t i o n s h i p , AG m=AH m-TAS m, t o g i v e t h e f r e e e n e r g y o f m i x i n g . In c o n s i d e r i n g t h e e n t h a l p y c o n t r i b u t i o n , t h e po l y m e r s o l u t i o n i s r e p r e s e n t e d by a number of l a t t i c e s i t e s w h i c h c a n be o c c u p i e d by a s o l v e n t m o l e c u l e o r a po l y m e r segment. E a c h l a t t i c e s i t e has c o o r d i n a t i o n number,z. The e n t h a l p y c hange on m i x i n g t h e s o l v e n t and p o l y m e r i s d e p e n d e n t on t h e e n e r g i e s o f c o n t a c t between t h e p o l y m e r segments and s o l v e n t m o l e c u l e s . The e n e r g y change a s s o c i a t e d w i t h t h e f o r m a t i o n o f a one p o l y m e r s e g m e n t - s o l v e n t m o l e c u l e c o n t a c t i s g i v e n by, AW 1 2=W 1 2-(W,,+W 2 2)/2 eqn [1] where W 1 2 = e n e r g y a s s o c i a t e d w i t h c o n t a c t between s o l v e n t and p o l y m e r W,, = s o l v e n t - s o l v e n t c o n t a c t e n e r g y W 2 2 = p o l y m e r - p o l y m e r c o n t a c t e n e r g y Summing t h e s e i n d i v i d u a l c o n t a c t e n e r g i e s f o r a p a r t i c u l a r o c c u p a n c y o f t h e l a t t i c e s i t e s o r c o m p o s i t i o n g i v e s t h e e n t h a l p y o f m i x i n g o f a p o l y m e r and s o l v e n t a s , A H ^ z A W ^ n , ^ eqn [ 2 ] 4 where z = l a t t i c e c o o r d i n a t i o n number n, = no. o f s o l v e n t m o l e c u l e s on t h e l a t t i c e <t>2 = f r a c t i o n of s i t e s o c c u p i e d by t h e p o l y m e r segments 0 2= n 2 P 2 / ( n i + n 2 P 2 ) where n 2 = no. o f p o l y m e r m o l e c u l e s on t h e l a t t i c e P 2 = no. o f segments p e r p o l y m e r m o l e c u l e The t e r m zAW 1 2 i s u s u a l l y r e p l a c e d by k T x t h e r e f o r e A H m = k T x i 2r>i02 eqn [3] where k = B o l t z m a n n s c o n s t a n t T = a b s o l u t e t e m p e r a t u r e . The term X12 i s t h e F l o r y i n t e r a c t i o n p a r a m e t e r and r e p r e s e n t s t h e maximum i n t e r a c t i o n e n e r g y a s o l v e n t m o l e c u l e c a n p o s s e s s i n a m i x t u r e , i . e . when c o m p l e t e l y s u r r o u n d e d by p o l y m e r s e g m e n t s . The e n t r o p y o f m i x i n g i s e x p r e s s e d i n t e r m s o f t h e number o f p o s s i b l e c o m b i n a t i o n s o f t h e s o l v e n t m o l e c u l e s and p o l y m e r segments on t h e l a t t i c e and i s c a l c u l a t e d f r o m t h e B o l t z m a n n r e l a t i o n s h i p , AS m=klnW eqn [4] A p p r o p r i a t e e v a l u a t i o n o f W l e a d s t o t h e F l o r y - H u g g i n s e q u a t i o n f o r t h e e n t r o p y of m i x i n g a p o l y m e r and s o l v e n t on t h e l a t t i c e , AS m=-k (n , ln^>,+n 2 l n ^ > 2 ) eqn [5] where 0, = n , / ( n , + n 2 P 2 ) . C o m b i n i n g t h e e n t h a l p y and e n t r o p y c o n s i d e r a t i o n s g i v e s t h e e x p r e s s i o n f o r t h e f r e e e n e r g y o f m i x i n g a p o l y m e r and s o l v e n t , 5 AG i n=kT(n 1ln0 1+n 2ln</» 2 + X i 2 n i 0 2 ) eqn [6] The <t>i and q>2 terms for the fracti o n of l a t t i c e s i t e s occupied by solvent molecules and polymer segments are p r a c t i c a l l y represented by the volume fractions of the solvent and polymer respectively. This assumes that solvent molecules and polymer segments are interchangeable with no effect on the l a t t i c e , i . e . no volume change on mixing, the most r e s t r i c t i v e aspect of the theory. From the expression for AGm, the theory can be extended to mixtures of more than two components. For example the free energy of mixing two polymers in a common solvent is given by, A G m = k T [ n 1 l n 0 1 + n 2 l n 0 2 + n 3 l n 0 3 + ( n i + n 2 P 2 + n 3 p 3 ) O i 0 2 X i 2 + 0itf»3Xi3 + 0 2 0 3 X 2 s ) ] eqn [7] where 1 denotes solvent, 2 and 3 are the two polymer species and 0 i=(n iP i)/(n,+n 2P 2+ n 3P 3) where i = 1,2 or 3 = number of molecules of i on the l a t t i c e X- • = zAW. ./kT ID 1D AW i j = W i j - ( W i i + W j j ) / 2 If two phases are present, then the equilibrium chemical potential of any component must be the same in each phase. The chemical potential of any of the three species i s given by, ( M J - M - ) = N AOAG m/9n. ) eqn [8] where = chemical potential of i when the volume f r a c t i o n i s <pi 6 / i ? = standard state chemical p o t e n t i a l . N A = Avogadro's number. As there must be the same value for the chemical p o t e n t i a l , , of the species in each phase, then two values for, <t>^, the equilibrium volume fr a c t i o n must e x i s t . Generally, the value of as a function of <j>2 w i l l decrease as 02 i s increased. Thus i f the chemical potential of the solvent, M,, i s plotted as a function of one of the polymer concentrations, c i 2 , then two values of <t>2 on the curve w i l l correspond to the same value of Mi i f two phases are present. In thi s case, the plot of M, against <J>2 must have a maximum, a minimum and an i n f l e c t i o n point. Obviously the closer the maximum and minimum, the closer the composition of the two phases. The c r i t i c a l conditions for m i s c i b i l i t y or appearance of two phases are represented when the maximum, minimum and point of i n f l e c t i o n a l l coincide. Mathematically, t h i s i s described by, 9MI/90 2 = d2u^/b2<t>2 = 0 Combining the above expression with eqn [7] allows the c r i t i c a l conditions for phase separation to be calculated, i . e . the values of <f>^ and x — which just produce phase separation in a two polymer one solvent phase system. If i t is assumed that X i 2 = X i 3 , i . e . both polymers are equally soluble in the solvent, and that P 2=P3 i . e . both have a similar number of segments per polymer molecule, then a solution of the above two equations gives the following values for c r i t i c a l conditions, 7 0 2 C = 0 3 C = ( 1 " 0 1 c ) / 2 X 2 3 C = 1 / P 2 0 2 C where c d e n o t e s c r i t i c a l c o n d i t i o n s . As P 2 i s v e r y l a r g e , t h i s i m p l i e s t h a t p h a s e s e p a r a t i o n w i l l r e s u l t even i f t h e i n t e r a c t i o n e n e r g y between segments i s o n l y s l i g h t l y p o s i t i v e . 2. THE PHASE DIAGRAM In an aqueous m i x t u r e o f two p o l y m e r s , a two ph a s e s y s t e m w i l l o n l y a r i s e when t h e c o n s t i t u e n t s e x c e e d a c e r t a i n r a n g e of c o n c e n t r a t i o n s a s d e f i n e d a b o v e . T h i s r a n g e may be r e p r e s e n t e d on a phase d i a g r a m ( F i g . 1 . ) . The c u r v e d l i n e d i v i d i n g t h e two a r e a s o f t h e ph a s e d i a g r a m i s t e r m e d t h e b i n o d i a l and a l l c o m p o s i t i o n s o f p o l y m e r A and B above t h e b i n o d i a l w i l l p r o d u c e two ph a s e s y s t e m s . P o i n t s a l o n g t h e b i n o d i a l o r nodes ( e g . B , B ' , C , C ) c a n be j o i n e d t o form t i e l i n e s ( e g . B - C , B ' - C ) . Any p o i n t a l o n g t h e t i e l i n e w i l l g i v e r i s e t o a s y s t e m w i t h t h e same phase c o m p o s i t i o n b u t t h e p h a s e v o l u m e s w i l l d i f f e r . The %w/w r a t i o o f b o t t o m t o t o p p h a s e i s e q u a l t o t h e r a t i o between t i e l i n e l e n g t h s ( e g . AC:BC). The b i n o d i a l c u r v e c a l c u l a t e d by F l o r y - H u g g i n s t h e o r y a s o u t l i n e d e a r l i e r a g r e e s q u a l i t a t i v e l y w i t h t h e e x p e r i m e n t a l b i n o d i a l c u r v e s u g g e s t i n g t h a t t h e F l o r y - H u g g i n s t h e o r y p r o v i d e s a r e a s o n a b l e d e s c r i p t i o n o f p h a s e s e p a r a t i o n . 8 C D cn LU >-_ J o POLYMER A F i g . 1 . G e n e r a l Phase D i a g r a m f o r a Two P o l y m e r / S o l v e n t Phase S y s t e m . The l i n e s BC o r B ' C c o n n e c t t h e p o i n t s r e p r e s e n t i n g t h e c o m p o s i t i o n o f t h e two p h a s e s a t e q u i l i b r i u m i . e . B r e p r e s e n t s t h e b o t t o m phase c o m p o s i t i o n and C t h e t o p p h a s e c o m p o s i t i o n . The t o t a l c o m p o s i t i o n i s g i v e n by A and t h e r a t i o o f AC:AB w i l l g i v e a w e i g h t r a t i o o f b o t t o m t o t o p p h a s e . 9 3. THE THEORY OF MOLECULAR PARTITION Due t o t h e l a r g e s i z e o f t h e m o l e c u l e s , p h a s e s e p a r a t i o n o c c u r s even w i t h v e r y s i m i l a r p o l y m e r s . T h i s means t h a t t h e r e i s a r a n g e o f i m m i s c i b l e s o l u t i o n s i n s o l v e n t s w i t h s i m i l a r p o l a r i t i e s . T h e s e i m m i s c i b l e s o l u t i o n s w o u l d be e x p e c t e d t o be s e l e c t i v e i n p a r t i t i o n i n g s u b s t a n c e s w h i c h d i s s o l v e i n t h e same s o l v e n t s . S i n c e aqueous s o l u t i o n s , w h i c h c a n be b u f f e r e d and made i s o t o n i c f o r c e l l s , w i l l form two p h a s e s y s t e m s , p a r t i t i o n i n g i n two p h a s e aqueous p o l y m e r s y s t e m s h a s p o t e n t i a l f o r s e p a r a t i n g p a r t i c l e s and m a c r o m o l e c u l e s of b i o l o g i c a l o r i g i n . I n a d d i t i o n many p o l y m e r s do n o t have a d e l e t e r i o u s e f f e c t on b i o l o g i c a l m a t e r i a l s ; i n f a c t p r o t e c t i v e e f f e c t s have been o b s e r v e d . U s i n g F l o r y - H u g g i n s t h e o r y , i t i s e a s y t o d e r i v e an e x p r e s s i o n f o r t h e p a r t i t i o n c o e f f i c i e n t o f a t h i r d p o l y m e r a d d e d a t low c o n c e n t r a t i o n t o a two p o l y m e r p h a s e s y s t e m f r o m eqn [7] ( 1 4 ) . A s s u m i n g X12 = X 1 3 = Xi«, t h e n t h e c h e m i c a l p o t e n t i a l o f t h e t h i r d p o l y m e r c a n be c a l c u l a t e d f r o m , ( M « - M 2 ) A T = 1 + l n f l > f t + P « C-0,+02 ( X 2 « - 1 / P 2 ) + « 3 ( X 3 < . - 1 / P 3 ) + X I 2 ( 0 i - 0 i 0 2 - 0 i 0 3 ) - X 2 3 0 2 0 3 } eqn [9] where ul r e f e r s t o t h e s t a n d a r d s t a t e c h e m i c a l p o t e n t i a l o f p u r e p o l y m e r b e f o r e m i x i n g w i t h 1,2 o r 3. I f t h e above e q u a t i o n f o r e a c h p h a s e i s e q u a t e d , s i n c e a t e q u i l i b r i u m t h e s o l u t e s i n e a c h p h a s e must have t h e same c h e m i c a l p o t e n t i a l , and s e c o n d o r d e r t e r m s a r e d r o p p e d t h e n , 10 K „ = e x p PflCUT-01?) ( 1 - X i • ) + («T-*?> (1/P2-X2<.) + ( 0 T - 0 B 3 ) ( 1 / P 3 - X 3 i . ) } eqn [10] T B where Kft = 0 „/0 „, t h e p a r t i t i o n c o e f f i c i e n t o f 4 and T/B d e n o t e t o p / b o t t o m p h a s e r e s p e c t i v e l y . T h i s e x p r e s s i o n i s s u p p o r t e d e x p e r i m e n t a l l y by s e v e r a l o b s e r v a t i o n s . The p a r t i t i o n c o e f f i c i e n t w i l l become more one s i d e d , t h e l a r g e r t h e m o l e c u l a r w e i g h t o f t h e p a r t i t i o n e d component o r t h e g r e a t e r t h e d i f f e r e n c e i n p o l y m e r c o n c e n t r a t i o n between t h e two p h a s e s . An i m p o r t a n t p o i n t i s t h a t t h e p a r t i t i o n of a s o l u t e i s d e t e r m i n e d by t h e i n t e r a c t i o n e n e r g i e s o f t h e p a r t i t i o n e d s o l u t e a n d t h e phase p o l y m e r s . M o r e o v e r , a s t h e p a r t i t i o n depends e x p o n e n t i a l l y on t h e s e p r o p e r t i e s t h e n p a r t i t i o n i n g would be e x p e c t e d t o be a v e r y s e n s i t i v e s e p a r a t i o n p r o c e d u r e . 4. THE THEORY OF ELECTROLYTE PARTITION P o l y m e r phase s y s t e m s need s m a l l amounts o f s a l t s o r s u c r o s e t o a c t a s a s u i t a b l e e n v i r o n m e n t f o r c e l l s . Systems c o n t a i n i n g n e u t r a l p o l y m e r s a r e l i t t l e a f f e c t e d by t h e a d d i t i o n o f most s a l t s , e g . a d e x t r a n - P E G s y s t e m has a c o n s t a n t volume r a t i o up t o 0.8M N a C l and 0.3M p h o s p h a t e but no t a c o n s t a n t c o m p o s i t i o n ( F i g . 2 . ) . The p a r t i t i o n i n g o f c e l l s , however, can be g r e a t l y a f f e c t e d by s m a l l amounts of s a l t s i f t h e s a l t s p a r t i t i o n u n e q u a l l y s i n c e i n s u c h c a s e s a p o t e n t i a l d i f f e r e n c e o c c u r s between t h e p h a s e s . The p o t e n t i a l d i f f e r e n c e between p h a s e s w i l l depend upon t h e d i f f e r e n c e i n p a r t i t i o n o f t h e a n i o n o r c a t i o n , F i g . 2 . Phase D i a g r a m s f o r D e x t r a n T500/PEG 8 0 0 0 / b u f f e r s y s t e m s . T = 2 2 ° C , pH=7.16. a) B u f f e r c o m p o s i t i o n 1OmM phosphate,130mM N a C l . b) B u f f e r c o m p o s i t i o n 11OmM p h o s p h a t e . A l l p o l y m e r c o m p o s i t i o n s i n %w/w ( 2 6 ) . 12 a c c o r d i n g t o t h e e q u a t i o n ( 1 5 ) : Ai// = [ R T / ( z + + z . ) F ] [ ( A M ? - A M + ° ) / R T + l n r . / r j eqn [11] T B where &\p = uV - i / / , t h e p o t e n t i a l d i f f e r e n c e between t h e p h a s e s z + / z . = n e t t c h a r g e o f c a t i o n / a n i o n F = F a r a d a y c o n s t a n t A M ? / A M ° = t h e d i f f e r e n c e i n s t a n d a r d s t a t e c h e m i c a l p o t e n t i a l between p h a s e s f o r a n i o n / c a t i o n r + or r . = f T / f ? o r f T / f B f + / f . = a c t i v i t y c o e f f i c i e n t s f o r a n i o n / c a t i o n . The p o t e n t i a l d i f f e r e n c e between t h e p h a s e s w i l l be d e t e r m i n e d p r i m a r i l y by t h e d e g r e e t o w h i c h t h e c a t i o n and a n i o n d i f f e r i n t h e i r i n t r i n s i c i n t e r a c t i o n s w i t h t h e p h a s e s a s d e s c r i b e d by d i f f e r e n c e s i n t h e i r s t a n d a r d s t a t e c h e m i c a l p o t e n t i a l s . However, f + o r f . c a n n o t be i n d e p e n d a n t l y m e a s u r e d . The p r e d i c t e d d e c r e a s e i n p o t e n t i a l d i f f e r e n c e between p h a s e s w i t h i n c r e a s e d c h a r g e p e r i o n i s n o t o b s e r v e d e x p e r i m e n t a l l y , p o s s i b l y due t o h i g h e r c h a r g e d i o n s i n t e r a c t i n g more s t r o n g l y w i t h t h e p hase p o l y m e r s , i n c r e a s i n g t h e m a g n i t u d e o f t h e Ajz° t e r m . A h i g h l y c h a r g e d m o l e c u l e s u c h as a p r o t e i n c a n have a s i g n i f i c a n t e f f e c t on t h e e l e c t r o s t a t i c p o t e n t i a l between p h a s e s . When t h e s a l t i s s u f f i c i e n t l y i n e x c e s s , t h e p o t e n t i a l i s d e t e r m i n e d by t h e e l e c t r o l y t e i n t e r a c t i o n s w i t h t h e p h a s e s but when t h e p r o t e i n i s i n e x c e s s , t h e p o t e n t i a l i s d e t e r m i n e d by t h e p r o t e i n i n t e r a c t i o n w i t h t h e p h a s e p o l y m e r s and t h e c a t i o n - p o l y m e r i n t e r a c t i o n s , e x p r e s s e d as f o l l o w s ( 1 4 ) , 13 4 0 = [ R T / O + z ) F ] [ ( A M ° ~ A M ? ) / R T + l n r / l n r + r Er tr + ( C ? / z C*) ( 1 - K . / K ) eqn [12] where p d e n o t e s p o l y m e r and T / B d e n o t e t o p / b o t t o m phase K. = cT/cB 1 i ' I I t i s e a s y t o a l t e r z by a l t e r i n g pH o r A \ / / by a l t e r i n g s a l t Er t y p e or i o n i c s t r e n g t h . 5. THE APPLICATIONS OF AFFINITY PARTITIONING T h e r e h as been some i n v e s t i g a t i o n i n t o t h e p o s s i b i l i t y o f i n c r e a s i n g t h e p a r t i t i o n o f a p a r t i c u l a r component o f a m i x t u r e w i t h an a f f i n i t y l i g a n d . An a f f i n i t y l i g a n d w i l l p a r t i t i o n p r e d o m i n a n t l y i n t o one o f t h e p h a s e s w h i l e bound t o one o f t h e components of a m i x t u r e , t h u s i n c r e a s i n g t h e p a r t i t i o n o f t h a t p a r t i c u l a r component. A s i m p l e example of an a f f i n i t y l i g a n d i s t h e e s t e r p r o d u c e d by c o n j u g a t i n g p a l m i t i c a c i d t o one o f t h e t e r m i n a l h y d r o x y l s o f PEG 8000. S m a l l amounts of t h i s e s t e r i n t h e phase s y s t e m i n c r e a s e t h e p a r t i t i o n o f e r y t h r o c y t e s i n t o t h e PEG r i c h p h a s e o f a d e x t r a n / P E G s y s t e m ( 1 6 , 1 7 ) . O t h e r examples o f a f f i n i t y l i g a n d s i n c l u d e P E G - t r i e t h y l a m i n e w h i c h a c t s a s a b i o s p e c i f i c l i g a n d u s e d t o p u r i f y c h o l i n e r g i c r e c e p t o r s s p e c i f i c f o r p a r t i c u l a r n e u r o t r a n s m i t t e r s ( 1 8 ) . Staphyl occus aureus has been m o d i f i e d w i t h PEG f o r use as an a f f i n i t y l i g a n d t o s e p a r a t e anti-S*a p h y l o c c u s aureus a n t i b o d i e s ( 1 9 ) . In t h i s c a s e an optimum d e g r e e o f m o d i f i c a t i o n e x i s t e d between i n c r e a s i n g t h e p a r t i t i o n o f t h e Staphyloccus aureus and l o s s o f a n t i b o d y b i n d i n g s i t e s due 1 4 t o s t e r i c i n t e r a c t i o n s w i t h t h e bound PEG. The p o s s i b i l i t y of u s i n g b i o s p e c i f i c a f f i n i t y l i g a n d s t o s e p a r a t e c e l l s on t h e b a s i s of c e l l s u r f a c e a n t i g e n s has been i n v e s t i g a t e d ( 2 0 ) . A model s y s t e m was u s e d t o s e l e c t i v e l y p a r t i t i o n human e r y t h r o c y t e s f r o m a m i x t u r e w i t h r a b b i t e r y t h r o c y t e s u s i n g a P E G - m o d i f i e d a n t i b o d y i n a d e x t r a n / P E G s y s t e m . The i n c r e a s e d p a r t i t i o n o f t h e human e r y t h r o c y t e s c o u l d be i n h i b i t e d by n a t i v e , u n m o d i f i e d a n t i b o d y , d e m o n s t r a t i n g t h a t t h e P E G - m o d i f i e d a n t i b o d y was, i n f a c t , an a f f i n i t y l i g a n d . I t was a l s o o b s e r v e d t h a t d e s p i t e a s i g n i f i c a n t d e c r e a s e i n t h e a b i l i t y of t h e a n t i b o d y t o a g g l u t i n a t e e r y t h r o c y t e s , u s u a l l y an i n d i c a t i o n o f a d e c r e a s e i n b i n d i n g a c t i v i t y , i t was s t i l l an e f f e c t i v e a f f i n i t y l i g a n d . I t was p o s t u l a t e d t h a t t h e h a e m a g g l u t i n a t i n g a c t i v i t y o f t h e a n t i b o d y was more s e n s i t i v e t o P E G - m o d i f i c a t i o n t h a n t h e b i n d i n g a b i l i t y , i . e . t h e h a e m a g g l u t i n a t i n g a c t i v i t y may n o t g i v e a good i n d i c a t i o n o f t h e b i n d i n g a c t i v i t y o f t h e P E G - m o d i f i e d p r o t e i n . S y n t h e s i s o f p r o t e i n - P E G c o n j u g a t e s i s an a c t i v e a r e a o f r e s e a r c h and a l t h o u g h a f f i n i t y p a r t i t i o n i n g i s n o t t h e most common use of t h e c o n j u g a t e s , work done on o t h e r u s e s o f t h e c o n j u g a t e s has r e s u l t e d i n a s u b s t a n t i a l i n c r e a s e i n t h e m e t h o d o l o g y of PEG m o d i f i c a t i o n o f p r o t e i n s . F o r example, P E G - m o d i f i e d a n t i g e n s a d m i n i s t e r e d t o a n i m a l s have r e s u l t e d i n i m m u n o s u p r e s s i o n ( 2 1 ) . A t t a c h i n g PEG t o enzymes p r o d u c e s a d e c r e a s e i n enzyme a n t i g e n i c i t y and 1 5 i m m u n o g e n i c i t y r e s u l t i n g i n an i n c r e a s e d serum l i f e t i m e w h i c h a l l o w s t h e c o n j u g a t e s t o be u s e d as d r u g s ( 2 2 , 2 3 ) . A d v a n t a g e o f t h e i n c r e a s e d serum l i f e t i m e e f f e c t i s a l s o t a k e n i n m o d i f y i n g h a e m o g l o b i n f o r use as a b l o o d s u b s t i t u t e . 6. THE THEORY OF AFFINITY PARTITION The p r e v i o u s d i s c u s s i o n s have c e n t r e d a r o u n d m o l e c u l a r p a r t i t i o n as d e t e r m i n e d by t h e c o m p a t i b i l i t y o f t h e p a r t i t i o n e d m a t e r i a l w i t h t h e p h a s e p o l y m e r s and t h e e l e c t r o s t a t i c p o t e n t i a l d i f f e r e n c e between p h a s e s . A f f i n i t y p a r t i t i o n i n g i s a n o t h e r method o f m a n i p u l a t i n g t h e c h e m i c a l p o t e n t i a l s o f t h e s o l u t e i n e a c h p h a s e . I f a l i g a n d w i t h a c e r t a i n p a r t i t i o n c o e f f i c i e n t a s s o c i a t e s s t r o n g l y w i t h a n o t h e r m o l e c u l e w i t h a d i f f e r e n t p a r t i t i o n c o e f f i c i e n t , t h e n t h e b i n d i n g w i l l change t h e p a r t i t i o n c o e f f i c i e n t o f t h e c o m p l e x . The b i n d i n g o f a l i g a n d t o a m a c r o m o l e c u l e w i t h n e q u i v a l e n t i n d e p e n d e n t b i n d i n g s i t e s , i . e . t h e n m i c r o s c o p i c e q u i l i b r i u m c o n s t a n t s a r e a l l i d e n t i c a l , can be c o n s i d e r e d i n t e r m s o f t h e s e q u e n t i a l a d d i t i o n o f t h e l i g a n d t o t h e b i n d i n g s i t e s . I f e q u a l s t h e c o n c e n t r a t i o n o f m a c r o m o l e c u l e s w i t h i s i t e s o c c u p i e d , t h e n t h e t o t a l c o n c e n t r a t i o n o f t h e m a c r o m o l e c u l e , Mfc ^ i s t h e sum o f a l l t h e s p e c i e s o f M., 1 n M. . = IM. t o t i = Q i 16 The p a r t i t i o n c o e f f i c i e n t o f t h e m a c r o m o l e c u l e i n t h e p r e s e n c e of l i g a n d , K m i s m t o t t o t I f t h e b i n o m i a l t h e o r y i s a p p l i e d t o t h e e q u a t i o n f o r M t Q t t h e n , n L M. = M 0(1+K L ) n i = 0 1 a T h e r e f o r e , t h e p a r t i t i o n c o e f f i c i e n t a t any s t a g e o f b i n d i n g i s g i v e n by, Km = K o ( _ 1 ^ V ( l + K B L B ) n a eqn [13] T B where K 0 = M 0/M 0, t h e p a r t i t i o n c o e f f i c i e n t o f u n o c c u p i e d m a c r o m o l e c u l e T B K ,K = m i c r o s c o p i c a s s o c i a t i o n c o n s t a n t f o r b i n d i n g of l i g a n d t o a s i t e i n t o p , b o t t o m phase T h i s model p r e d i c t s t h a t an a f f i n i t y l i g a n d w i l l i n c r e a s e t h e p a r t i t i o n o f a bound m o l e c u l e , p r o v i d i n g i t has a p a r t i t i o n c o e f f i c i e n t g r e a t e r t h a n 1. I t c a n n o t be assumed t h a t t h e a s s o c i a t i o n c o n s t a n t s i n upper and l o w e r p h a s e s a r e t h e same, a s K , i n g e n e r a l , w i l l be i n c r e a s e d i n t h e phase i n w h i c h t h e l i g a n d i s a t l o w e r c o n c e n t r a t i o n . When t h e l i g a n d i s bound t o one o f t h e ph a s e p o l y m e r s and t h u s 17 d i r e c t e d i n t o t h e p h a s e i n w h i c h t h a t p o l y m e r p r e d o m i n a t e s , t h e n i t i s i m p l i e d t h a t t h e s t a n d a r d s t a t e c h e m i c a l p o t e n t i a l d i f f e r s i n d i f f e r e n t p h a s e s , t h u s a f f e c t i n g K . 3 T h i s w i l l d e c r e a s e t h e e f f e c t of t h e a f f i n i t y l i g a n d by T B r e d u c i n g t h e r a t i o , K K,/K . The model assumes t h a t t h e l i g a n d i n t e r a c t s r e v e r s i b l y w i t h t h e m a c r o m o l e c u l e , but a f f i n i t y e f f e c t s a r e a l s o o b s e r v e d i f one o f t h e p hase p o l y m e r s i s c o v a l e n t l y l i n k e d T B t o t h e a f f i n i t y l i g a n d . In t h i s c a s e K and K become v e r y 3 3 l a r g e and, K = K 0K, m 1 In f a c t t h e d ependence o f K m on t h e number of l i g a n d b i n d i n g s i t e s m e asured e x p e r i m e n t a l l y i s l e s s t h a n p r e d i c t e d t h e o r e t i c a l l y . T h i s i s p r o b a b l y b e c a u s e t h e bound l i g a n d s do not behave i d e a l l y as assumed t h r o u g h o u t t h e d e r i v a t i o n . A more a c c u r a t e model would t a k e i n t o a c c o u n t an a c t i v i t y c o e f f i c i e n t f o r t h e c o m p l e x e d l i g a n d s w h i c h i n t e r a c t w i t h e a c h o t h e r as t h e amount of bound l i g a n d i n c r e a s e s . 7. THE THEORY OF CELL AND PARTICLE PARTITION The t h e o r y d e v e l o p e d f o r s o l u t e p a r t i t i o n i s not as u s e f u l t o d e s c r i b e c e l l and p a r t i c l e p a r t i t i o n . T h i s i s b e c a u s e a p a r t i c l e o v e r a c e r t a i n s i z e d o e s n o t d i f f u s e f r e e l y between t h e p h a s e s , but t e n d s t o c o l l e c t a t t h e i n t e r f a c e o f t h e s y s t e m where i t has l e a s t f r e e e n e r g y . In t h e c a s e o f a s o l u t e , t h e f r e e e n e r g y o f i n t e r f a c i a l a d s o r p t i o n i s s m a l l compared t o t h a t o f a p a r t i c l e b e c a u s e a 18 p a r t i c l e w i l l d e s t r o y a g r e a t e r a r e a o f t h e i n t e r f a c e on a d s o r p t i o n t h a n a p a r t i c l e , t h u s h a v i n g a g r e a t e r f r e e e n e r g y o f a d s o r p t i o n t h a n a s o l u t e . A t h e o r y c a n be w r i t t e n s t a r t i n g w i t h t h e B o l t z m a n n e q u a t i o n w h i c h r e l a t e s t h e p r o b a b i l i t i e s o f a p a r t i c l e b e i n g i n e i t h e r o f two compartments, d e s i g n a t e d 1 and 2, t o t h e f r e e e n e r g y r e q u i r e d t o move t h e p a r t i c l e between c o m p a r t m e n t s , AE. However, t h e major drawback i s t h e a s s u m p t i o n o f f r e e d i f f u s i o n . K = n , / n 2 = C,/C 2 = e x p ( - A E / k T ) eqn [14] A n o t h e r p r o b l e m w i t h t h e use o f t h e above e q u a t i o n i s t h a t t h e c o n c e p t o f v o l u m e t r i c c o n c e n t r a t i o n of p a r t i c l e s i s d i f f i c u l t t o a p p l y when t h e p a r t i c l e s a r e p a r t i t i o n i n g between t h e i n t e r f a c e and one of t h e p h a s e s . In f a c t numbers of p a r t i c l e s s h o u l d be c o n s i d e r e d ( 2 4 ) . I f a p a r t i c l e i s l o c a t e d a t t h e l i q u i d - l i q u i d i n t e r f a c e , t h e n t h e i n t e r f a c i a l a r e a i s r e d u c e d by t h e c r o s s - s e c t i o n a l a r e a of t h e p a r t i c l e . The d e c r e a s e i n a r e a p r o d u c e s a p r o p o r t i o n a t e d e c r e a s e i n f r e e e n e r g y , t h u s s t a b i l i s i n g p a r t i c l e s a d s o r b e d a t t h e i n t e r f a c e . I f a p a r t i c l e has e q u a l a f f i n i t y f o r b o t h p h a s e s , t h e n a d s o r p t i o n a t t h e i n t e r f a c e w i l l be s i g n i f i c a n t when t h e f r e e e n e r g y a s s o c i a t e d w i t h a d s o r p t i o n i s of t h e o r d e r of t h e a v e r a g e t h e r m a l e n e r g y o f a p a r t i c l e . F o r example, a t y p i c a l s u r f a c e t e n s i o n i n a phase s y s t e m o f 5 x 1 0 " 3 e r g / c m 2 w o u l d a d s o r b p a r t i c l e s w i t h a d i a m e t e r o v e r 320A. The g r e a t e r t h e s u r f a c e t e n s i o n , t h e s m a l l e r t h e p a r t i c l e s a d s o r b e d a t t h e 19 i n t e r f a c e . The work done on moving a p a r t i c l e f r o m t h e i n t e r f a c e i n t o t h e t o p p h a s e i s t h e sum of two components, A E T I = A T B 7 T B - A B A 7 where A T B 7 T B * s a c o n t r i b u t i o n f r o m t h e n e t t e n e r g y change a s s o c i a t e d w i t h t h e i n c r e a s e i n i n t e r f a c i a l s u r f a c e a r e a , and A „ A 7 i s a c o n t r i b u t i o n from t h e e n e r g y r e q u i r e d t o D t r a n s f e r a p o r t i o n o f t h e p a r t i c l e s u r f a c e a r e a , A f i, from b o t t o m p h a s e t o t o p p h a s e . V a l u e s f o r A__ and A_, a r e o b t a i n e d f r o m c o n t a c t a n g l e measurements. The c o n t a c t a n g l e method r e s u l t s i n t h e e x p r e s s i o n f o r p a r t i c l e p a r t i t i o n as f o l l o w s ( 2 5 ) , l n K = - A E T I / k T eqn [15] o r l n K = - 7 T B n a 2 ( l - A 7 / 7 T B ) 2 / k T eqn [16] where a = p a r t i c l e r a d i u s A 7 = d i f f e r e n c e i n p a r t i c l e s u r f a c e f r e e e n e r g y i n t o p and b o t t o m p h a s e . 7,p B = i n t e r f a c i a l t e n s i o n between t o p and b o t t o m p h a s e s When t y p i c a l v a l u e s a r e s u b s t i t u t e d i n t o t h i s e q u a t i o n , i t i s f o u n d t h a t A E ^ j i s v e r y much l a r g e r t h a n kT s u g g e s t i n g t h a t few p a r t i c l e s s h o u l d p a r t i t i o n i n t o t h e u p p e r p h a s e . E x p e r i m e n t a l l y t h i s i s n o t o b s e r v e d . However, q u a l i t a t i v e l y i t w o u l d s t i l l be e x p e c t e d t h a t p a r t i c l e p a r t i t i o n w o u l d d e p e n d on t h e s u r f a c e p r o p e r t i e s o f t h e p a r t i c l e , t h e i r s i z e and a r e a , t h e t e m p e r a t u r e and t h e i n t e r f a c i a l t e n s i o n 20 between t h e p h a s e s . In f a c t an e x p o n e n t i a l dependance of K on A E T I has been o b s e r v e d ( 2 6 ) . As i n d i c a t e d above t h e i n t e r f a c i a l t e n s i o n i s e x t r e m e l y i m p o r t a n t i n p a r t i c l e p a r t i t i o n . To m a x i m i s e t h e e f f e c t o f t h e a f f i n i t y l i g a n d , a s y s t e m w i t h a low i n t e r f a c i a l t e n s i o n , i n t h e o r d e r o f 4x10"* e r g / c m 2 , was u s e d t h r o u g h o u t t h i s s t u d y . However, a s t h e i n t e r f a c i a l t e n s i o n v a r i e s w i t h t i e l i n e l e n g t h ( 7 T B = T L L a , where a = 3.4 t o 4.2 i n d i f f e r e n t s y s t e m s ) , t h i s meant t h a t t h e s y s t e m was c l o s e t o t h e c r i t i c a l p o i n t , making i t a d i f f i c u l t s y s t e m t o m a n i p u l a t e . The s y s t e m was a f f e c t e d g r e a t l y by s m a l l n o n - s y s t e m a t i c e r r o r s s u c h a s t h e p r e c i s e s a l t c o n c e n t r a t i o n , t h e r e f o r e a l l e x p e r i m e n t s were ru n s i m u l t a n e o u s l y w i t h c o n t r o l s . F o r example, N a C l has l i t t l e e f f e c t on e r y t h r o c y t e p a r t i t i o n but p h o s p h a t e has a l a r g e e f f e c t , e s p e c i a l l y i n s y s t e m s c l o s e t o t h e c r i t i c a l p o i n t a s u s e d i n t h i s c a s e . T h i s i s a c o n s e q u e n c e o f b o t h t h e i n c r e a s e d t i e l i n e l e n g t h (and t h e r e f o r e i n c r e a s e d i n t e r f a c i a l t e n s i o n ) and t h e e l e c t r o s t a t i c p o t e n t i a l between p h a s e s t h a t p h o s p h a t e p r o d u c e s . A n o t h e r p r o b l e m r e s u l t i n g f r o m t h e c l o s e n e s s of t h e s y s t e m t o t h e c r i t i c a l c o m p o s i t i o n was t h a t a s m a l l change r e s u l t e d i n t h e f o r m a t i o n o f a one phase s y s t e m . I f a d d i t i o n o f t h e a f f i n i t y l i g a n d formed a one p h a s e s y s t e m , t h e a d d i t i o n o f a s m a l l amount of PEG 8000 r e f o r m e d t h e p h a s e s . 21 8. THE THEORY OF MONOCLONAL ANTIBODIES In t h i s s t u d y , i t was aimed t o p r o d u c e an a f f i n i t y l i g a n d w h i c h combined t h e s p e c i f i c i t y o f a n t i b o d i e s w i t h t h e p o t e n t i a l t o be a p p l i e d t o t h e s e p a r a t i o n o f many d i f f e r e n t c e l l t y p e s . The a p p r o a c h t a k e n n e c e s s i t a t e d t h e use o f a m o n o c l o n a l a n t i b o d y , t h e t h e o r y o f w h i c h f o l l o w s . The m a j o r i t y o f t h e e x p o s e d s u r f a c e o f a p r o t e i n i s a n t i g e n i c (27,28) and a t y p i c a l immune r e s p o n s e w i l l r e s u l t i n h u n d r e d s o r t h o u s a n d s o f c l o n e s o f a n t i b o d y - p r o d u c i n g l y m p h o c y t e s r e c o g n i z i n g many d i f f e r e n t a n t i g e n i c d e t e r m i n a n t s . Of p a r t i c u l a r i n t e r e s t h e r e a r e a n t i b o d i e s r a i s e d a g a i n s t human e r y t h r o c y t e s of M o r N s p e c i f i c i t i e s . The a n t i g e n s o f t h e MN b l o o d g r o u p s y s t e m a r e c a r r i e d by t h e e r y t h r o c y t e membrane s i a l o g l y c o p r o t e i n , g l y c o p h o r i n A. E r y t h r o c y t e s f r o m homozygotes c a r r y m o l e c u l e s o f MM o r NN s p e c i f i c i t y w h i l e h e t e r o z y g o t e s have b o t h m o l e c u l e s i n e q u a l amounts ( 2 9 ) . The M and N s p e c i f i c i t i e s seem t o be due t o d i f f e r e n t t e r m i n a l amino a c i d s e q u e n c e s ( 3 0 , 3 1 ) . I m m u n i s a t i o n w i t h t h e M or N a n t i g e n w i l l r e s u l t i n many c l o n e s of a n t i b o d y - p r o d u c i n g l y m p h o c y t e s , some r e c o g n i z i n g a n t i g e n i c d e t e r m i n a n t s s p e c i f i c t o M o r N e r y t h r o c y t e s b u t o t h e r s r e c o g n i z i n g a n t i g e n i c d e t e r m i n a n t s common t o M and N g l y c o p h o r i n A, i . e . a p o l y c l o n a l r e s p o n s e . D e s p i t e t h e d i v e r s i t y o f t h e o v e r a l l immune r e s p o n s e , e a c h i n d i v i d u a l c l o n e of l y m p h o c y t e s i s c o m m i t t e d t o s e c r e t i n g one a n t i b o d y t y p e , f o r i n s t a n c e IgG, s p e c i f i c f o r one a n t i g e n i c d e t e r m i n a n t a s d e s c r i b e d by t h e c l o n a l 22 selection theory (Fig.3). Hybridoma technology uses c e l l culture techniques to propagate and select individual clones of lymphocytes to provide an "immortal" supply of antibody of the selected s p e c i f i c i t y . The demonstration that i t was possible to fuse two d i f f e r e n t plasma c e l l tumour l i n e s with retention of both antibody products (32) was the s t a r t of hybridoma technology. However, prior to t h i s , important relevant discoveries included, proof of the clonal selection theory (33), development of c e l l fusion techniques (34,35), the a r t i f i c i a l induction of myelomas (36) and their adaptation to tissue culture (37). Myeloma or plasmacytoma c e l l s a r i s e from a tumor of malignant, antibody producing, plasma c e l l s . The Structure of Immunoglobulin G Immunoglobulin G (IgG) i s a symmetrical molecule made up of two i d e n t i c a l glycosylated heavy chains (Mf 50,000 -75,000) and two i d e n t i c a l non-glycosylated l i g h t chains (M r approx. 25,000) as shown in F i g . 4. The heavy chains are joined by two disulphide bonds to each other in the non-variable or F c region of the molecule. Each l i g h t chain is joined by disulphide bonds to one of the heavy chains. Enzymic digestion by papain s p l i t s the molecule into one F c and two F ^ fragments. The F ^ fragment consists of one l i g h t chain and part of a heavy chain, i t includes the F v or variable region of the molecule which binds the antigen and i s d i f f e r e n t in IgG molecules with d i f f e r e n t A Clone of Lymphocytes 1 1 1 1 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Antibodies F i g . 3 . The C l o n a l S e l e c t i o n T h e o r y . I n t e r a c t i o n o f a n t i g e n ( A ) w i t h t h e r e c e p t o r i m m u n o g l o b u l i n s on t h e s u r f a c e o f c l o n e 2 l e a d s t o p r o l i f e r a t e d d i f f e r e n t i a t i o n i n t o c e l l s w h i c h s e c r e t e a n t i b o d y of t h e same s p e c i f i c i t y a s t h e r e c e p t o r . 24 LIGHT CHAIN HEAVY CHAIN PAPAIN CLEAVAGE MEMBRANE E X T E N S I O N F i g . 4 . S t r u c t u r e o f t h e IgG M o l e c u l e . R e d u c t i o n of t h e d i s u l p h i d e c r o s s l i n k s w i l l s p l i t t h e m o l e c u l e i n t o h eavy and l i g h t c h a i n s . The enzyme p a p a i n w i l l c l e a v e t h e m o l e c u l e i n t o F ^ and F c f r a g m e n t s a s i n d i c a t e d d u r i n g l i m i t e d d i g e s t i o n . 25 s p e c i f i c i t i e s ( 3 8 ) . The F c f r a g m e n t c o n s i s t s of o n l y heavy c h a i n and i s c o n s t a n t i r r e s p e c t i v e o f t h e s p e c i f i c i t y o f t h e IgG m o l e c u l e a s i t i s n o t i n v o l v e d w i t h a n t i g e n b i n d i n g . A l t h o u g h t h e F c f r a g m e n t l a c k s t h e a b i l i t y t o b i n d a n t i g e n , i t p o s s e s s e s o t h e r b i o l o g i c a l l y a c t i v e s i t e s , s u c h as t h o s e c o n c e r n e d w i t h complement f i x a t i o n , membrane t r a n s m i s s i o n , macrophage f i x a t i o n and c a t a b o l i c r e g u l a t i o n . The p a p a i n d i g e s t i o n o f IgG p r o d u c e s f r a g m e n t s w i t h r e t e n t i o n of b i o l o g i c a l a c t i v i t y making i t p r e f e r a b l e o v e r c h e m i c a l d e g r a d a t i o n . However p r o l o n g e d e x p o s u r e l e a d s t o a r ange o f s u b - f r a g m e n t s . S o u r c e o f t h e Myeloma C e l l s A c e l l l i n e w h i c h s e c r e t e s m o n o c l o n a l a n t i b o d i e s i s t h e r e s u l t o f t h e f u s i o n o f a myeloma o r p l a s m a c y t o m a c e l l a n d a s p l e e n c e l l t o p r o d u c e a h y b r i d o m a . A s c h e m a t i c d i a g r a m o f t h e p r o c e d u r e t o p r o d u c e h y b r i d o m a s a s d e s c r i b e d i n t h e f o l l o w i n g p a g e s i s g i v e n i n F i g . 5. M i n e r a l o i l o r 2 , 4 , 6 , 1 O - t e t r a m e t h y l p e n t a d e c a n e ( p r i s t a n e ) a r e p o t e n t i n d u c e r s o f myeloma i n BALB/c m i c e . S e v e r a l o f t h e s e myelomas have been a d a p t e d t o c o n t i n u o u s t i s s u e c u l t u r e l i n e s (39) u s e d t o f u s e w i t h s p l e e n c e l l s and t h u s g e n e r a t e h y b r i d o m a s . The most commonly u s e d myeloma c e l l l i n e i s d e s i g n a t e d P3-NS1 Ag4-1 ( N S 1 ) . T h i s c e l l l i n e s y n t h e s i z e s some IgG l i g h t c h a i n s but t h e s e a r e b r o k e n down i n t e r n a l l y and n o t s e c r e t e d . The i m p l i c a t i o n o f t h i s i s t h a t t h e gene f o r l i g h t c h a i n s y n t h e s i s i s p r e s e n t i n NS1 c e l l s and may be e x p r e s s e d i n t h e r e s u l t i n g h y b r i d o m a . 26 Another useful outcome of well characterised mouse myelomas has been an increased knowledge of immunoglobulin structure, biosynthesis and genetics (40). Spleen c e l l s used in the fusion are from immunised mice; these provide the genes coding for IgG synthesis of the selected s p e c i f i c i t y . , Fusion of C e l l s Aqueous solutions of PEG w i l l fuse almost a l l c e l l types at a s u f f i c i e n t l y high concentration. The fusing concentration of PEG is usually above 30% w/w and the optimum concentration i s determined by the balance between increasing fusion e f f i c i e n c y and minimising c e l l damage. The concentration dependence seems to be related to the a b i l i t y of the PEG to decrease the water a c t i v i t y of the solution since solutions inducing maximum fusion have v i r t u a l l y no water unassociated with the polymer (41). Many other polymers and viruses, in p a r t i c u l a r , Sendai virus which was used in early hybridoma work (42) w i l l induce c e l l fusion. When spleen and myeloma c e l l s are treated with a fusogen, such as PEG 3500, the i r membranes fuse and multinucleate c e l l s or heterokaryons are formed (42). At the next c e l l d i v i s i o n the nuclei of the heterokaryons fuse and the c e l l then divides to produce daughter c e l l s with an equal share of the genetic material and double the number of chromosomes of the parent c e l l s . 27 Immune mouse i Spleen Normal antibody-forming c e l l s o O O O O O O O O O + Plasmacytoma c e l l s Polyethylene g l y c o l J Unfused plasmacytoma c e l l s Heterokaryons Unfused spleen c e l l s 8806688 o o o o o Hat medium Die i n hat medium Hybrids © © O 0) Die i n culture Clone by l i m i t i n g d i l u t i o n 7 7 \ \ Clone 1 Clone 2 o^o aJa Clone 3 Clone 4 F i g . 5 . P r o d u c t i o n o f H y b r i d o m a s ( s e e n e x t page f o r l e g e n d ) . 28 F i g . 5. ( p r e v i o u s page) P r o d u c t i o n of h y b r i d o m a s . S p l e e n c e l l s from immune mice are. f u s e d w i t h myeloma ( p l a s m a c y t o m a ) c e l l s l a c k i n g i n HGPRT u s i n g PEG 3500. The b i n u c l e a t e f u s i o n p r o d u c t s a r e c a l l e d h e t e r o k a r y o n s . A t t h e n e x t c e l l d i v i s i o n , t h e n u c l e i f u s e g e n e r a t i n g h y b r i d c e l l s w h i c h grow i n HAT medium a s t h e genes f o r HGPRT s y n t h e s i s have been s u p p l i e d by t h e s p l e e n c e l l h a l f o f t h e f u s i o n . U n f u s e d myeloma c e l l s d i e i n HAT medium, and u n f u s e d s p l e e n c e l l s w i l l o n l y s u r v i v e a few d a y s i n c u l t u r e . H y b r i d s a r e s e l e c t e d f o r t h e d e s i r e d s p e c i f i c i t y and r e c l o n e d by l i m i t i n g d i l u t i o n . F u s i o n i s a p o o r l y c o n t r o l l e d e v e n t and i n a d d i t i o n t o t h e r e q u i r e d s p l e e n - m y e l o m a f u s i o n , t h e r e w i l l be s p l e e n - s p l e e n and myeloma-myeloma c e l l f u s i o n s o r m u l t i p l e s of t h e s e . A l t h o u g h s p l e e n c e l l s w i l l d i e w i t h i n a few d a y s i n c u l t u r e , t h e myeloma c e l l s w i l l c o n t i n u e t o m u l t i p l y a n d must be d e s t r o y e d i n o r d e r t o c u l t u r e a l o n g t e r m h y b r i d o m a l i n e . S e l e c t i o n o f h y b r i d o m a c e l l s i s u s u a l l y by t h e HAT p r o c e d u r e . HAT S e l e c t i o n T h i s i s a method w h i c h s e l e c t s h y b r i d o m a s by c u l t u r i n g i n a medium w h i c h o n l y s u p p o r t s h y b r i d o m a g r o w t h and not myeloma g r o w t h . I t depends on t h e f a c t t h a t t h e main b i o s y n t h e t i c pathway t o g u a n o s i n e i s b l o c k e d by a m i n o p t e r i n , a f o l i c a c i d a n t a g o n i s t . T h e r e i s an a l t e r n a t i v e " s a l v a g e 29 5-AMINO-IMIDAZOLE-4-CARBOXY RIBONUCLEOTIDE 5-FORMIDO-IMIDAZOLE-4-CARBOXAMIDE RIBONUCLEOTIDE PRPP PP HYPOXANTHINE INOSINE MONOPHOSPHATE Hypoxanthine Guanine Phosphoribosyl Transferase (HGPRT) GUANINE (or 6-THIOGUANINE) GUANOSINE MONOPHOSPHATE (GMP) PRPP PP GDP -•dGDP THYMIDINE Thymidine Kinase RNA* GTP dGTP IP «* dTDP Thymidylate Synthetase •dTTP —**DNA dCTP dATP UDP •dUDP »"dUMP F i g . 6 . M e t a b o l i c pathways r e l e v a n t t o h y d r i d o m a s e l e c t i o n i n HAT medium. C e l l s l a c k i n g HGPRT a r e s e l e c t e d by g r o w t h i n medium c o n t a i n i n g t o x i c g u a n i n e a n a l o g u e s , 6 - t h i o g u a n i n e o r 8 - a z o g u a n i n e 30 pathway" i n w h i c h h y p o x a n t h i n e o r g u a n i n e a r e c o n v e r t e d t o g u a n i n e monophosphate v i a t h e enzyme h y p o x a n t h i n e g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e (HGPRT) as shown i n F i g . 6. Myeloma c e l l s l a c k i n g i n HGPRT a r e c h e c k e d p r i o r t o f u s i o n by g r o w t h i n HAT medium, w h i c h c o n t a i n s h y p o x a n t h i n e , a m i n o p t e r i n and t h y m i d i n e . C e l l l i n e s w h i c h d i e i n t h i s medium a r e u n a b l e t o c o n v e r t h y p o x a n t h i n e o r g u a n i n e i n t o g u a n o s i n e s i n c e t h e y l a c k HGPRT. A f t e r t h e s e myeloma c e l l s l a c k i n g i n HGPRT a r e f u s e d w i t h s p l e e n c e l l s w h i c h s y n t h e s i z e HGPRT, t h e h y b r i d o m a c a n s y n t h e s i z e HGPRT and t h u s s u r v i v e i n HAT medium b u t u n f u s e d myeloma c e l l s w i l l d i e . T hymocyte F e e d e r C e l l s L y mphoid c e l l s o f t e n grow p o o r l y or d i e a t low c e l l p o p u l a t i o n d e n s i t y . The r e a s o n s a r e not w e l l u n d e r s t o o d but may be due t o t o x i n s f r o m t i s s u e c u l t u r e v e s s e l s o r r e q u i r e m e n t s f o r i l l d e f i n e d g r o w t h f a c t o r s . T h e s e p r o b l e m s c a n be p a r t i a l l y overcome by c u l t u r i n g t h e l y m p h o i d c e l l s t o g e t h e r w i t h slow o r n o n - g r o w i n g c e l l s t e r m e d f e e d e r c e l l s . Commonly u s e d f e e d e r c e l l s i n c l u d e t h y m o c y t e s ( 4 3 ) , normal s p l e e n c e l l s (44) o r p e r i t o n e a l c e l l s ( 4 5 ) . The f e e d e r c e l l s do n o t have t o be h i s t o c o m p a t i b l e w i t h t h e h y b r i d o m a s so t h y m o c y t e s from c h e a p e r S w i s s mice were u s e d i n t h i s s t u d y . R e c l o n i n g o f H y b r i d o m a s 31 O f t e n s e v e r a l c e l l s grow i n t h e same w e l l o f t h e c e l l c u l t u r e p l a t e . F o r an a n t i b o d y t o be m o n o c l o n a l i t must d e s c e n d f r o m a s i n g l e s p l e e n - m y e l o m a c e l l f u s i o n . R e c l o n i n g e n s u r e s t h a t t h e a n t i b o d y i s , i n f a c t , m o n o c l o n a l by d i l u t i n g and r e p l a t i n g t h e h y b r i d o m a s s u c h t h a t e a c h w e l l c o n t a i n s a s i n g l e c e l l . E v e n s i n g l e c l o n e s s h o u l d be r e c l o n e d a t l e a s t t w i c e t o e n s u r e g e n e t i c s t a b i l i t y . A h y b r i d o m a c e l l has a h i g h p r o b a b i l i t y o f s p o n t a n e o u s chromosome l o s s a s s o c i a t e d w i t h a l o s s o f a n t i b o d y p r o d u c i n g a b i l i t y . A f t e r r e c l o n i n g , t h e r a t e o f chromosome l o s s i s s m a l l b ut o v e r g r o w t h by a n o n - p r o d u c i n g mutant i s a l w a y s a p o t e n t i a l h a z a r d . The most common method of r e c l o n i n g i s by l i m i t i n g d i l u t i o n ( 4 6 ) . I f c e l l s a r e grown i n s m a l l numbers, t h e f r a c t i o n of w e l l s i n t h e c e l l c u l t u r e p l a t e c o n t a i n i n g g r o w t h s h o u l d f o l l o w t h e P o i s s o n d i s t r i b u t i o n , F 0 = e where F 0 i s t h e f r a c t i o n of c e l l s w i t h no g r o w t h and X i s t h e a v e r a g e number o f c l o n e s p e r w e l l . I f X = 1 t h e n F 0 = 0.37, t h i s means t h a t t o o b t a i n 0.95 p r o b a b i l i t y t h a t w e l l s w i t h g r o w t h a r e m o n o c l o n a l , a t l e a s t 37% of t h e w e l l s s h o u l d have no c l o n e g r o w t h . The T y p e s o f A n t i b o d y S e c r e t e d by t h e Hybridoma A l t h o u g h o n l y 1% o f s p l e e n c e l l s a c t i v e l y s e c r e t e a n t i b o d y , a b o u t 10% of h y b r i d o m a s s e c r e t e a n t i b o d y . T h e r e seems t o be a p r e f e r e n c e f o r myeloma c e l l s t o f u s e w i t h 32 a c t i v a t e d B c e l l s . An a c t i v a t e d B c e l l i s a l y m p h o c y t e w h i c h has been s t i m u l a t e d t o p r o d u c e a n t i b o d y ( 4 7 ) . A n o t h e r p o s s i b i l i t y i s t h a t myeloma c e l l s may a c t i v a t e n o n - s e c r e t i n g B c e l l s t o r a p i d s e c r e t i o n ( 4 8 ) . The o r i g i n a l myeloma c e l l l i n e , MOPC-21, u s e d by K o h l e r and M i l s t e i n (49) s e c r e t e d IgG, w i t h K l i g h t c h a i n s . T h i s meant t h a t t h e h y b r i d o m a s e c r e t e d mixed m o l e c u l e s made up fr o m v a r i o u s c o m b i n a t i o n s o f t h e two heavy a n d l i g h t c h a i n s ( s e e F i g . 4 f o r d e t a i l s of c h a i n s ) . I f a s s o c i a t i o n o f t h e c h a i n s were random, o n l y a s m a l l m i n o r i t y of IgG m o l e c u l e s would o r i g i n a t e e n t i r e l y f r o m t h e s p l e e n c e l l s . A b i n d i n g s i t e made up o f myeloma heavy o r l i g h t c h a i n ( o r b o t h ) would no t be e x p e c t e d t o b i n d a n t i g e n . T h i s means t h a t many s e c r e t e d IgG m o l e c u l e s would be c o m p l e t e l y i n a c t i v e , w h i l e some wo u l d p o s s e s s o n l y one a c t i v e b i n d i n g s i t e and a m i n o r i t y would be c o m p l e t e l y a c t i v e . More r e c e n t l y t h e most commonly us e d f u s i o n p a r t n e r i s NS1 . NS1 c e l l s s y n t h e s i z e l i g h t c h a i n s o n l y so t h e a n t i b o d i e s s e c r e t e d by a h y b r i d o m a a r i s i n g f r o m an NS1 c e l l f u s i o n i n c l u d e more a c t i v e m o l e c u l e s t h a n a h y b r i d o m a f r o m MOPC-21. I f hea v y and l i g h t c h a i n p a i r i n g i s random and r a t e s o f s y n t h e s i s e q u a l , t h e n 25% o f t h e IgG s h o u l d have o n l y s p l e e n c e l l l i g h t c h a i n s (two b i n d i n g s i t e s ) , 50% s h o u l d have one s p l e e n c e l l a nd one NS1 c e l l l i g h t c h a i n (one b i n d i n g s i t e ) a nd 25% s h o u l d have two NS1 c e l l l i g h t c h a i n s (0 b i n d i n g s i t e s ) , i . e . 75% o f t h e s e c r e t e d a n t i b o d y w o u l d be a c t i v e . R e c e n t l y n o n - p r o d u c i n g v a r i a n t s o f myeloma 33 c e l l s have been a d a p t e d t o c o n t i n u o u s c e l l c u l t u r e ( 5 0 ) . H y b r i d o m a s o f t h e s e myeloma c e l l s s e c r e t e 100% a c t i v e a n t i b o d y . Aims o f t h e S t u d y A l t h o u g h t h e P E G - m o d i f i e d IgG u s e d by S h a r p e t a l (20) showed a s i g n i f i c a n t a b i l i t y t o i n c r e a s e t h e p a r t i t i o n of e r y t h r o c y t e s , t h e s y s t e m was l i m i t e d i n t h a t t h e P E G - m o d i f i e d a n t i b o d y c o u l d o n l y be u s e d t o a l t e r e r y t h r o c y t e p a r t i t i o n a s t h e e r y t h r o c y t e was t h e a n t i g e n u s e d t o r a i s e t h e a n t i b o d y . The m o d i f i c a t i o n o f an a n t i b o d y f o r use a s an a f f i n i t y l i g a n d p o s e s s e v e r a l p r o b l e m s w i t h r e s p e c t t o t h e optimum d e g r e e of a n t i b o d y m o d i f i c a t i o n . The optimum d e g r e e o f m o d i f i c a t i o n i s t h a t w h i c h p r e s e r v e s t h e b i n d i n g a c t i v i t y o f t h e a n t i b o d y , w h i l e s t i l l a t t a c h i n g s u f f i c i e n t PEG t o make an e f f e c t i v e a f f i n i t y l i g a n d w i t h a h i g h p a r t i t i o n c o e f f i c i e n t . Once one p a r t i c u l a r a n t i b o d y i s m o d i f i e d s u c e s s f u l l y t h e r e i s no r e a s o n t o e x p e c t a d i f f e r e n t a n t i b o d y t o r e s p o n d t o m o d i f i c a t i o n i n t h e same way. The d i v e r s i t y o f t h e immune r e s p o n s e g i v e s r i s e t o a h e t e r o g e n e o u s m i x t u r e o f a n t i b o d i e s d i r e c t e d a g a i n s t many d i f f e r e n t a n t i g e n i c d e t e r m i n a n t s o f t h e a n t i g e n m o l e c u l e . T h i s c a u s e s b a t c h v a r i a t i o n i n t h e p u r i f i e d a n t i b o d y s o l u t i o n a n d e a c h d i f f e r e n t a n t i b o d y may r e s p o n d d i f f e r e n t l y t o c h e m i c a l m o d i f i c a t i o n l e a d i n g t o p r o b l e m s w i t h b o t h u s i n g and c h a r a c t e r i s i n g t h e m o d i f i e d a n t i b o d y . However a 34 m o n o c l o n a l a n t i b o d y s i m p l i f i e s t h e s e p r o b l e m s b e c a u s e i t i s a homogeneous sample o f a n t i b o d y m o l e c u l e s , a l l w i t h t h e same s p e c i f i c i t y and t h e same r e s p o n s e t o c h e m i c a l t r e a t m e n t s . I t i s a p u r e c h e m i c a l r e a g e n t w h i c h i s r a r e l y f o u n d i n b i o l o g i c a l i s o l a t e s . In t h i s s t u d y , a P E G - m o d i f i e d m o n o c l o n a l a n t i b o d y was u s e d as a s e c o n d a f f i n i t y l i g a n d i n a s y s t e m c o n t a i n i n g human e r y t h r o c y t e s a s d e s c r i b e d i n F i g . 7. The m o n o c l o n a l a n t i b o d y was d i r e c t e d a g a i n s t t h e F c r e g i o n o f t h e r a b b i t IgG m o l e c u l e , w h i c h i s n o t i n v o l v e d i n t h e b i n d i n g of a n t i g e n and i s c o n s t a n t i r r e s p e c t i v e of t h e a n t i b o d y s p e c i f i c i t y . T h i s meant t h a t t h e f i r s t a n t i b o d y s p e c i f i c i t y c o u l d be a l t e r e d b u t t h e same P E G - m o d i f i e d m o n o c l o n a l a n t i b o d y c o u l d be u s e d a s t h e a f f i n i t y l i g a n d . By b i n d i n g t h e s e c o n d , m o n o c l o n a l a n t i b o d y t o t h e F c r e g i o n o f t h e r a b b i t IgG, i t was hoped t h a t t h e b i n d i n g o f t h e f i r s t a n t i b o d y t o t h e e r y t h r o c y t e would be a f f e c t e d m i n i m a l l y . A n o t h e r p o t e n t i a l a d v a n t a g e o f u s i n g t h e s e c o n d a n t i b o d y i s t h a t i f t h e same a n t i g e n i c d e t e r m i n a n t i s r e p e a t e d a l o n g t h e F c f r a g m e n t t h e n s e v e r a l m o l e c u l e s o f s e c o n d a n t i b o d y may b i n d e a c h f i r s t a n t i b o d y , t h u s i n c r e a s i n g t h e amount o f PEG bound and c o n s e q u e n t l y t h e p a r t i t i o n c o e f f i c i e n t of t h e f i r s t a n t i b o d y compared t o d i r e c t m o d i f i c a t i o n o f t h e f i r s t a n t i b o d y . A n o t h e r i n t e r e s t i n g outcome o f t h e s t u d y i s t h e e x a m i n a t i o n o f t h e p o s s i b i l i t y o f u s i n g m o n o c l o n a l a n t i b o d i e s as a f f i n i t y l i g a n d s . I f t h e t e c h n i q u e i s 35 Mouse monoclonal IgG modified with PEG 1900 NN Glycophorin A F i g . 7 a . A S c h e m a t i c r e p r e s e n t a t i o n o f PEG 1900 m o d i f i e d mouse m o n o c l o n a l a n t i F c f r a g m e n t o f r a b b i t IgG bound t o an e r y t h r o c y t e v i a a n t i - N N g l y c o p h o r i n A r a b b i t IgG. F i g . 7 b . The PEG 1900 c o a t e d e r y t h r o c y t e w i l l p a r t i t i o n p r e d o m i n a n t l y i n t o t h e PEG 8000 r i c h upper p h a s e o f a 5,3.4 s y s t e m . 36 s u c e s s f u l , t h e n t h e r e e x i s t s g r e a t p o t e n t i a l t o s e p a r a t e c l o s e l y r e l a t e d c e l l t y p e s , s u c h a s s u b s e t s of l y m p h o c y t e s , w h i c h a r e i n d i s t i n g u i s h a b l e u s i n g c o n v e n t i o n a l p o l y c l o n a l a n t i b o d i e s . A summary o f t h e p o t e n t i a l a d v a n t a g e s o f u s i n g a s e c o n d , m o n o c l o n a l a n t i b o d y a r e a s f o l l o w s : 1. The same m o n o c l o n a l a n t i b o d y c an be u s e d w i t h d i f f e r e n t f i r s t a n t i b o d i e s . 2. A m o n o c l o n a l a n t i b o d y i s a p u r e r e a g e n t . 3. The s e c o n d a n t i b o d y t e c h n i q u e may a t t a c h more PEG t o t h e e r y t h r o c y t e t h a n by d i r e c t l y m o d i f y i n g t h e a n t i b o d y . 4. A m o n o c l o n a l a n t i b o d y c a n be s e l e c t e d t o b i n d t h e F c r e g i o n o f IgG w h i c h s h o u l d have l e s s e f f e c t on t h e b i n d i n g s i t e of t h e IgG t h a n d i r e c t m o d i f i c a t i o n w i t h PEG. 9. ANALYTICAL TECHNIQUES USED IN THE STUDY F a s t P r o t e i n L i q u i d C h r o m a t o g r a p h y (FPLC) S e p a r a t i o n by i o n exchange c h r o m a t o g r a p h y i s o b t a i n e d by r e v e r s i b l e a d s o r p t i o n . D i f f e r e n t s u b s t a n c e s have d i f f e r e n t a f f i n i t i e s f o r t h e i o n e x c h a n g e r due t o c h a r g e d i f f e r e n c e s , w h i c h c a n be c o n t r o l l e d by v a r y i n g pH and i o n i c s t r e n g t h . O t h e r t y p e s o f b i n d i n g s u c h as Van d e r Waals o r p o l a r i n t e r a c t i o n s may o c c u r b u t have a s m a l l e f f e c t . I n i t i a l l y t h e i o n e x c h a n g e r , w h i c h c a n be e i t h e r an a n i o n o r a c a t i o n i s a t e q u i l i b r i u m w i t h i t ' s c o u n t e r i o n . On a p p l i c a t i o n of t h e sample, c o u n t e r i o n s a r e e x c h a n g e d f o r t h e sample i o n s . The sample i o n s a r e t h e n e l u t e d by an i n c r e a s i n g i o n i c s t r e n g t h g r a d i e n t o f c o u n t e r i o n s . 37 The FPLC s y s t e m i s s i m i l a r t o an HPLC s y s t e m e x c e p t t h a t i t i s a d a p t e d f o r s e p a r a t i o n of b i o l o g i c a l m a t e r i a l s by u s i n g a medium p r e s s u r e , i n t h e o r d e r o f 0-4 MPa, and a s l o w e r f l o w r a t e , i n t h e o r d e r o f 2 m l / m i n u t e . A l s o no p o t e n t i a l l y d e n a t u r i n g m e t a l i o n s a r e f o u n d w i t h i n t h e s y s t e m . The mono Q column u s e d w i t h t h e FPLC s y s t e m i n t h i s s t u d y c o n s i s t s o f a q u a t e r n a r y ammonium s a l t a n i o n e x c h a n g e r ( - C H 2 N + ( C H 3 ) 3 ) bound t o a m o n o d i s p e r s e beaded r e s i n w i t h a p a r t i c l e d i a m e t e r o f lOjim. Sodium D o d e c y l S u l p h 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 p h o r e s i s  (SDS-PAGE) A p o l y a c r y l a m i d e g e l i s a l a t t i c e d s t r u c t u r e w i t h p o r e s o f m o l e c u l a r d i m e n s i o n s . The p o r e s i z e c a n be c o n t r o l l e d by t h e monomer ( a c r y l a m i d e ) c o n c e n t r a t i o n u s e d when t h e g e l i s p o l y m e r i z e d . I f a p r o t e i n i s p o s i t i o n e d i n t h e g e l and a v o l t a g e a p p l i e d a c r o s s t h e g e l , i t w i l l m i g r a t e t o w a r d s t h e anode w i t h a m o b i l i t y d e p e n d e n t on i t ' s n e t t c h a r g e . I f t h e p o r e s a r e o f a s i m i l a r s i z e t o t h e p r o t e i n , t h e y can impose f r i c t i o n a l r e s i s t a n c e t o t h e p a s s a g e o f t h e p r o t e i n . I f s o d i um d o d e c y l s u l p h a t e (SDS), an a n i o n i c d e t e r g e n t , i s i n t h e g e l o r t h e b u f f e r , t h e b i n d i n g o f SDS by t h e p r o t e i n r e n d e r s a l l t h e m o l e c u l e s s i m i l a r l y c h a r g e d . T h i s r e s u l t s i n s e p a r a t i o n by a s i e v i n g e f f e c t , d e p e n d e n t m o s t l y on s i z e . In t h i s s t u d y , s l a b g e l s c o n t a i n i n g SDS i n t h e b u f f e r o n l y and r o d g e l s c o n t a i n i n g SDS i n b o t h t h e r u n n i n g b u f f e r and t h e g e l were u s e d . T h i s meant t h a t s e p a r a t i o n was m o s t l y s i z e d e p e n d e n t . 38 D i f f u s i o n o f t h e p r o t e i n w i l l r e s u l t i n s p r e a d i n g of t h e p r o t e i n bands and a d e c r e a s e i n r e s o l u t i o n . T h i s c an be m i n i m i s e d by u s i n g a s t a c k i n g g e l . The s t a c k i n g g e l has a l a r g e r p o r e s i z e and a l o w e r pH t h a n t h e r u n n i n g g e l . The sample c o n t a i n s p r o t e i n and an a n i o n , i n t h i s c a s e g l y c i n e , w h i c h m i g r a t e s s l o w e r t h a n t h e p r o t e i n i n t h e s t a c k i n g g e l and f a s t e r i n t h e r u n n i n g g e l . When a v o l t a g e i s a p p l i e d a c r o s s t h e s t a c k i n g g e l , t h e i o n s become s t a c k e d i n o r d e r o f t h e i r r e l a t i v e m o b i l i t i e s , i n d e p e n d e n t o f t h e i r o r i g i n a l c o n c e n t r a t i o n s , r e d u c i n g t h e t h i c k n e s s o f t h e sample s t a r t i n g z o n e . The i o n m o b i l i t y o r c o n d u c t i v i t y (K) w i t h i n a. g e l depends on t h e c u r r e n t ( i ) and t h e e l e c t r i c f i e l d ( E ) , i . e . E=i//c. The pH o f t h e s t a c k i n g g e l i s c l o s e t o t h e p i o f t h e g l y c i n e a n d c o n s e q u e n t l y i t has a low m o b i l i t y . I n t h e r u n n i n g g e l , however, t h e g l y c i n e i s above i t ' s p i and has a h i g h m o b i l i t y , r e s u l t i n g i n a low e l e c t r i c f i e l d i n r e g i o n s where g l y c i n e d o m i n a t e s . E a c h p r o t e i n band e n t e r s t h e g e l i n t h e o r d e r i n w h i c h i t was s t a c k e d . A low e l e c t r i c f i e l d i s p r e s e n t b o t h b e f o r e and a f t e r t h e p r o t e i n band due t o t h e h i g h m o b i l i t y of t h e g l y c i n e . T h i s means t h a t t h e t r a i l i n g edge o f t h e p r o t e i n w i l l m i g r a t e q u i c k l y i n t h e h i g h e l e c t r i c f i e l d o f t h e p r o t e i n band d i r e c t l y i n f r o n t o f i t , c a t c h i n g up t o t h e l e a d i n g edge o f t h e p r o t e i n band. The l e a d i n g edge o f t h e p r o t e i n band w i l l be s l o w e d a s i t moves i n t o t h e low e l e c t r i c f i e l d c a u s e d by t h e g l y c i n e . T h i s h as t h e e f f e c t of s h a r p e n i n g t h e p r o t e i n band. substrate(OPD) (colourless) 2> products (yellow) H R P -anti-mouse IgG conj ugate PVC. microtitre plate •ANTIBODY "ANTIGEN (eg. fragment) F i g . 8 . The E n z y m e - l i n k e d Immunosorbent A s s a y ( E L I S A ) CO VO 40 The E n z y m e - l i n k e d Immunosorbent A s s a y ( F i g . 8 . ) The ELISA i s b a s e d on t h e f a c t t h a t p o l y v i n y l c h l o r i d e (PVC) s u r f a c e s w i l l t i g h t l y a d s o r b nanogram amounts of p r o t e i n . The a n t i g e n - c o n t a i n i n g s o l u t i o n i s p i p e t t e d i n t o a PVC m i c r o E L I S A p l a t e w h i c h c o n t a i n s 96 2 5 0 u l w e l l s and l e f t o v e r n i g h t a t 4°C. Unbound m a t e r i a l i s washed o u t and t h e a n t i b o d y s o l u t i o n , s p e c i f i c f o r t h e bound a n t i g e n , i s a d d e d . A g a i n unbound a n t i b o d y i s washed o u t and any r e m a i n i n g n o n - s p e c i f i c p r o t e i n b i n d i n g s i t e s a r e s a t u r a t e d w i t h b o v i n e serum a l b u m e n ( B S A ) . F i n a l l y a r e v e a l i n g a g e n t i s added t o g i v e an i n d i c a t i o n o f t h e amount of a n t i b o d y bound. T h i s i s an e n z y m e - a n t i b o d y c o n j u g a t e s p e c i f i c f o r t h e p r i m a r y a n t i b o d y . When t h e enzyme s u b s t r a t e i s a d d e d t o t h e p l a t e , f o r m a t i o n o f t h e p r o d u c t s o f t h e r e a c t i o n c a t a l y s e d by t h e enzyme i n d i c a t e t h a t t h e p r i m a r y a n t i b o d y has bound t o t h e a n t i g e n . 41 B. MATERIALS AND METHODS 1. IMMUNISATION OF THE RABBITS Two a d u l t New Z e a l a n d W h i t e male r a b b i t s were i n j e c t e d s u b c u t a n e o u s l y w i t h NN g l y c o p h o r i n A (50 ug) i n c o m p l e t e F r e u n d s a d j u v e n t ( 1 : 1 e m u l s i o n ) . The NN g l y c o p h o r i n A was i s o l a t e d f r o m human b l o o d by John Cavanagh by p h e n o l e x t r a c t i o n of t h e l i t h i u m i o d o s a l i c y l i c a c i d e x t r a c t o f human e r y t h r o c y t e g h o s t s ( 5 1 ) . The r a b b i t s were r e i n j e c t e d one month l a t e r w i t h NN g l y c o p h o r i n A (50ug) i n p h o s p h a t e b u f f e r e d s a l i n e (PBS, 16.7mM N a 2 H P O „ , 3.3mM NaH 2PO,, 130.4mM NaCl,pH 7 . 2 ) . A f t e r one week a p p r o x i m a t e l y 30ml o f b l o o d was drawn f r o m an e a r v e i n . A c o n t i n u o u s s o u r c e o f a n t i g e n was m a i n t a i n e d by b o o s t e r i n j e c t i o n s once a month. 2. PURIFICATION OF THE RABBIT ANTI-NN GLYCOPHORIN A  IMMUNOGLOBULIN G The b l o o d was a l l o w e d t o c l o t a t room t e m p e r a t u r e and t h e serum o b t a i n e d by d e c a n t a t i o n and c e n t r i f u g a t i o n . A s o l u t i o n o f s a t u r a t e d ( N H f l ) 2 S O « was added d r o p w i s e t o t h e s l o w l y s t i r r e d serum a t room t e m p e r a t u r e . A t a b o u t 40% s a t u r a t i o n most o f t h e i m m u n o g l o b u l i n s were p r e c i p i t a t e d and t h e s u s p e n s i o n was s t i r r e d s l o w l y f o r a b o u t 30 m i n u t e s . I f t o o much ( N H f l ) 2 S O a was added t h e sample would be c o n t a m i n a t e d w i t h t r a n s f e r r i n and a l b u m i n . The s u s p e n s i o n was c e n t r i f u g e d a t I0,000g f o r 10 m i n u t e s and t h e p e l l e t washed t h r e e t i m e s w i t h 40% s a t u r a t e d ( N H , ) 2 S 0 4 s o l u t i o n . 42 The p e l l e t was d i a l y s e d a g a i n s t w a t e r and t h e p r e c i p i t a t e d l i p o p r o t e i n and IgM removed by c e n t r i f u g a t i o n . The IgG was d i a l y s e d a g a i n s t 1OmM p h o s p h a t e b u f f e r , pH 8 and s e p a r a t e d f r o m t h e r e m a i n i n g IgM and o t h e r c o n t a m i n a t i n g p r o t e i n s on a P h a r m a c i a FPLC s y s t e m u s i n g a mono Q a n i o n e x c h a n g e column w i t h a 0-1M N a C l i o n i c s t r e n g t h g r a d i e n t e l u t i o n i n 1OmM p h o s p h a t e , pH 8 ( F i g . 9 . ) . SDS-PAGE was u s e d t o c h e c k t h e p u r i t y o f t h e IgG ( F i g . 1 5 . ) and i d e n t i f y t h e f r a c t i o n s . 3. MICROTITRE OF THE ANTI-NN GLYCOPHORIN A IMMUNOGLOBULIN G The a b i l i t y o f t h e a n t i - N N g l y c o p h o r i n A IgG t o a g g l u t i n a t e human e r y t h r o c y t e s was a s s a y e d v i a a m i c r o t i t r e e x p e r i m e n t . T h i s a l s o gave an i n d i c a t i o n o f t h e r e l a t i v e a f f i n i t i e s o f t h e a n t i b o d y f o r MN, NN and MM e r y t h r o c y t e s . Two f o l d s e r i a l d i l u t i o n s o f 50 u l of IgG s o l u t i o n (1mg/ml) were made up i n PBS i n a m i c r o t i t r e p l a t e . An e q u a l volume o f 1% h a e m a t o c r i t f r e s h e r y t h r o c y t e s u s p e n s i o n w i t h known NN, MN o r MM s p e c i f i c i t y was a d d e d t o e a c h w e l l of t h e m i c r o t i t r e p l a t e . The s u s p e n s i o n s were mixed and e x a m i n e d a f t e r f o u r h o u r s . In t h e w e l l s o f t h e m i c r o t i t r e p l a t e c o n t a i n i n g a g g l u t i n a t i n g c o n c e n t r a t i o n s o f IgG t h e c e l l s were c l u m p e d t o g e t h e r i n a b u t t o n a t t h e b o t t o m of t h e w e l l , w hereas i n t h o s e c o n t a i n i n g s u b - a g g l u t i n a t i n g IgG c o n c e n t r a t i o n s t h e c e l l s were u n i f o r m l y d i s t r i b u t e d o v e r t h e b o t t o m o f t h e w e l l s . The r e s u l t s a r e shown i n T a b l e 1., p.73. The c o n c e n t r a t i o n d e p e n d e n c e o f t h e b i n d i n g o f e r y t h r o c y t e s by a n t i NN g l y c o p h o r i n A IgG was a l s o examined 43 F i g . 9 . F a s t P r o t e i n L i q u i d C h r o m a t o g r a p h y p r o f i l e of t h e serum p r o t e i n s o f an immunised r a b b i t p a r t i a l l y p u r i f i e d by p r e c i p i t a t i o n w i t h ( N H 2 ) « S O „ on a mono Q column w i t h an i o n i c s t r e n g t h g r a d i e n t o f 0-1M N a C l i n 1OmM p h o s p h a t e b u f f e r , pH 8. The p e a k s were d e t e c t e d by a b s o r p t i o n a t 280nm and i d e n t i f i e d by SDS-PAGE. 44 u s i n g an E L I S A ( F i g . 1 6 ) . 4. FRAGMENTATION OF RABBIT IMMUNOGLOBULIN G BY PAPAIN T h i s i s an a d a p t a t i o n o f t h e methods of P o r t e r (52) and U t s u m i ( 5 3 ) . R a b b i t IgGdOmg) i n p h o s p h a t e buf fer(50mM) , pH 8 c o n t a i n i n g c y s t e i n e ( 1 O m M ) and EDTA(2mM), was i n c u b a t e d w i t h a c t i v a t e d m e r c u r i p a p a i n ( 1 5 m i n s , 37° C) i n t h e same b u f f e r a t 37° C. The m e r c u r i p a p a i n was o b t a i n e d f r o m BDH. A f t e r 16-20 h o u r s t h e r e a c t i o n was s t o p p e d by i r r e v e r s i b l e a l k y l a t i o n o f t h e enzyme s u l p h y d r y l g r o u p s u s i n g i o d o a c e t a m i d e , f i n a l c o n c e n t r a t i o n 20mM, and t h e m i x t u r e was h e l d on i c e , p r o t e c t e d f r o m l i g h t , f o r one h o u r . The m i x t u r e was d i a l y s e d a g a i n s t sodium a c e t a t e (lOmM), pH 5.5 a t 4°C, t h e n f r a c t i o n a t e d on t h e FPLC mono Q column w i t h an i o n i c s t r e n g t h g r a d i e n t f r o m 0.01-1M sodium a c e t a t e , pH 5.5 ( F i g . 1 0 ) . The f r a c t i o n s were examined w i t h SDS-PAGE ( F i g . 1 1 ) . The F c f r a g m e n t was a l s o c r y s t a l l i s e d f r o m t h e d i g e s t m i x t u r e by d i a l y s i s a g a i n s t s o d i u m borate(1OmM), pH 8.6 ( 5 4 ) . T h i s y i e l d e d a p u r e r sample o f F c f r a g m e n t but t h e y i e l d was t o o low f o r p r e p a r a t i v e u s e . 5. SODIUM DODECYL SULPHATE POLYACRYLAMIDE GEL  ELECTROPHORESIS Two t y p e s of SDS-PAGE were u s e d , s l a b g e l s w i t h no SDS i n t h e g e l and r o d g e l s w i t h SDS. A l l c o n c e n t r a t i o n s a r e w e i g h t r a t i o s u n l e s s o t h e r w i s e s p e c i f i e d . The s l a b g e l s c o n s i s t e d o f 10% a c r y l a m i d e : N , N ' - m e t h y l e n e - b i s - a c r y l a m i d e 45 F i g . 1 0 . FPLC p r o f i l e of t h e p a p a i n d i g e s t o f IgG on a mono Q column w i t h an i o n i c s t r e n g t h g r a d i e n t o f 0.01-1M sodium a c e t a t e , pH 5.5. The p e a k s were d e t e c t e d by a b s o r p t i o n a t 280nm and i d e n t i f i e d by SDS-PAGE. 46 Mol.Wt.Std. 1 2 3 IgG, IgG2 94 K — 67K — 45 K — 30K — — — 20 K — 14.4K — — -F i g . 11. D i a g r a m of 10% s l a b g e l of r e d u c e d r a b b i t IgG and t h e p a p a i n d i g e s t of IgG f r a c t i o n a t e d by FPLC. l a n e 1. Whole p a p a i n d i g e s t , l a n e 2. F a b f r a g m e n t , l a n e 3. F c f r a g m e n t . l a n e 4. F c f r a g m e n t c r y s t a l l i s e d by d i a l y s i s a g a i n s t N a 2 B a 0 7 (0.01M, pH 8.6) 47 (30:0.8) r e s o l v i n g g e l w i t h a 5% a c r y l a m i d e : N , N ' - m e t h y l e n e - b i s - a c r y l a m i d e ( b i s ) s t a c k i n g g e l . The r o d g e l s were 3% a c r y l a m i d e : b i s ( 3 0 : 1 . 2 ) . B o t h t y p e s o f g e l were u s e d f o r n a t i v e and r e d u c e d p r o t e i n . Method f o r S l a b G e l s T h i s i s an a d a p t a t i o n of t h e methods o f D a v i s (55) and O r n s t e i n ( 5 6 ) . The s t o c k s o l u t i o n s were made up a s f o l l o w s : 1. A c r y l a m i d e : b i s (30:0.8) - A c r y l a m i d e s o l u t i o n (60g i n 200ml of w a t e r ) c o n t a i n i n g c h a r c o a l was h e a t e d t o 60°C, t h e n h e l d a t 56°C f o r 30 m i n u t e s p r o t e c t e d f r o m l i g h t . The s o l u t i o n was f i l t e r e d and b i s (1.6g p e r 200ml) was adde d . The s o l u t i o n was s t o r e d a t 4°C i n t h e d a r k . 2. 1.875M T r i s ( h y d r o x y m e t h y l ) a m i n o e t h a n e ( t r i s - b a s e ) , pH 8.8. 3. 1M [ T r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e h y d r o c h l o r i d e ] ( t r i s - H C l ) , pH 6.8. 4. Sample p r e p a r a t i o n s o l u t i o n ( 1 0 % SDS) - The f o l l o w i n g were c o m b i n e d : S D S ( 0 . 4 1 g ) , g l y c e r o l ( 1 2 . 3 m l ) , t r i s - H C l ( 1 . 2 8 m l ) , w a t e r ( 1 2 . 3 m l ) a n d bromophenol b l u e dye t o c o l o u r . 5. R u n n i n g b u f f e r - G l y c i n e ( 5 7 . 6 g ) and SDS(4g) were added t o 4L of t r i s - b a s e ( 0 . 0 5 M ) , pH 8.3. 6. C o o m a s s i e s t a i n - C o o m a s s i e b l u e d y e ( 0 . 4 g ) was d i s s o l v e d i n i s o p r o p a n o l ( 3 0 m l ) , w a t e r ( 1 3 0 m l ) and a c e t i c a c i d ( 3 0 m l ) . 7. D e s t a i n s o l u t i o n - An aqueous s o l u t i o n o f 35%v/v e t h a n o l and 10%V / V a c e t i c a c i d was u s e d . 48 P o u r i n g and r u n n i n g t h e g e l A c r y l a m i d e : b i s ( 1 0 m l ) , t r i s - b a s e ( 6 m l ) , 0.2M E t h y l e n e d i a m i n e t e t r a a c e t a t e ( E D T A , 0.3ml) and w a t e r ( 1 3 . 4 m l ) were m i x e d and d e g a s s e d . N , N , N ' , N ' - T e t r a m e t h y l e t h y l e n e d i a m i n e (TEMED, 0.015ml) and f r e s h l y made 10% ( N H , ) 2 S 2 0 8 ( 0 . 3 m l ) were added and t h e r e s o l v i n g g e l was p o u r e d i n t o a H o e f f l e r g e l e l e c t r o p h o r e s i s a p p a r a t u s . Water was l a y e r e d o v e r t h e g e l s u r f a c e and p o l y m e r i z a t i o n was c o m p l e t e w i t h i n 30-40 m i n u t e s . The p r e v i o u s p r o c e d u r e was r e p e a t e d t o p r e p a r e t h e s t a c k i n g g e l u s i n g , a c r y l a m i d e : b i s ( 2 . 5 m l ) , t r i s - H C l ( 1 , 8 8 m l ) , 0.2M E D T A ( 0 . 1 5 m l ) , w a t e r ( 1 0 . 3 m l ) , T E M E D ( 7 . 5 u l ) , and 10% ( N H „ ) 2 S 2 0 8 ( 0 . 1 5 m l ) . The w a t e r was removed f r o m t h e s u r f a c e of t h e r e s o l v i n g g e l , a sample comb i n s e r t e d and t h e s t a c k i n g g e l p o u r e d . A g a i n p o l y m e r i z a t i o n was c o m p l e t e d i n 30-40 m i n u t e s . E a c h p r o t e i n sample ( l 0 - 2 0 u g ) was m i x e d w i t h an e q u a l volume o f sample p r e p a r a t i o n s o l u t i o n and r e d u c e d w i t h m e r c a p t o e t h a n o l ( 5 u l ) a t 100°C f o r one m i n u t e . The s a m p l e s were a p p l i e d t o t h e g e l , a i r b u b b l e s removed and a c o n s t a n t c u r r e n t o f 75mA was a p p l i e d u n t i l t h e t r a c k i n g dye r e a c h e d t h e end o f t h e g e l . The g e l was s t a i n e d f o r two h o u r s , d e s t a i n e d o v e r s e v e r a l h o u r s , s o a k e d i n 10% g l y c e r o l s e v e r a l h o u r s , t h e n d r i e d s i x h o u r s on a B i o r a d 224 g e l d r i e r . 49 Rod G e l s The a c r y l a m i d e s y s t e m i s a m o d i f i c a t i o n of t h a t u s e d by F a i r b a n k s e t a l ( 5 7 ) . S t o c k s o l u t i o n s were made up as f o l l o w s : 1. 30% A c r y l a m i d e : b i s (30:1.2) was made up as f o r s l a b g e l s 2. 10X B u f f e r - 1M t r i s - H C l , 2M sodium a c e t a t e , 0.2M EDTA, pH 7.4 3. C o o m a s s i e b l u e s t a i n - as f o r s l a b g e l s 4. D e s t a i n s o l u t i o n - a s f o r s l a b g e l s 5. R u n n i n g b u f f e r - 100ml 10X b u f f e r , 50ml SDS(4%) were made up t o 1L w i t h w a t e r 6. Sample p r e p a r a t i o n s o l u t i o n - 20mM t r i s - H C l , 2mM EDTA, 2% SDS, 14% g l y c e r o l , 20mg/ml p y r o n i n Y, pH 8. F o r r e d u c i n g c o n d i t i o n s , 50mM m e r c a p t o e t h a n o l was u s e d . The r e a g e n t s were m i x e d a s f o l l o w s , t h e m i x t u r e was d e g a s s e d p r i o r t o a d d i t i o n o f ( N H A ) 2 S 2 0 8 . a. 30% A c r y l a m i d e : b i s (3ml) b. 10x B u f f e r (3ml) c. 4% SDS (1.3ml) d. 0.5%v/v TEMED (1.5ml) e. Water (18ml) f . 1.5% ( N H , ) 2 S 2 0 8 (3ml) The m i x t u r e was c a s t a s r o d g e l s i n 125x5mm i n s i d e d i a m e t e r t u b e s . The r o d s were mounted i n a B i o - r a d model 150A e l e c t r o p h o r e s i s chamber and run a t an i n i t i a l c u r r e n t o f 0.5mA p e r t u b e u n t i l t h e sample e n t e r e d t h e g e l , t h e n t h e c u r r e n t was i n c r e a s e d t o 8mA p e r t u b e . When t h e t r a c k i n g dye 50 reached the end of the gel, the gels were removed and the tracking dye marked with Indian ink. The gels were stained and fixed for one hour in Coomassie stain, then destained one hour. F i n a l clearing was in 7% acetic acid for one hour. 6. IMMUNISATION OF BALB/C MICE BALB/c mice were injected subcutaneously with 80 ug of rabbit IgG in complete Freunds adjuvent. At two week intervals two further subcutaneous injections of rabbit IgG in incomplete adjuvent were administered. After the f i r s t three injections, a t r i a l bleed from the r e t r o - o r b i t a l sinus was checked for production of IgG against rabbit IgG by an ELISA assay. F i n a l l y an intraperitoneal boost of the F c fragment of rabbit IgG was administered four days prior to fusion. The i n i t i a l injections were of whole IgG even though a monoclonal antibody against the F c fragment was required. This was due to the p o s s i b i l i t y of the antigenic determinants of the F c fragment being altered by the papain digestion. The monoclonal antibody was required to recognize the F c region of the whole IgG molecule. The f i n a l boost of F c fragment was to p r e f e r e n t i a l l y stimulate lymphocytes secreting a n t i - F c fragment antibodies over those secreting antibodies directed against other areas of the IgG molecule. 51 7. CULTURE OF NS1 CELLS P r e p a r a t i o n of S t o c k S o l u t i o n s a. Heat i n a c t i v a t e d f e t a l c a l f serum ( F C S ) . The FCS was i n c u b a t e d one h o u r a t 56°C, c e n t r i f u g e d a t 500g t h e n f i l t e r e d t h r o u g h 0.45 and 0.22um s t e r i l e N a l g e n e f i l t e r s . The FCS was s t o r e d a t - 2 0 ° C . b. L - G l u t a m i n e . A s o l u t i o n of L - g l u t a m i n e (30mg/ml) was a u t o c l a v e d , t h e n f i l t e r e d t h r o u g h a s t e r i l e 0.2 um membrane f i l t e r . I t was s t o r e d i n 1ml a l i q u o t s a t - 2 0 ° C . c . Growth medium. RPMI-1640 was o b t a i n e d from F i s h e r , made up a s d i r e c t e d and f i l t e r e d t h r o u g h 0.45 and 0.22um s t e r i l e N a l g e n e f i l t e r s . RPMI-1640 (100ml) was m i x e d w i t h h e a t i n a c t i v a t e d FCS (10ml) and L - g l u t a m i n e ( 1 m l ) . d. S e r u m - f r e e medium. As above w i t h o u t FCS. T h a w ing and C u l t u r e o f NS1 C e l l s The NS1 c e l l s were thawed o v e r f i v e m i n u t e s a t room t e m p e r a t u r e , t h e n added t o c o l d s e r u m - f r e e medium ( 1 0 m l ) . A 100 u l a l i q u o t was s t a i n e d w i t h e r y t h r o s i n B dye and t h e v i a b i l i t y o f t h e c e l l s was c h e c k e d by c o u n t i n g t h e dead s t a i n e d c e l l s on a h a e m o c y t o m e t e r . E r y t h r o s i n B o n l y s t a i n s d e a d c e l l s . I f t h e v i a b i l i t y o f t h e c e l l s was o v e r 80%, t h e c e l l s were c e n t r i f u g e d (300g, 5min) and t h e medium a s p i r a t e d o f f . The c e l l s were r e s u s p e n d e d i n g r o w t h medium t o g i v e a f i n a l c o n c e n t r a t i o n of 10 5 c e l l s p e r m l . I n c u b a t i o n was i n T25 f l a s k s a t 37°C, 5% C 0 2 and 100% h u m i d i t y . At a p o p u l a t i o n d e n s i t y o f a p p r o x i m a t e l y 10 6 c e l l s p e r ml, t h e c e l l s were d i v i d e d and an e q u a l volume o f f r e s h g r o w t h 52 medium a d d e d . F r e e z i n g o f NS1 c e l l s A p p r o x i m a t e l y 10 s c e l l s were r e s u s p e n d e d i n 1ml of f r e e z i n g medium ( 1 0 % V / V DMSO, 40%v/v FCS, 50%v/v RPMI-1640). The c e l l s were f r o z e n i n i t i a l l y a t -70°C o v e r n i g h t , f o l l o w e d by l o n g t e r m s t o r a g e i n l i q u i d n i t r o g e n . S e l e c t i o n o f HAT S e n s i t i v e NS1 C e l l s T h i s i s n e c e s s a r y t o e n s u r e t h a t o n l y h y b r i d o m a s s u r v i v e a f t e r f u s i o n . The HAT medium was made up by a d d i n g 1ml o f lOOxHAT (1OmM h y p o x a n t h i n e , 0.04mM a m i n o p t e r i n , 1.6mM t h y m i d i n e ) t o 100ml of g r o w t h medium. NS1 c e l l s were p l a t e d i n t o HAT medium and a l l d i e d w i t h i n 24 h o u r s . 8. PREPARATION OF CELLS FOR FUSION The NS1 c e l l s were grown from f r o z e n s t o r a g e . A p p r o x i m a t e l y 10 7 c e l l s were u s e d i n t h e f u s i o n . The Thymocyte F e e d e r C e l l s The thymuses were removed f r o m s i x week o l d S w i s s m i c e . Two t o t h r e e thymuses were p l a c e d i n serum f r e e medium and washed w e l l . The t h y m o c y t e s were t e a s e d o u t o f t h e thymus w i t h f o r c e p s , t h e n drawn up and down i n a s y r i n g e t o b r e a k up c l u m p s . The c e l l s u s p e n s i o n was t r a n s f e r r e d t o a t e s t t u b e , a l l o w e d t o s e t t l e , t h e n t r a n s f e r r e d a g a i n t o l e a v e any l a r g e c lumps of c e l l s b e h i n d . The c e l l s were c o u n t e d and r e s u s p e n d e d i n 130ml o f HAT medium. The f i n a l t h y m o c y t e c o n c e n t r a t i o n was 5 x 1 0 s c e l l s p e r m l . 53 The S p l e e n C e l l s The s p l e e n was removed from an immunized BALB/c mouse and washed w i t h s e r u m - f r e e medium. The s p l e e n was t e a s e d a p a r t i n 5ml of s e r u m - f r e e medium and p a s s e d t h r o u g h a s t e r i l e s i e v e . T h i s was r e p e a t e d and l a r g e p i e c e s o f s p l e e n were g r o u n d w i t h a s y r i n g e p l u n g e r . The s u s p e n s i o n was c e n t r i f u g e d (300g, 5min) and t h e medium was a s p i r a t e d o f f . E r y t h r o c y t e s were l y s e d i n 5ml of N H „ C 1 ( 0 . 1 7 M ) f o r 5 min, t h e n c e n t r i f u g e d (300g, 5 m i n ) . The NH f lCl was a s p i r a t e d o f f and t h e c e l l s were r e s u s p e n d e d i n 5ml o f s e r u m - f r e e medium. A p p r o x i m a t e l y 10 s c e l l s were u s e d i n t h e f u s i o n . P r e p a r a t i o n o f t h e PEG 3500 S o l u t i o n PEG 3500 ( I 0 g ) was a u t o c l a v e d , c o o l e d , t h e n s e r u m - f r e e medium (10ml) was added w h i l e t h e PEG 3500 was s t i l l l i q u i d . The pH was s l i g h t l y a l k a l i n e so t h e s o l u t i o n was p i n k i n c o l o u r . 9. FUSION PROTOCOL The s p l e e n c e l l s ( 1 0 8 i n 5ml) and NS1 c e l l s ( 1 0 7 i n 1ml) were mixed, c e n t r i f u g e d (300g, 5min) and t h e medium was a s p i r a t e d o f f . S t e r i l e PEG 3500 ( i g / m l ) i n s e r u m - f r e e medium was add e d (1ml, 0.1ml e v e r y 6 s e c o n d s ) w i t h s t i r r i n g , t h e n s t i r r e d f o r one m i n u t e . Next s e r u m - f r e e medium was added (5ml, 0.2ml e v e r y 12 s e c o n d s f o r t h e f i r s t 2ml, t h e n 0.5ml e v e r y 30 s e c o n d s f o r t h e n e x t 3ml) w i t h s t i r r i n g . The c e l l s were r e s u s p e n d e d by p i p e t t i n g and t h e f i n a l volume made up t o 10ml. The c e l l s were c e n t r i f u g e d (300g, 5min) and 54 r e s u s p e n d e d i n t h y m o c y t e " f e e d e r " medium ( 5 m l ) . The r e m a i n i n g f e e d e r medium was added t o make a f i n a l volume o f 130ml. The c e l l s were p l a t e d i n t o 96 w e l l p l a t e s , 3 d r o p s p e r w e l l . L i p o p o l y s a c c h a r i d e (LPS, 0.025mg/ml, 1drop p e r w e l l ) , a g r o w t h s t i m u l a n t was a d d e d t o h a l f t h e p l a t e s t o make a c o m p a r i s o n . The c e l l s were i n c u b a t e d a t 37°C, 5% C 0 2 , 100% h u m i d i t y f o r f o u r d a y s , t h e n e a c h w e l l was f e d w i t h t h y m o c y t e s i n HAT medium. A f t e r one week HAT s e l e c t i o n was t e r m i n a t e d and t h e c e l l s were c u l t u r e d i n HT medium (lOOxHT, 1OmM h y p o x a n t h i n e , 1.6mM t h y m i d i n e ) f o r s e v e r a l d a y s t o e n s u r e t h e c o m p l e t e r e m o v a l o f a m i n o p t e r i n p r i o r t o c u l t u r e i n n o r m a l g r o w t h medium. The i n h i b i t i o n c o n s t a n t o f a m i n o p t e r i n f o r d i h y d r o f o l a t e r e d u c t a s e i s l e s s t h a n 10" 9M (58) and t h e c o n c e n t r a t i o n o f a m i n o p t e r i n i n HAT medium i s 4X10" 7M t h e r e f o r e t h e c o n c e n t r a t i o n must d e c r e a s e 400 f o l d b e f o r e t h e enzyme r e g a i n s a c t i v i t y . A m i n o p t e r i n i s m e t a b o l i s e d e x t r e m e l y s l o w l y and t h e main r o u t e of r e m o v a l from c e l l c u l t u r e s i s by d i l u t i o n . F o r t h i s r e a s o n s l o w g r o w i n g c e l l l i n e s were c u l t u r e d i n HT medium f o r s e v e r a l weeks. 10. THE ENZYME-LINKED IMMUNOSORBENT ASSAY(ELISA) Goat a n t i - m o u s e I g G - h o r s e r a d i s h p e r o x i d a s e (igG-HRP) was o b t a i n e d f r o m Sigma. A 96 w e l l PVC m i c r o E L I S A p l a t e was c o a t e d w i t h a n t i g e n s o l u t i o n ( l u g / m l i n 15mM N a 2 C 0 3 , 35mM NaHC0 3, pH 9.6) a t 2 0 0 u l p e r w e l l and l e f t a t 4°C o v e r n i g h t . The a n t i g e n s u s e d i n t h i s s t u d y were r a b b i t IgG, F and F , 55 fragments of rabbit IgG, and NN,MM or MN glycophorin A. The plate was washed three times with PBS-Tween (0.5% Tween 80) and blotted dry. An aliquot of antibody solution was added (200 ul per well) and incubated for one hour at 37°C. The antibody solutions used were monoclonal antibody in ascites f l u i d , culture supernatant or FPLC p u r i f i e d monoclonal antibody and rabbit IgG. The antibody solution was removed and the plate was washed three times with PBS-Tween. A solution of BSA (0.5%) in PBS-Tween was added (200 ul per well) as a blocking agent and incubated 30 minutes at 37°C. The BSA solution was removed and the plate was washed three times with PBS-Tween. A solution of goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) in PBS was added to each well (200 ul per wel l ) . The anti-mouse IgG-HRP conjugate was a 1:8000 d i l u t i o n of a 0.5mg/ml stock solution and the enzyme:anti-mouse IgG molar r a t i o was 4:1. The plate was incubated for two hours at 37°C. The excess conjugate was removed by washing the plate three times with PBS-Tween. O-Phenylenediamine solution (OPD, 0.04% in 25mM c i t r i c acid, 5lmM Na2HPOa, pH 5), containing 0.12% H 20 2 added just prior to use, was added to each well (200ul per well). The colour was allowed to develop in the dark u n t i l the reaction was stopped by addition of H 2SO„ (4M, 50 ul per wel l ) . The absorbance of each well was measured at 492nm with a 690nm reference beam on a Titertek Multiskan MC. 56 11. RECLONING BY LIMITING DILUTION B o t h c e l l s p r o d u c i n g a n t i - r a b b i t Fc and a n t i - w h o l e r a b b i t IgG were r e c l o n e d . An a l i q u o t of c e l l s was d i l u t e d 100 t i m e s i n t o HT medium and c o u n t e d . The c e l l s were d i l u t e d i n HT medium t o a c o n c e n t r a t i o n o f a p p r o x i m a t e l y 10 c e l l s p e r ml and p l a t e d i n t o a 96 w e l l c e l l c u l t u r e p l a t e . The c e l l s were grown as b e f o r e w i t h t h y m o c y t e f e e d e r c e l l s , t h e n e x amined by E L I S A f o r F c s p e c i f i c i t y . 12. ANTIBODY PRODUCTION AND PURIFICATION M o n o c l o n a l A n t i b o d y P r o d u c t i o n v i a A s c i t e s F l u i d In most c a s e s i n j e c t i o n o f 1 0 6 — 1 0 7 h i s t o c o m p a t i b l e h y b r i d c e l l s i n t o m ice w i l l r e s u l t i n tumor f o r m a t i o n w i t h i n two t o f o u r weeks. I n t r a p e r i t o n e a l i n j e c t i o n o f h y b r i d o m a s o f t e n r e s u l t s i n t h e d e v e l o p m e n t o f a s c i t e s c o n t a i n i n g 5-15mg/ml of a n t i b o d y , a s i m i l a r l e v e l t o t h a t f o u n d i n t h e serum. A s c i t e s y i e l d p e r mouse i s 2-5ml whereas serum y i e l d i s o n l y 0.5-1ml, t h e r e f o r e g e n e r a t i o n o f a s c i t e s i s a r a p i d method t o p r o d u c e m o n o c l o n a l a n t i b o d i e s . BALB/c m i c e were i n j e c t e d w i t h p r i s t a n e (0.5ml p e r mouse) i n t r a p e r i t o n e a l l y ( I / P ) and t h r e e t o f o u r d a y s l a t e r i n j e c t e d w i t h 10 6 h y b r i d o m a c e l l s I/P i n 1ml o f s e r u m - f r e e medium. A f t e r s e v e n t o t e n d a y s , t h e a s c i t e s f l u i d was d r a i n e d by i n s e r t i n g a n e e d l e I/P a n d t h i s was r e p e a t e d e v e r y two d a y s u n t i l d e a t h o f t h e mouse. E a c h mouse t y p i c a l l y y i e l d e d 5-6ml. The a s c i t e s was c e n t r i f u g e d t o remove t h e c e l l s , t h e n s t o r e d a t - 2 0 ° C u n t i l p u r i f i c a t i o n . 57 F i g . 1 2 . FPLC p r o f i l e o f m o n o c l o n a l IgG p r e c i p i t a t e d by s a t u r a t e d (NH ( t ) 2 S O , s o l u t i o n . E l u t i o n was w i t h a - 0 . 0 l - 0 . 3 M p h o s p h a t e i o n i c s t r e n g t h g r a d i e n t , pH 8 on a mono Q co l u m n . A s i n g l e peak was o b t a i n e d due t o t h e m o n o c l o n a l o r i g i n o f t h e a n t i b o d y . 58 P u r i f i c a t i o n o f A n t i b o d i e s f r o m A s c i t e s F l u i d and C e l l  C u l t u r e S u p e r n a t a n t A s c i t e s f l u i d ( t y p i c a l l y 2mg/ml o f IgG) o r c e l l c u l t u r e s u p e r n a t a n t ( t y p i c a l l y I0ug/ml o f IgG) were p r e c i p i t a t e d w i t h ( N H f l ) 2 S O o and washed t h r e e t i m e s w i t h 40% s a t u r a t e d ( N H q ) 2 S O i , a s d e s c r i b e d e a r l i e r . The p e l l e t was d i a l y s e d a g a i n s t 1OmM p h o s p h a t e , pH 8 and p u r i f i e d on a FPLC mono Q column, w i t h a 0.01-0.3M i o n i c s t r e n g t h g r a d i e n t and a f l o w r a t e o f 2ml/min. T y p i c a l l y one m a j o r peak was e l u t e d w i t h t r a c e s o f m u r i n e p r o t e i n s i n t h e c a s e o f t h e a s c i t e s p r e p a r a t i o n ( F i g . 1 2 ) . 13. FREEZING AND THAWING OF HYBRIDOMA CELLS A p p r o x i m a t e l y 10 7 c e l l s were r e s u s p e n d e d i n 1ml o f h y b r i d o m a f r e e z i n g medium ( 9 0 % v / v FCS, 10%v/v DMSO) and f r o z e n a t - 7 0 ° C o v e r n i g h t . The v i a l s were s t o r e d i n l i q u i d n i t r o g e n . The c e l l s were thawed o v e r f i v e m i n u t e s , t h e n a d d e d t o c o l d HT medium. O f t e n t h e c e l l s were s l o w t o b e g i n t o d i v i d e and r e q u i r e d t h y m o c y t e f e e d e r c e l l s . 14. CHARACTERISATION OF THE PURIFIED MONOCLONAL ANTIBODY BY  A WESTERN BLOT The s p e c i f i c i t y o f t h e m o n o c l o n a l a n t i b o d y f o r heavy and l i g h t c h a i n s o f IgG and t h e p a p a i n d i g e s t f r a c t i o n s was e x a m i n e d u s i n g t h e W e s t e r n b l o t t e c h n i q u e w h i c h i d e n t i f i e s p r o t e i n s a f t e r e l e c t r o p h o r e t i c s e p a r a t i o n ( 5 9 ) . The p r o t e i n s 59 a r e e l u t e d e l e c t r o p h o r e t i c a l l y f r o m t h e p o l y a c r y l a m i d e g e l o n t o a membrane made o f n i t r o c e l l u l o s e w h i c h b i n d s p r o t e i n t i g h t l y . The membrane i s t h e n e x p o s e d t o a n t i b o d y . U s u a l l y t h e d e v e l o p i n g a n t i b o d y i s r a d i o a c t i v e b u t i n t h e p r e s e n t c a s e t h e bound a n t i b o d y was r e v e a l e d w i t h HRP a n t i - m o u s e IgG c o n j u g a t e i n a s i m i l a r way t o t h e E L I S A . P o l y a c r y l a m i d e g e l s of I g G 1 f I g G 2 , F ^ and F c f r a g m e n t s were r u n i n a 10% s l a b g e l a s d e s c r i b e d e a r l i e r . Two p i e c e s o f S-S f i l t e r p a p e r , a n i t r o c e l l u l o s e s h e e t and a S c o t c h b r i t e pad were s o a k e d i n d e g a s s e d t r a n s f e r b u f f e r ( t r i s - b a s e ( 1 2 . 1 g ) , g l y c i n e ( 5 7 . 6 g ) , m e t h a n o l ( 8 0 0 m l ) , made up t o 4L, pH 8 . 3 ) . A " s a n d w i c h " was b u i l t up i n t h e f o l l o w i n g o r d e r : s c o t c h b r i t e p a d, f i l t e r p a p e r , n i t r o c e l l u l o s e p a p e r , g e l , f i l t e r p a p e r and s c o t c h b r i t e pad ( F i g . 2 1 b ) . The s a n d w i c h was c l i p p e d t o g e t h e r and p l a c e d i n a t r a n s - b l o t c e l l s u c h t h a t t h e n i t r o c e l l u l o s e p a p e r was between t h e g e l and t h e anode. The c e l l was f i l l e d w i t h t r a n s f e r b u f f e r and t h e p r o t e i n s were t r a n s f e r r e d o v e r s i x t e e n t o twenty f o u r h o u r s a t 12v w i t h c o o l i n g . I m m e d i a t e l y a f t e r t r a n s f e r t h e n i t r o c e l l u l o s e s h e e t was immersed i n a s o l u t i o n o f t r i s - B S A (0.9%w/v N a C l , 5%w/v f r a c t i o n V BSA, 1OmM t r i s - H C l , pH 7.4) and i n c u b a t e d a t 37°C f o r 90 m i n u t e s t o s a t u r a t e r e m a i n i n g b i n d i n g s i t e s on t h e n i t r o c e l l u l o s e . The n i t r o c e l l u l o s e was t h e n immersed i n t r i s - B S A c o n t a i n i n g m o n o c l o n a l a n t i b o d y (50 ug/ml) and i n c u b a t e d f o r 90 m i n u t e s a t 37°C . D u r i n g b o t h i n c u b a t i o n s t h e b l o t t e d s i d e o f t h e n i t r o c e l l u l o s e was f a c i n g upwards. A f t e r i n c u b a t i o n t h e n i t r o c e l l u l o s e was 60 washed w i t h s h a k i n g f o r t e n m i n u t e s i n t r i s - s a l i n e (0.9%w/v N a C l , 1OmM t r i s - H C l , pH 7 . 4 ) , t w e n t y m i n u t e s i n two l o t s o f t r i s - s a l i n e c o n t a i n i n g 0.05%w/v N o n i d e t P-40 and t e n m i n u t e s i n t r i s - s a l i n e . T h i s was f o l l o w e d by i m m e r s i o n i n a s o l u t i o n o f a n t i - m o u s e IgG-HRP c o n j u g a t e , a w a s h i n g s t e p , t h e n OPD s o l u t i o n ( a s d e s c r i b e d f o r E L I S A ) . F i n a l l y t h e n i t r o c e l l u l o s e was washed w i t h t r i s - s a l i n e and a i r - d r i e d a t room t e m p e r a t u r e . The d a r k b a n d s were marked i n c a s e of f a d i n g . 15. A F F I N I T Y CHROMATOGRAPHY OF ANTIBODIES a . P r e p a r a t i o n o f t h e S e p h a r o s e - P r o t e i n C o n j u g a t e C N B r - a c t i v a t e d S e p h a r o s e 4B was o b t a i n e d from P h a r m a c i a . A n t i - r a b b i t IgG ( g o a t ) was o b t a i n e d from C a p p e l . C N B r - a c t i v a t e d S e p h a r o s e 4B (1g) was s w o l l e n and washed i n HC1 (0.001M, 2 0 0 m l ) . R a b b i t IgG o r a n t i - r a b b i t IgG (30mg) i n NaHC0 3 b u f f e r (0.1M, pH 8.3) c o n t a i n i n g N a C l (0.5M) was add e d t o t h e s w o l l e n g e l and m i x e d , e n d - o v e r - e n d f o r 2 h o u r s a t room t e m p e r a t u r e . The s u s p e n s i o n was washed w i t h NaHC0 3 b u f f e r (0.1M, pH 8 . 3 ) , t h e n m i x e d e n d - o v e r - e n d i n g l y c i n e b u f f e r (0.2M, pH 8.0) f o r 2 h o u r s a t room t e m p e r a t u r e . The s u s p e n s i o n was washed 5 t i m e s w i t h a c y c l e o f NaHC0 3 (0.1M, pH 8.3) f o l l o w e d by CH 3COONa (0.1M, pH 4 . 0 ) . The c o n j u g a t e was s t o r e d a t "4°C i n b u f f e r c o n t a i n i n g s o d ium a z i d e (3.6mM). b. Use of t h e S e p h a r o s e - P r o t e i n C o n j u g a t e A b a t c h p r o c e s s was u s e d f o r c o n v e n i e n c e . The sample, e i t h e r r a b b i t IgG o r m o n o c l o n a l a n t i b o d y , was added t o t h e 61 S e p h a r o s e s u s p e n s i o n and i n c u b a t e d a t 37°C f o r 2 h o u r s , t h e n washed w i t h PBS. The s u s p e n s i o n was r e s u s p e n d e d i n HC1 (0.001M) f o r 15 m i n u t e s and t h e s u p e r n a t a n t c o l l e c t e d i n b u f f e r . T h i s s t e p was r e p e a t e d and t h e comb i n e d s u p e r n a t a n t was d i a l y s e d a g a i n s t t h e r e q u i r e d b u f f e r . 16. MODIFICATION OF THE MONOCLONAL ANTIBODY WITH PEG 1900 The u s u a l a p p r o a c h t o p r e p a r i n g p r o t e i n - P E G c o n j u g a t e s i s t o s y n t h e s i z e an " a c t i v a t e d " PEG w i t h a r e a c t i v e f u n c t i o n a l g r o u p t h a t w i l l r e a d i l y c o u p l e w i t h a p r o t e i n f u n c t i o n a l g r o u p , u s u a l l y a p r i m a r y amine. I n t h i s s t u d y , PEG 1900 was a c t i v a t e d u s i n g c y a n u r i c c h l o r i d e ( 6 0 ) . S y n t h e s i s of 2 ( A l k o x y p o l y e t h y l e n e g l y c o x y ) - 4 , 6  d i c h l o r o t r i a z i n e (PCC) A r e a c t i o n scheme i s g i v e n i n F i g . 13. NaH ( 0 . l 8 g ) was s u s p e n d e d i n a n h y d r o u s benzene (50ml) and t h e s y s t e m was p u r g e d w i t h n i t r o g e n . PEG-1900 ( I 0 g ) i n a n h y d r o u s benzene (50ml) was ad d e d o v e r f i f t e e n m i n u t e s . The r e a c t i o n m i x t u r e was s t i r r e d f o r one hour u n d e r n i t r o g e n f l o w a n d one hour under a s t a t i o n a r y n i t r o g e n a t m o s p h e r e . A f o u r f o l d m o l a r e x c e s s o f c y a n u r i c c h l o r i d e (3.9g) was added a n d r e f l u x e d f o r t w e l v e h o u r s . The r e a c t i o n m i x t u r e was p o u r e d i n t o p e t r o l e u m e t h e r ( b . p . f r a c . 6 0 - 8 0 ° C , 150ml) a t 0°C and t h e p r e c i p i t a t e d p r o d u c t c o l l e c t e d by f i l t r a t i o n on Whatman #42 p a p e r . The p r o d u c t was r e d i s s o l v e d i n a minimum o f a n h y d r o u s a c e t o n e and r e p r e c i p i t a t e d w i t h two volumes o f p e t r o l e u m e t h e r a t 0°C. The r e p r e c i p i t a t i o n was r e p e a t e d t h r e e t i m e s . 62 PEG-OH • NaH RT. N2,2hr PEG-0"Ncf+ H-PEfr-O"* x N N II 12hr P E G - O ^ N ^ C l NaCl 1 P E G - O ^ N ^ C l 0 M b o r a , e - R l f 4 0 m m P E G - 0 ^ N ^ N H R Fig.13. Reaction scheme for protein modification v i t h PES vi a an activated cyanuric chloride derivative of PEG. 63 The PCC was vacuum d r i e d and s t o r e d a t -70°C under n i t r o g e n . The r e a c t i o n e f f i c i e n c y was d e t e r m i n e d by a s s a y i n g t h e h y d r o l y s a b l e c h l o r i d e s o f t h e PCC. D e t e r m i n a t i o n of H y d r o l y s a b l e C h l o r i d e s of PCC The B u c h l e r C o t l o v e C h l o r i d e T i t r a t o r u s e s t h e c o u l o m e t r i c p r i n c i p l e of t i t r a t i o n . S i l v e r i o n s a r e g e n e r a t e d a t t h e anode w h i c h r e a c t w i t h t h e c h l o r i d e i o n s and p r e c i p i t a t e o u t . When a l l t h e f r e e c h l o r i d e has r e a c t e d , t h e i n c r e a s e i n c u r r e n t i s d e t e c t e d as t h e end p o i n t . Of f o u r s a m p l e s o f a p p r o x i m a t e l y 40mg of PCC, two were h y d r o l y s e d i n N a 2 B M 0 7 (0.1M, pH 10) f o r a t l e a s t two h o u r s . The o t h e r two s a m p l e s were d i s s o l v e d i n t h e same b u f f e r , pH 9, i m m e d i a t e l y p r i o r t o t i t r a t i o n . A s o l u t i o n of n i t r i c a c i d (0.1M) and 10% a c e t i c a c i d (4ml) and 0.62% g e l a t i n ( 2 0 0 u l ) was added t o e a c h sample, a b l a n k and a N a C l s t a n d a r d . The f r e e c h l o r i d e s were t i t r a t e d u s i n g t h e c h l o r i d o m e t e r . R e a c t i o n o f PCC w i t h t h e M o n o c l o n a l A n t i b o d y P r e v i o u s s t u d i e s on m o d i f i c a t i o n o f a n t i b o d i e s w i t h PCC f o r use a s a f f i n i t y l i g a n d s f o u n d t h a t a 3:1 m o l a r r a t i o o f P C C : l y s i n e i n t h e r e a c t i o n m i x t u r e gave t h e b e s t compromise between a h i g h p a r t i t i o n c o e f f i c i e n t and r e t e n t i o n o f b i n d i n g a c t i v i t y i n t h e m o d i f i e d a n t i b o d y ( 2 0 ) . F o r t h i s r e a s o n t h e 3:1 r a t i o was u s e d t h r o u g h o u t t h i s s t u d y and i t was f o u n d more e f f e c t i v e t h a n t h e 1:1 r a t i o w h i c h was a l s o t r i e d . M o n o c l o n a l IgG ( a p p r o x i m a t e l y 2-8mg/ml f i n a l c o n c e n t r a t i o n ) was made up i n N a 2 B f l 0 7 (0.1M, pH 9) and PCC 64 was added t o g i v e a 3:1 P C C : l y s i n e m o l a r r a t i o ( a s s u m i n g 90 l y s i n e s p e r IgG m o l e c u l e (6.1). A f t e r f o r t y m i n u t e s a t room t e m p e r a t u r e , t h e r e a c t i o n was s t o p p e d by f r e e z i n g t h e m i x t u r e o r r e m o v a l of t h e u n r e a c t e d PEG by d i a l y s i s o r u l t r a f i l t r a t i o n , a l t h o u g h most o f t h e PCC was h y d r o l y s e d anyway. The d e g r e e of p r i m a r y amine m o d i f i c a t i o n was a s s a y e d by t r i n i t r o b e n z e n e s u l p h o n i c a c i d (TNBS) o r 4 p h e n y l s p i r o [ f u r a n - 2 H ( 3 H ) , 1 ' - p h t h a l a n ] - 3 , 3 ' d i o n e ( f l u o r e s c a m i n e ) . 17. DEGREE OF PEG 1900 MODIFICATION OF THE MONOCLONAL  ANTIBODY As PCC r e a c t s p r i n c i p a l l y w i t h t h e p r i m a r y amines o f a p r o t e i n , t h e amount o f PEG a t t a c h e d can be e s t i m a t e d by a s s a y i n g t h e p r i m a r y a m i n e s b e f o r e and a f t e r r e a c t i o n w i t h PCC. The most commonly u s e d method i s t h e TNBS a s s a y ( 6 2 ) , a c o l o u r i m e t r i c p r o c e d u r e . A n o t h e r a s s a y u s e s f l u o r e s c a m i n e w h i c h forms s t a b l e f l u o r o p h o r s w i t h p r i m a r y a m i n e s . The TNBS A s s a y To 1ml o f p r o t e i n s o l u t i o n (0-1mg/ml) were added 4% NaHC0 3, pH 9.5 (1ml) and 0.1% TNBS ( 1 m l ) . The s a m p l e s were v o r t e x e d t h e n i n c u b a t e d a t 37°C f o r two h o u r s . A f t e r i n c u b a t i o n 10% SDS (1ml) and HC1 (0.5M, 1ml) were a d d e d t o e a c h sample, v o r t e x e d and t h e a b s o r b a n c e r e a d on a H e w l e t t P a c k a r d 8450A u v / v i s s p e c t r o p h o t o m e t e r by t a k i n g t h e a v e r a g e a b s o r b a n c e o v e r a 330-340nm r a n g e . 65 The Fluorescamine Assay Fluorescamine reacts rapidly with primary amines at pH 8-9 to y i e l d highly fluorescent products (63, Fig.14a). The reaction i s complete within seconds and the excess reagent hydrolysed within a minute (64). The reagent i s water insoluble so i s added to a buffered protein solution in acetone or dioxane. The addition of organic solvents to polymer solutions often p r e c i p i t a t e s the polymer. This i s the case with dextran and the assay cannot be used for protein estimation in dextran-containing solutions such as the dextran-rich phase of two phase systems. Obviously amine-containing buffers cannot be used and the best buffers are phosphate or borate s a l t s . The widely used ninhydrin method for detection of amines uses the fluorescent ninhydrin derivatives of phenyl acetaldehyde, which i s formed in situ from phenylalanine and ninhydrin, and primary amine (65). Fluorescamine, however, reacts d i r e c t l y with primary amines to form the same fluorophors (66). To 1.5ml of protein solution (0-1uug) in 0.1M phosphate, pH 8, fluorescamine (0.5ml, 0.3mg/ml) in spectroscopic grade acetone was added while vortexing. After a minimum of seven minutes the fluorescence of the solution was measured on a Turner model 430 spectrofluorometer with an excitation wavelength of 390nm and emission detected at 475nm. A standard immunoglobulin solution was assayed simultaneously as the fluorescence reading depends on the 66 RNH-FLUORESCAMINE + H 2 0 COOH FLUOROPHOR HYDROLYSIS PRODUCTS F i g . 1 4 a . The r e a c t i o n o f f l u o r e s c a m i n e w i t h p r i m a r y amines t o y i e l d a h i g h l y f l u o r e s c e n t d e r i v a t i v e . RNH-RNH 0 2 N V ^ > - N 0 ^ NO-TNBS F i g . 1 4 b . The r e a c t i o n o f TNBS w i t h p r i m a r y a m i n e s t o y i e l d a d e r i v a t i v e w i t h a b s o r b a n c e a t 335nm. m a c h i n e s e n s i t i v i t y s e t t i n g . 18. ASSAY OF THE BINDING ACTIVITY OF THE PEG-ANTIBODY  CONJUGATE The E L I S A showed a n e g a t i v e r e s u l t f o r t h e P E G - m o d i f i e d a n t i b o d y . T h i s c o u l d be due e i t h e r t o t h e P E G - m o d i f i e d a n t i b o d y no l o n g e r b i n d i n g t h e r a b b i t IgG, o r t h e H R P - a n t i mouse IgG c o n j u g a t e no l o n g e r b i n d i n g t h e P E G - m o d i f i e d m o n o c l o n a l a n t i b o d y . To compare t h e b i n d i n g o f r a b b i t IgG by P E G - m o d i f i e d and n a t i v e m o n o c l o n a l a n t i b o d y , t h e a n t i b o d y was removed f r o m t h e m i c r o E L I S A p l a t e by d i s s o c i a t i n g t h e a n t i g e n - a n t i b o d y bond and a s s a y i n g t h e a n t i b o d y c o n c e n t r a t i o n by f l u o r e s c a m i n e . The method was t h e same a s t h e ELISA, e x c e p t t h e BSA b l o c k i n g s t e p was o m i t t e d , u n t i l t h e a d d i t i o n o f t h e H R P - a n t i mouse IgG c o n j u g a t e . I n s t e a d o f a d d i n g t h e c o n j u g a t e , 10% d i o x a n e i n H C l (0.01M, 200 u l p e r w e l l ) was added t o d i s s o c i a t e t h e r a b b i t I g G - m o n o c l o n a l a n t i b o d y bond ( 6 7 ) . A f t e r f i f t e e n m i n u t e s a t room t e m p e r a t u r e , t h e c o n t e n t s o f e a c h w e l l was p i p e t t e d i n t o 1.1ml p h o s p h a t e (0.1M), pH 8 and 0.2ml NaOH (0.01M). The f l u o r e s c a m i n e a s s a y was u s e d t o e s t i m a t e t h e bound p r o t e i n and an E L I S A a s s a y was r u n s i m u l t a n e o u s l y . The NaOH was added t o m a i n t a i n pH 8 s i n c e t h e r a t e o f t h e f l u o r e s c a m i n e r e a c t i o n i s pH d e p e n d e n t . 6 8 19. SINGLE TUBE PARTITION EXPERIMENTS The Two Phase System The following stock solutions were required to make up the 5% dextran, 3.4% PEG 8000 phase system. Dextran T500 (M =494,000, M =181,200) was obtained from Pharmacia and PEG . w n 8000 (Mw=6,000-7,000) was obtained from Union Carbide 1. Dextran. Dextran T500 (22g) was made up to I00g with water and s t i r r e d for two hours. The %w/v was determined by polarimetry, using a Steeg and Reuter polarimeter, on a 12.5 times d i l u t i o n of the stock solution using [ a ] D 5 = +199° 2. PEG. PEG 8000 (30g) was made up to 100ml with water. The %w/w was determined by measuring the r e f r a c t i v e index of a 6.25 times d i l u t i o n of the stock solution using a Bausch and Lomb "Serum Protein" Meter. (%w/w = RRI/SRRI.p.6.25 where RRI = r e f r a c t i v e index of PEG solution r e l a t i v e to water and SRRI = the s p e c i f i c r e f r a c t i v e index of PEG, p = density of PEG solution) 3. NaCl (0.6M) 4. Sodium phosphate buffer (0.3M, pH 7.2) The stock solutions and water were mixed to give a f i n a l o v e r a l l composition of 5%w/v dextran, 3.4%w/w PEG, 130mM NaCl and 1OmM phosphate (a 5,3.4 system). The phases were allowed to separate overnight or by centrifugation (ten minutes at 200g), then stored as upper and lower phase separately. 69 P a r t i t i o n o f t h e N a t i v e and M o d i f i e d A n t i b o d i e s V a r i o u s amounts of a n t i b o d y (up t o 200 u l ) , b o t h n a t i v e r a b b i t IgG and m o n o c l o n a l IgG o r P E G - m o d i f i e d m o n o c l o n a l IgG, were add e d t o 4ml o f 5,3.4 t o t a l phase s y s t e m . I f t h e r e s u l t i n g d i l u t i o n o f t h e phase s y s t e m f o r m e d one p h a s e , s t o c k PEG (approx.30%w/w) was added t o r e f o r m t h e p h a s e s (10-20 u l ) . The p h a s e s were m i x e d t w i c e a t e i g h t m i n u t e i n t e r v a l s and a f t e r e i g h t m i n u t e s , 2 0 u l s a m p l e s were t a k e n from t h e upper p h a s e . The s y s t e m was made i n t o one phase by t h e a d d i t i o n o f p h o s p h a t e b u f f e r (200 u l ) and 20 u l samples o f t h e t o t a l p h a s e s y s t e m were t a k e n . The p r o t e i n i n t h e upper p h a s e and t o t a l p h a s e s was a s s a y e d by f l u o r e s c a m i n e . P r e p a r a t i o n of E r y t h r o c y t e s f o r P a r t i t i o n i n g B l o o d was c o l l e c t e d i n t o 0.38% N a 3 C 6 H 5 0 7 ( f i n a l c o n c e n t r a t i o n ) from human o r r a b b i t d o n o r s . The e r y t h r o c y t e s were washed t h r e e t i m e s i n 10 volumes of PBS, t h e n f i x e d i n 1% g l u t a r a l d e h y d e f o r l a t e r use o r u s e d i m m e d i a t e l y . F i x e d c e l l s were washed t h r e e t i m e s i n t e n volumes o f PBS b e f o r e u s e . P a r t i t i o n o f C e l l s A s m a l l a l i q u o t of f r e s h o r f i x e d c e l l s was washed and r e s u s p e n d e d i n u p p e r p h a s e . The c e l l c o n c e n t r a t i o n i n t h e l o a d mix was d e t e r m i n e d by impedance c e l l c o u n t i n g on a P a r t i c l e D a t a I n c . E l e c t r o z o n e C e l l o s c o p e and was t y p i c a l l y 2 x 1 0 s c e l l s p e r m l . A p p r o x i m a t e l y 10 7 c e l l s were added t o 1ml o f p h a s e s y s t e m from t h e l o a d mix. O t h e r a d d i t i v e s i n c l u d e d v a r i o u s c o m b i n a t i o n s of a n t i b o d i e s o r b u f f e r s w h i c h 70 r e s u l t e d i n t h e f o r m a t i o n o f one p h a s e . An a l i q u o t was removed f o r c o u n t i n g t h e t o t a l c e l l c o n c e n t r a t i o n i n t h e phase s y s t e m and 10-20 u l o f s t o c k PEG s o l u t i o n was added t o r e f o r m t h e p h a s e s . The s y s t e m s were m i x e d t w i c e a t e i g h t m i n u t e i n t e r v a l s , s e t t l e d , t h e n t h e t o p p h a s e was s a m p l e d and t h e c e l l s c o u n t e d . The % o f c e l l s r e t u r n i n g t o t h e upper phase was c a l c u l a t e d and e x p r e s s e d as a p a r t i t i o n c o e f f i c i e n t . 700K FIBRINOGEN - f l ^ Q ^ ~ • H A P T O G L O B I N 2 - 1 200K • 19G 1 6 0 K — ^ f " T R A N S F E R R I N A L B U M I N 65K 1 • 1! • iTfzl F i g . 15. 3% SDS-PAGE of n a t i v e and reduced a n t i - N N g l y c o p h o r i n A r a b b i t IgG and mouse monoc lona l IgG. l a n e 1. S t a n d a r d m o l e c u l a r weight m a r k e r s . l ane 2. N a t i v e mouse monoc lona l I g G . l ane 3. Reduced mouse monoc lona l I g G . l ane 4. N a t i v e r a b b i t IgG . l ane 5. Reduced r a b b i t IgG. 72 C. RESULTS AND DISCUSSION 1. RABBIT ANTI-NN GLYCOPHORIN A The r a b b i t s r e s p o n d e d w e l l t o t h e NN g l y c o p h o r i n A i m m u n i s a t i o n and y i e l d e d a p p r o x i m a t e l y 40mg o f IgG on e a c h 40ml b l e e d . P r e c i p i t a t i o n of IgG by ( N H a ) 2 S O f l r e s u l t e d i n t h e c o - p r e c i p i t a t i o n o f c o n t a m i n a t i n g p r o t e i n s , m o s t l y IgM and f i b r i n o g e n . T h i s was m i n i m i s e d by a d d i n g t h e ( N H a ) 2 S 0 1 , as a s o l u t i o n r a t h e r t h a n a s o l i d t o a v o i d h i g h l o c a l c o n c e n t r a t i o n s c a u s i n g p r e c i p i t a t i o n , o f c o n t a m i n a t i n g p r o t e i n s . The w a s h i n g s t e p w i t h 40% s a t u r a t e d ( N H f l ) 2 S 0 i , a l s o r e d u c e d the l e v e l of c o n t a m i n a t i n g p r o t e i n s . D i a l y s i s a g a i n s t w a ter p r e c i p i t a t e d l i p o p r o t e i n s and t h e b u l k of t h e IgM. F i n a l p u r i f i c a t i o n on t h e FPLC mono Q column ( F i g . 9 ) shows t h a t t h e IgG was e l u t e d a s two p e a k s a t 8% a n d 12% N a C l . The f i r s t peak i s I g G 2 and t h e s e c o n d peak i s IgG, w h i c h i s t h e more b a s i c of t h e two IgG c l a s s e s t h u s b i n d i n g t o t h e column more s t r o n g l y and r e q u i r i n g a g r e a t e r i o n i c s t r e n g t h f o r e l u t i o n . The c l a s s of an IgG m o l e c u l e i s d e t e r m i n e d by t h e heavy c h a i n s t r u c t u r e and d e f i n e d by s i z e and e l e c t r o p h o r e t i c m o b i l i t y . T h r o u g h o u t t h i s s t u d y I g G 2 was u s e d due t o t h e p o s s i b i l i t y t h a t t h e m o n o c l o n a l a n t i b o d y r e c o g n i z e d a heavy c h a i n d e t e r m i n a n t o n l y p r e s e n t on I g G 2 , a s t h i s was t h e c l a s s u s e d i n t h e BALB/c mouse i m m u n i s a t i o n . In f a c t no d i f f e r e n c e was o b s e r v e d i n t h e a f f i n i t y o f t h e m o n o c l o n a l a n t i b o d y f o r I g G 2 o v e r IgG,. The IgM bound s t r o n g l y t o t h e column and was e l u t e d a t 55% N a C l . 73 The mono Q column i s a s t r o n g a n i o n e x c h a n g e r compared t o a DEAE c e l l u l o s e column w h i c h e x p l a i n s t h e h i g h c o n c e n t r a t i o n of N a C l r e q u i r e d t o e l u t e t h e IgG and IgM from t h e mono Q column compared t o t h e f o r m e r . The A c t i v i t y of t h e A n t i - N N G l y c o p h o r i n A IgG The a c t i v i t y o f t h e IgG was a s s a y e d by d i r e c t h a e m a g l u t i n a t i o n i n a m i c r o t i t r e p l a t e ( T a b l e 1 ) . B l o o d t y p e I n i t i a l r a b b i t Maximum Minimum IgG e r y t h r o c y t e e r y t h r o c y t e c o n e . ( m g / m l ) . a g g l u t i n a t i o n a g g r e g a t i o n d i l u t i o n c o n e , (mg/ml) NN 4.0 2" 9 0.008 MN 4.0 2" 8 0.016 MM 4.0 2~ 5 0.125 T a b l e 1. R e s u l t s o f m i c r o t i t r e o f r a b b i t a n t i - N N g l y c o p h o r i n IgG and human e r y t h r o c y t e s o f NN,MM o r MN s p e c i f i c i t i e s . E r y t h r o c y t e s were add e d a t 1% h a e m a t o c r i t . P r o t e i n c o n c e n t r a t i o n s d e t e c t e d by O D 2 8 o . The a n t i - N N g l y c o p h o r i n A IgG c a u s e d a g g r e g a t i o n o f NN e r y t h r o c y t e s a t a c o n c e n t r a t i o n f i f t e e n t i m e s as d i l u t e t h a n t h a t w h i c h c a u s e d a g g l u t i n a t i o n o f MM e r y t h r o c y t e s and t w i c e 74 as d i l u t e a s t h a t w h i c h c a u s e d a g g l u t i n a t i o n o f MN e r y t h r o c y t e s . T h i s d e g r e e o f c r o s s r e a c t i v i t y was e x p e c t e d w i t h a p o l y c l o n a l a n t i b o d y b e c a u s e t h e M and N a n t i g e n s s h a r e many common a n t i g e n i c d e t e r m i n a n t s . P r e v i o u s s t u d i e s by S h a r p (20) u s e d r a b b i t IgG w i t h a minimum a g g r e g a t i o n c o n c e n t r a t i o n o f 0.03mg/ml compared t o t h e v a l u e o f 0.008mg/ml f o r a n t i - N N g l y c o p h o r i n A IgG and NN e r y t h r o c y t e s ( T a b l e 1 ) . T h i s may be b e c a u s e t h e IgG i n t h i s s t u d y was r a i s e d a g a i n s t i s o l a t e d NN g l y c o p h o r i n A, a known s u r f a c e a n t i g e n , whereas t h e a n t i g e n u s e d by S h a r p was r a i s e d a g a i n s t whole e r y t h r o c y t e s . The p o s s i b i l i t y o f c o n t a m i n a t i n g a n t i g e n s t o d i l u t e t h e c e l l s u r f a c e a n t i g e n s i s f a r . g r e a t e r i n a sample o f e r y t h r o c y t e s t h a n i n a p u r i f i e d homogeneous p r o t e i n s a m p l e . A l s o t h e s h e a r f o r c e s i n p a s s i n g an e r y t h r o c y t e t h r o u g h a n e e d l e d u r i n g i m m u n i s a t i o n c o u l d r u p t u r e t h e c e l l s e x p o s i n g new a n t i g e n s . The b i n d i n g o f a n t i - N N g l y c o p h o r i n A IgG t o g l y c o p h o r i n A was a l s o e x amined u s i n g an E L I S A ( F i g . 1 6 ) . The g l y c o p h o r i n A (1 ug/ml) a d s o r b e d t o t h e m i c r o E L I S A p l a t e was bound by r a b b i t a n t i - N N g l y c o p h o r i n A, w h i c h was i n t u r n bound by m o n o c l o n a l a n t i - r a b b i t Fc f r a g m e n t and r e v e a l e d by t h e H R P - a n t i mouse IgG c o n j u g a t e . S i n c e t h e m o n o c l o n a l a n t i b o d y was u s e d w e l l above s a t u r a t i o n l e v e l , t h e a b s o r b a n c e a t 492nm g i v e s an i n d i c a t i o n of t h e d e g r e e o f b i n d i n g o f g l y c o p h o r i n A by a n t i - N N g l y c o p h o r i n A IgG. A t t h e minimum a g g r e g a t i o n c o n c e n t r a t i o n o f 0.008mg/ml from t h e m i c r o t i t r e r e s u l t s ( T a b l e 1 ) , t h e EL I S A a b s o r b a n c e i s 0.3, o n l y j u s t 75 1.0 2 . 0 3 . 0 4 . 0 5 . 0 6 . 0 7 . 0 8 . 0 9 . 0 L O G 2 DILUTION FACTOR F i g . 16. C o n c e n t r a t i o n d e pendence of b i n d i n g o f MN and NN g l y c o p h o r i n A by a n t i - N N g l y c o p h o r i n A r a b b i t IgG. The m i c r o E L I S A p l a t e was c o a t e d a t 1ug/ml a n d t h e i n i t i a l r a b b i t IgG c o n c e n t r a t i o n was 1mg/ml. The bound a n t i b o d y was r e v e a l e d u s i n g m o n o c l o n a l mouse a n t i - r a b b i t F c f r a g m e n t IgG, f o l l o w e d by HRP-anti-mouse IgG c o n j u g a t e and OPD. The ELISA a b s o r b a n c e i s p r o p o r t i o n a l t o t h e bound a n t i b o d y . 76 a b o v e b a c k g r o u n d a b s o r p t i o n . T h i s d e m o n s t r a t e s t h a t o n l y s m a l l amounts o f IgG need t o b i n d t h e e r y t h r o c y t e t o c a u s e a g g r e g a t i o n . The E L I S A was r e v e a l e d by b i n d i n g t h e m o n o c l o n a l a n t i b o d y t o t h e r a b b i t IgG, f o l l o w e d by t h e H R P - a n t i mouse IgG c o n j u g a t e . T h i s d e m o n s t r a t e d t h a t t h e m o n o c l o n a l a n t i b o d y would b i n d t h e r a b b i t IgG w h i c h was bound t o g l y c o p h o r i n A, s u g g e s t i n g b i n d i n g w o u l d a l s o o c c u r when e r y t h r o c y t e s were u s e d . SDS-PAGE of t h e R a b b i t IgG The SDS-PAGE r o d g e l s o f IgG a r e shown i n F i g . 1 5 i n two d i f f e r e n t a r r a n g e m e n t s t o f a c i l i t a t e c o m p a r i s o n s o f b o t h t h e n a t i v e and r e d u c e d IgG and t h e m o n o c l o n a l and p o l y c l o n a l IgG. The n a t i v e r a b b i t IgG r a n a s two bands i n SDS-PAGE ( F i g . 1 5 , l a n e 4 ) . The more i n t e n s e band h a s an a p p r o x i m a t e m o l e c u l a r w e i g h t o f 150,000 so i s l i k e l y t o be whole IgG. The l e s s i n t e n s e band has an a p p r o x i m a t e m o l e c u l a r w e i g h t of 300,000 and i s l i k e l y t o be a d i m e r o f IgG. The p r o b a b i l i t y t h a t t h e band a t 300K i s a d i m e r i s s u p p o r t e d by t h e f a c t t h a t t h e r e d u c e d IgG r u n s a s two bands, c o r r e s p o n d i n g t o h e a v y and l i g h t c h a i n s of IgG ( F i g . 1 5 , l a n e 5 ) . P r e s u m a b l y t h e d i m e r d i s s o c i a t e s on r e d u c t i o n . However, no c o n t a m i n a t i n g f i b r i n o g e n o r IgM bands were o b s e r v e d so t h e s a m p l e s were assumed t o be homogeneous. No d i f f e r e n c e s were o b s e r v e d i n SDS-PAGE o f IgG, a n d I g G 2 ( F i g . 1 1 ) . 77 2. PAPAIN DIGESTION OF RABBIT IMMUNOGLOBULIN G P a p a i n c l e a v e s p o l y p e p t i d e s and p r o t e i n s , n o n - s p e c i f i c a l l y a t t h e p e p t i d e bond one r e s i d u e removed t o w a r d s t h e C t e r m i n u s from a p h e n y l a l a n i n e r e s i d u e ( 6 8 ) , e.g. i H 2 N - A l a - P h e - T y r - L y s - A l a - C O O H L i m i t e d d i g e s t i o n a l l o w s c l e a v a g e below t h e h i n g e r e g i o n of IgG b u t above t h e hea v y c h a i n d i s u l p h i d e l i n k a g e ( F i g . 4 ) . However p r o l o n g e d e x p o s u r e of F c f r a g m e n t s t o p a p a i n y i e l d s a r a n g e o f s u b - f r a g m e n t s . P a p a i n i s a t h i o l p r o t e a s e w h i c h means t h a t t h e c y s t e i n e s u l p h y d r y l i n t h e a c t i v e s i t e must be i n t h e r e d u c e d f o r m f o r a c t i v i t y . Due t o t h i s t h e r e a c t i o n was c a r r i e d o u t under r e d u c i n g c o n d i t i o n s and t h e IgG was d i s s o c i a t e d i n t o heavy and l i g h t c h a i n s . The r e a c t i o n was m o n i t o r e d by FPLC b ut i t was not i m m e d i a t e l y o b v i o u s f r om t h e FPLC p r o f i l e whether t h e two pe a k s were F c and F ^ o r IgG hea v y and l i g h t c h a i n s . I t was f o u n d t h a t s t o p p i n g t h e d i g e s t i o n p r i o r t o c o m p l e t e f r a g m e n t a t i o n o f t h e IgG y i e l d e d t h e most F c f r a g m e n t . I f t h e d i g e s t i o n was c o n t i n u e d t o c o m p l e t i o n , a r a n g e o f low m o l e c u l a r w e i g h t s u b - f r a g m e n t s was f o r m e d . R e m a i n i n g u n d i g e s t e d IgG was removed d u r i n g FPLC s e p a r a t i o n o f t h e f r a g m e n t s ( F i g . 1 0 ) . FPLC F r a c t i o n a t i o n o f t h e P a p a i n D i g e s t F r a c t i o n s The p a p a i n d i g e s t f r a c t i o n s were f r a c t i o n a t e d on t h e mono Q a n i o n e x c h a n g e r w i t h an i o n i c s t r e n g t h g r a d i e n t o f 0.01-1M sodium a c e t a t e , pH 5.5 ( F i g . 1 0 ) . The F Q b f r a g m e n t o n l y bound t o t h e column a t low c o n c e n t r a t i o n s and i n 78 p r e p a r a t i v e c h r o m a t o g r a p h y was e l u t e d by t h e s t a r t i n g b u f f e r . The F c f r a g m e n t was e l u t e d l a s t and was u n a v o i d a b l y c o n t a m i n a t e d w i t h whole IgG when ex a m i n e d by SDS-PAGE ( F i g . 1 1 ) . S u b s e q u e n t t o t h i s p a r t o f t h e s t u d y , C l e a r d i n e t a l . ( 6 9 ) u s e d t h e FPLC mono Q column t o s e p a r a t e mouse IgG p a p a i n d i g e s t f r a g m e n t s and f o u n d a s i m i l a r c o n t a m i n a t i o n w i t h whole IgG. The F c f r a g m e n t f r o m t h e FPLC c a n o n l y be c o n s i d e r e d e n r i c h e d i n F c f r a g m e n t . P a p a i n d i d n o t b i n d t o t h e column and was e l u t e d i n t h e v o i d volume. C r y s t a l l i s a t i o n o f t h e F c f r a g m e n t y i e l d e d a homogeneous sample w h i c h r a n a s one band i n SDS-PAGE ( F i g . 1 1 ) but y i e l d s were t o o low f o r p r e p a r a t i v e u s e . As t h e h y b r i d o m a s were s e l e c t e d a s F ^ n o n - b i n d i n g as w e l l as F c b i n d i n g , t h e p r e s c e n c e o f whole IgG i n t h e F c f r a c t i o n was n o t a p r o b l e m . T h i s was d e m o n s t r a t e d by t h e W e s t e r n b l o t ( F i g . 2 1 a ) d i s c u s s e d l a t e r . 3. PRODUCTION AND SELECTION OF HYBRIDOMAS F u s i o n and g r o w t h o f c l o n e s p r o c e e d e d a s d e s c r i b e d e a r l i e r . H a l f t h e c l o n e s were c u l t u r e d i n medium c o n t a i n i n g LPS, a m i t o g e n w h i c h s t i m u l a t e s l y m p h o c y t e p r o l i f e r a t i o n ( 7 1 ) . The c e l l s c u l t u r e d i n LPS were s l i g h t l y s l o w e r t o b e g i n d i v i d i n g t h a n t h o s e w i t h o u t LPS, but d i v i d e d more r a p i d l y a f t e r t h e i n i t i a l l a g p h a s e . However no d i f f e r e n c e i n t h e t o t a l number o f c l o n e s was o b s e r v e d w i t h o r w i t h o u t LPS. Fig.10 CELL CULTURES ( x1000) 79 P 3 - N S 1 - A g 4 - 1 MYELOMA CELLS F i g . 1 7 . C e l l c u l t u r e s s h o w i n g d i v i d i n g m y e l o m a a n d h y d r i d o m a c e l l s , f i b r o b l a s t c o n t a m i n a t i o n a n d t h y m o c y t e f e e d e r c e l l s . 80 I n i t i a l l y 960 w e l l s were p l a t e d w i t h t h e f u s i o n p r o d u c t s and t h y m o c y t e f e e d e r c e l l s . Of t h e s e 240 showed h y b r i d o m a c l o n e s , many w e l l s c o n t a i n e d f r o m two t o t e n c l o n e s i n a s i n g l e w e l l . One h u n d r e d c l o n e s were e x a m i n e d by E L I S A f o r a n t i - r a b b i t IgG a c t i v i t y , f i f t y o f w h i c h were p o s i t i v e . T h e s e f i f t y c l o n e s were c u l t u r e d f o r one week, t h e n examined f o r F s p e c i f i c i t y . Ten c l o n e s bound t h e F c c f r a g m e n t but n o t t h e F a ^ f r a g m e n t . Over t h e n e x t two weeks t h r e e c l o n e s were l o s t a s a r e s u l t of o v e r g r o w t h by f i b r o b l a s t s ( s e e l a t e r i n d i s c u s s i o n ) and two c e l l l i n e s s t o p p e d p r o d u c i n g a n t i b o d y , p r e s u m a b l y due t o s p o n t a n e o u s chromosome l o s s . The f i v e r e m a i n i n g c l o n e s were d e s i g n a t e d A6,B4,D1,E4 and F1 by t h e n o r m a l c o n v e n t i o n of naming c e l l l i n e s a f t e r t h e c o o r d i n a t e s o f t h e w e l l s i n w h i c h t h e y o r i g i n a t e d . Ten o f t h e a n t i - w h o l e IgG p r o d u c i n g c l o n e s were r e c l o n e d by l i m i t i n g d i l u t i o n a s i t was n o t known i f t h e a n t i b o d y was c r o s s r e a c t i n g o r s e v e r a l c l o n e s were p r o d u c i n g d i f f e r e n t a n t i b o d i e s i n t h e same w e l l , i . e . t h e a n t i b o d y was n o t m o n o c l o n a l . A l t h o u g h F 0 was 0.54, w e l l above t h e 0.37 r e q u i r e d t o e n s u r e 95% p r o b a b i l i t y t h a t g r o w t h o r i g i n a t e d f r o m one ce lKsee r e c l o n i n g i n i n t r o d u c t i o n ) , no f u r t h e r c l o n e s were f o u n d t o be F c s p e c i f i c so i t was assumed t h a t t h e a n t i b o d i e s were c r o s s r e a c t i v e . A l l t h e a n t i - r a b b i t F c p r o d u c i n g c l o n e s were r e c l o n e d t o e n s u r e g e n e t i c s t a b i l i t y . D u r i n g t h e e a r l y s t a g e s o f c u l t u r e , f i b r o b l a s t c o n t a m i n a t i o n was o b s e r v e d i n t h e c e l l c u l t u r e s ( F i g . 1 7 ) . 1 6 0 K I B G — - f t P FT T 1 7 #. • k* i Ln i * f 1 2 I 3 I f 5 6 F i g . 1 8 . 3% SDS-PAGE o f n a t i v e p u r i f i e d m o n o c l o n a l a n t i b o d i e s . l a n e 1. P o l y c l o n a l r a b b i t I gG. l a n e 2. M o n o c l o n a l A6 f r o m c e l l c u l t u r e s u p e r n a t a n t l a n e 3. M o n o c l o n a l A6 f r o m a s c i t e s . l a n e 4. M o n o c l o n a l B4 f r o m c e l l c u l t u r e s u p e r n a t a n t l a n e 5. M o n o c l o n a l B4 f r o m a s c i t e s . l a n e 6. M o n o c l o n a l E6 f r o m a s c i t e s . l a n e 7. M o l e c u l a r w e i g h t m a r k e r s . ( 7 0 ) 82 The thymuses of v e r y young mice a r e r i c h i n f i b r o b l a s t s and t h e s o u r c e o f t h e c o n t a m i n a t i o n was t h e f o u r week o l d mice u s e d as t h y m o c y t e d o n o r s . F i b r o b l a s t s w i l l grow c o n t i n u o u s l y i n c u l t u r e and have t h e p o t e n t i a l t o o v e r g r o w c l o n e s . They s e t t l e t o t h e b o t t o m o f t h e c e l l c u l t u r e w e l l so r e p e a t e d c a r e f u l t r a n s f e r w i l l remove h y b r i d o m a s f r o m f i b r o b l a s t s . S e v e r a l a l i q u o t s o f e a c h a n t i - F c p r o d u c i n g c e l l l i n e were s t o r e d i n l i q u i d n i t r o g e n and two samp l e s of e a c h a n t i - w h o l e IgG p r o d u c i n g c e l l l i n e . 4. PURIFICATION OF THE MONOCLONAL ANTIBODY The m o n o c l o n a l a n t i b o d y was p u r i f i e d f r o m b o t h c u l t u r e s u p e r n a t a n t and a s c i t e s f l u i d by p r e c i p i t a t i o n w i t h (NHflJjSOfl, f o l l o w e d by FPLC p u r i f i c a t i o n on t h e mono Q column w i t h a 0.01-0.03M p h o s p h a t e , pH 8, i o n i c s t r e n g t h g r a d i e n t ( F i g . 1 2 ) . The p r o t e i n was e l u t e d a s one peak w i t h l i t t l e o r no c o n t a m i n a t i o n . Any m u r i n e c o n t a m i n a n t s f r o m t h e a s c i t e s were removed by t h i s p r o c e d u r e . The m o n o c l o n a l a n t i b o d i e s r a n as s i n g l e bands when n o n - r e d u c e d i n SDS-PAGE ( F i g . 1 8 ) , w i t h a p p r o x i m a t e m o l e c u l a r w e i g h t s o f 146,000. The c e l l l i n e B4 e i t h e r d i e d o r m u t a t e d w h i l e i n t h e mouse p e r i t o n e u m ( F i g . 1 8 , l a n e 5 ) , as t h e p r o t e i n p u r i f i e d f r o m B4 a s c i t e s had a M r i n e x c e s s o f 700,000 w h i c h had no a f f i n i t y f o r r a b b i t F c f r a g m e n t when a s s a y e d by E L I S A . I t c o u l d be n o n - s p e c i f i c IgM p r o d u c e d by t h e a s c i t e s d o n o r . 0.8 F i g . 1 9 . C o n c e n t r a t i o n d e p e n d e n c e o f t h e b i n d i n g o f r a b b i t F c by u n p u r i f i e d m o n o c l o n a l a n t i - r a b b i t F . The m i c r o E L I S A p l a t e was c o a t e d w i t h 1ug/ml o f r a b b i t F . The m o n o c l o n a l a n t i b o d y was u n p u r i f i e d i n c e l l c u l t u r e s u p e r n a t a n t ( a p p r o x . I0ug/ml) i n i t i a l l y d i l u t e d 200 f o l d . The h i g h e s t d i l u t i o n w i t h no a p p a r e n t d e c r e a s e i n b i n d i n g i s 6400 f o l d ( 1 . 5 n g / m l ) . 84 F i g . 2 0 . C o n c e n t r a t i o n d e p e n d e n c e o f t h e t h e b i n d i n g o f r a b b i t F c by p u r i f i e d m o n o c l o n a l a n t i b o d y . The p l a t e was c o a t e d a t 1ug/ml and t h e i n i t i a l m o n o c l o n a l a n t i b o d y c o n c e n t r a t i o n was 0.5mg/ml. The maximum d i l u t i o n w i t h no a p p a r e n t d e c r e a s e i n b i n d i n g i s 15ug/ml. 85 The m o n o c l o n a l a n t i b o d y p u r i f i e d from a s c i t e s had a g r e a t e r m o l e c u l a r w e i g h t t h a n t h a t p u r i f i e d f r o m c e l l c u l t u r e s u p e r n a t a n t (compare l a n e s 2 & 4 w i t h l a n e s 3 & 6, F i g . 1 8 ) . T h i s c o u l d e i t h e r be due t o t h e a s c i t e s o r i g i n a t i n g a n t i b o d y b e i n g bound t o a low m o l e c u l a r w e i g h t c o n t a m i n a n t or t h e c u l t u r e s u p e r n a t a n t o r i g i n a t i n g a n t i b o d y b e i n g c l e a v e d by n o n - s p e c i f i c p r o t e a s e s . The l a t t e r seems most l i k e l y a s t h e r e i s a b l u r r i n g o f t h e p r o t e i n band w h i c h i s c h a r a c t e r i s t i c of p r o t e o l y s i s . 5. ACTIVITY OF THE PURIFIED MONOCLONAL ANTIBODY The a c t i v i t y of t h e m o n o c l o n a l a n t i b o d i e s d e c r e a s e d a f t e r p u r i f i c a t i o n . T h i s i s d e m o n s t r a t e d by c o m p a r i n g an E L I S A o f p u r i f i e d and n o n - p u r i f i e d a n t i b o d y ( F i g s . 1 9 & 2 0 ) . The n o n - p u r i f i e d a n t i b o d y c o u l d be d i l u t e d s i g n i f i c a n t l y compared t o t h e p u r i f i e d a n t i b o d y b e f o r e b i n d i n g below s a t u r a t i o n l e v e l o f t h e r a b b i t F . T h i s l o s s i n a c t i v i t y may have been due t o n o n - s p e c i f i c p r o t e a s e s i n t h e a n t i b o d y s o l u t i o n o r t o t h e e f f e c t s o f p r e c i p i t a t i o n w i t h ( N H f t ) 2 S O i , . Samples o f a n t i b o d y were l e f t a t room t e m p e r a t u r e w i t h and w i t h o u t p r o t e a s e i n h i b i t o r , PMSF ( 7 2 , 7 3 ) , f o r one t o two d a y s . No f u r t h e r d e c r e a s e i n a c t i v i t y was o b s e r v e d i n samples w i t h or w i t h o u t p r o t e a s e i n h i b i t o r s s u g g e s t i n g t h a t p e r h a p s t h e p r e c i p i t a t i o n w i t h ( N H „ ) 2 S O , c a u s e d t h e l o s s i n a c t i v i t y . No f u r t h e r l o s s i n a c t i v i t y o c c u r r e d d u r i n g s t o r a g e a t - 7 0 ° C . 8 6 P r e c i p i t a t i o n w i t h ( N H i , ) 2 S 0 4 i s c o n s i d e r e d a g e n t l e , e f f e c t i v e and s i m p l e method o f r e d u c i n g t h e p r o t e i n l o a d on t h e i o n e x c h a n g e c o l u m n . The ( N H a ) 2 S O „ i o n s remove w a t e r o f h y d r a t i o n f r o m t h e p r o t e i n , e x p o s i n g h y d r o p h i l i c i o n i c g r o u p s t h u s d e c r e a s i n g t h e p r o t e i n s o l u b i l i t y . Such a p r o c e d u r e seems u n l i k e l y t o c a u s e a l a r g e l o s s o f a c t i v i t y b u t s e v e r a l s t u d i e s o f m o n o c l o n a l a n t i b o d i e s s u g g e s t t h a t t h e y may be p a r t i c u l a r l y s u s c e p t i b l e t o d e n a t u r a t i o n by ( N H « ) 2 S O « s o l u t i o n ( 7 4 , 7 5 ) . A n o t h e r p o s s i b i l i t y i s t h a t d e g r a d a t i o n and c o r r e s p o n d i n g l o s s o f a c t i v i t y o c c u r r e d d u r i n g p u r i f i c a t i o n , p r i o r t o r e m o v a l of p r o t e a s e s by FPLC. The S p e c i f i c i t y of t h e M o n o c l o n a l A n t i b o d y by t h e W e s t e r n  B l o t T e c h n i q u e The r e s u l t s a r e shown i n F i g . 2 l a . The m o n o c l o n a l a n t i b o d y r e c o g n i z e s t h e F c f r a g m e n t and h e a v y c h a i n , but n o t t h e F ^ f r a g m e n t o r l i g h t c h a i n o f r a b b i t IgG. T h i s i s as e x p e c t e d s i n c e t h e h e a v y c h a i n s i n c l u d e t h e F c r e g i o n o f t h e IgG m o l e c u l e . The l i g h t c h a i n i s n o t r e c o g n i z e d by t h e m o n o c l o n a l a n t i b o d y s i n c e t h i s i s n o t p a r t o f t h e F c f r a g m e n t . The m o n o c l o n a l a n t i b o d y d o e s n o t r e c o g n i z e t h e s m a l l f r a g m e n t s ( M r < l 5 K ) ) a r i s i n g f r o m t h e p r o l o n g e d e x p o s u r e o f r a b b i t IgG t o p a p a i n . 87 M o l . Wt. Std. 1 2 3 IgG, IgG2 94 K — 67 K — _ v N 45 K — 30K — v_ _ _ _ ' — *—^ 20 K — U.4K — — — F i g . 2 1 a . A W e s t e r n b l o t o f m o n o c l o n a l a n t i - F c f r a g m e n t a g a i n s t p a p a i n d i g e s t f r a g m e n t s of r a b b i t IgG and whole IgG. Bands r e c o g n i z e d by t h e a n t i b o d y a r e i n d i c a t e d 'zzz^ l a n e 1. P a p a i n d i g e s t m i x t u r e , l a n e 2. F ^ f r a g m e n t , l a n e 3. F c f r a g m e n t . l a n e 4. F f r a g m e n t by c r y s t a l l i s a t i o n . DIRECTION OF ^ PROTEIN MIGRATION F i g . 2 1 b . S c h e m a t i c r e p r e s e n t a t i o n o f e l e c t r o p h o r e t i c t r a n s f e r of p r o t e i n bands from a p o l y a c r y a m i d e g e l t o n i t r o c e l l u l o s e p a p e r . 88 6. SYNTHESIS AND ANALYSIS OF THE PEG-MONOCLONAL ANTIBODY  Analysis of PCC The PCC synthesis was in two steps, f i r s t the formation of the sodium salt of PEG-OH followed by a nucleophilic displacement of one of the chlorides of cyanuric chloride (Fig.13). The substitution of the f i r s t chloride deactivates the other two chlorides to nucleophilic substitution. The degree of substitution of cyanuric chloride was assayed by hydrolysis of the remaining chlorides and t i t r a t i o n against AgN03. The results are shown in Table 2. Sample Sample T i t r e T i t r e for % Free Mod of Wt. (mg) (mM CI') 100% C l " i n cyanuric y i e l d sample chloride PCC PCC PCC (H) PCC (H) 37.3 42. 1 37.2 39.9 NaCl Std. 0.1 (107mM) Blank 82 90 188 210 107 364 41 1 363 389 1 07 22.5 21.9 51.8 54.0 30.7% (H)=hydrolysed sample Table 2. Results of Free Chloride T i t r a t i o n of PCC 1900. 89 The t i t r a t i o n r e s u l t s i n T a b l e 2 i n d i c a t e t h a t 30.7% of t h e PEG 1900 was m o d i f i e d t o t h e e x t e n t of one c y a n u r i c c h l o r i d e m o l e c u l e a d d e d . The PCC was s t o r e d under n i t r o g e n a t - 7 0 ° C . Slow h y d r o l y s i s o c c u r e d and maximum s t o r a g e t i m e was one y e a r . R e a c t i o n o f PCC w i t h t h e M o n o c l o n a l A n t i b o d y The p r i m a r y amines of t h e a n t i b o d y e f f e c t e d a n u c l e o p h i l i c s u b s t i t u t i o n o f one of t h e r e m a i n i n g c h l o r i d e s on t h e PCC ( F i g . 1 3 ) . The r e m a i n i n g c h l o r i d e i s much l e s s r e a c t i v e b u t t h e r e does a p p e a r t o be some r e a c t i o n a t t h i s s i t e c r o s s - l i n k i n g t h e PEG t o t h e p r o t e i n ( 7 6 ) . The maximum r e a c t i o n t i m e i s 40 m i n u t e s b e c a u s e t h e c o m p e t i n g h y d r o l y s i s r e a c t i o n d e s t r o y s t h e f r e e PCC i n t h a t t i m e . T h e r e i s a l s o some e v i d e n c e t h a t t h e r e m a i n i n g c h l o r i d e o f t h e P C C - p r o t e i n c o n j u g a t e i s h y d r o l y s e d ( 7 7 ) . The d e g r e e of m o d i f i c a t i o n o f t h e m o n o c l o n a l a n t i b o d y was e s t i m a t e d by a s s a y i n g t h e p r i m a r y amines b e f o r e and a f t e r t h e l i n k a g e r e a c t i o n u s i n g t h e f l u o r e s c a m i n e and TNBS a s s a y s . 7. RESULTS OF THE TNBS AND FLUORESCAMINE ASSAYS B o t h f l u o r e s c a m i n e and TNBS a s s a y s gave l i n e a r s t a n d a r d c u r v e s f o r n a t i v e and m o d i f i e d a n t i b o d y ( F i g s . 2 2 a , 2 2 b ) . The c o r r e l a t i o n c o e f f i c i e n t s f o r b o t h a s s a y s a r e s i g n i f i c a n t a t t h e 0.95 c o n f i d e n c e l e v e l and t h e r e g r e s s i o n e q u a t i o n s a r e shown. The d e g r e e o f m o d i f i c a t i o n was c a l c u l a t e d f r o m t h e g r a d i e n t s o f t h e s t a n d a r d c u r v e s . 90 3.0 PROTEIN CONCENTRATION ug'ml F i g . 2 2 a . L i n e a r r e g r e s s i o n a n a l y s i s o f f l u o r e s c a m i n e s t a n d a r d c u r v e s f o r n a t i v e and P E G - m o d i f i e d mouse IgG. The r e g r e s s i o n e q u a t i o n s a r e Y=0.02+0.368X f o r n a t i v e mouse IgG and Y=0.047+0.24X f o r P E G - m o d i f i e d mouse IgG, where Y = f l u o r e s c e n c e i n t e n s i t y and X = p r o t e i n c o n c e n t r a t i o n ( u g / m l ) . The c o r r e l a t i o n c o e f f i c i e n t s a r e 0.95 and 0.96 r e s p e c t i v e l y . * — * N a t i v e mouse IgG P E G - m o d i f i e d mouse IgG 91 D e g . M o d n ( f l u o r e s c a m i n e ) = 1 - ( 0 . 2 4 0 / 0 . 3 6 8 ) = 0.35 Deg.Mod n(TNBS)=1-(1.248/1.754) = 0.29 The TNBS a s s a y was f o u n d t o p r o d u c e s y s t e m a t i c a l l y l o w e r r e s u l t s t h a n t h e f l u o r e s c a m i n e a s s a y . T h i s i s most l i k e l y due t o TNBS r e a c t i n g w i t h o t h e r n u c l e o p h i l e s on t h e p r o t e i n , s u c h a s s u l p h y d r y l g r o u p s ( 7 8 ) . PCC w i l l a l s o r e a c t w i t h o t h e r n u c l e o p h i l e s , however i t i s u n l i k e l y t o r e a c t t o t h e same e x t e n t as TNBS. The TNBS a s s a y has s e v e r a l p r a c t i c a l d i s a d v a n t a g e s i n t h a t t h e a s s a y u s e s l a r g e amounts of p r o t e i n ( i n t h e o r d e r of m i l l i g r a m s ) and i s n o t e a s i l y r e p r o d u c i b l e . The f l u o r e s c a m i n e a s s a y , however, u s e s s m a l l amounts o f p r o t e i n ( i n t h e o r d e r o f m i c r o g r a m s ) and i s r e p r o d u c i b l e . I t has been o b s e r v e d t h a t p r o t e i n amino g r o u p s may f a i l t o r e a c t w i t h TNBS and some of t h e t r i n i t r o p h e n y l a t e d g r o u p s on p r o t e i n s a r e i n e n v i r o n m e n t s w h i c h p e r t u r b t h e i r a b s o r p t i o n s p e c t r u m ( 7 9 ) . T h i s may a c c o u n t f o r some of t h e p r o b l e m s w i t h r e p r o d u c i b i l i t y . The f l u o r e s c a m i n e a s s a y has a 80-95% y i e l d o f p r i m a r y amine f l u o r e s c e n t d e r i v a t i v e s ( 8 0 ) . A n o t h e r a d v a n t a g e o f t h e f l u o r e s c a m i n e a s s a y i s t h a t f r e e PEG up t o a c o n c e n t r a t i o n o f 0.08% f i n a l c o n c e n t r a t i o n i n t h e a s s a y m i x t u r e does not i n t e r f e r e . T h i s means t h e f r e e PEG d o e s n o t have t o be removed f r o m t h e sample p r i o r t o t h e a s s a y . The a s s a y t h e r e f o r e c an be u s e d t o e s t i m a t e c o n c e n t r a t i o n s o f p r o t e i n i n p h a s e s y s t e m s . F r e e PEG doe s i n t e r f e r e w i t h t h e TNBS a s s a y and must be removed f r o m s a m p l e s p r i o r t o a s s a y ( 8 1 ) . 92 1.8 0.0 0 .2 0.4 0.6 0.8 1.0 PROTEIN CONCENTRATION mg/ml F i g . 2 2 b . L i n e a r r e g r e s s i o n a n a l y s i s o f TNBS s t a n d a r d c u r v e s f o r n a t i v e and P E G - m o d i f i e d mouse IgG. The r e g r e s s i o n e q u a t i o n s a r e Y=0.092+2.165X f o r n a t i v e mouse IgG and Y=0.032+1.547X f o r P E G - m o d i f i e d mouse IgG, where Y = a b s o r b e n c e and X = p r o t e i n c o n c e n t r a t i o n (mg/ml). The c o r r e l a t i o n c o e f f i c i e n t s a r e 1.00 and 0.99 r e s p e c t i v e l y . *--* N a t i v e mouse IgG <£--<£> P E G - m o d i f i e d mouse IgG 93 The mole r a t i o o f PEG 1900:IgG c a n be c a l c u l a t e d u s i n g a n o m i n a l r a t i o o f 90 l y s i n e s p e r mole ( 6 1 ) . The d e g r e e of m o d i f i c a t i o n o f 35% (by f l u o r e s c a m i n e ) r e p r e s e n t s t h e a t t a c h m e n t o f 31-32 m o l e c u l e s of PEG 1900 t o e a c h m o l e c u l e of IgG. However, t h i s d o e s n o t t a k e i n t o a c c o u n t any PEG 1900 bound t o o t h e r g r o u p s . 8. THE ACTIVITY OF THE PEG-MODIFIED ANTIBODY The b i n d i n g a c t i v i t y o f t h e m o d i f i e d a n t i b o d y was a s s a y e d a s d e s c r i b e d e a r l i e r . The amount o f m o d i f i e d a n t i b o d y bound was a s s a y e d by f l u o r e s c a m i n e and compared w i t h t h e r e s u l t s o f an E L I S A r u n s i m u l t a n e o u s l y . The r e s u l t s a r e shown i n F i g . 2 3 . A l t h o u g h t h e amounts o f a n t i b o d y bound were v e r y s m a l l , i t a p p e a r e d t h a t t h e n a t i v e m o n o c l o n a l a n t i b o d y had a 50 t i m e s g r e a t e r a f f i n i t y f o r t h e F c f r a g m e n t t h a n d i d t h e P E G - m o d i f i e d a n t i b o d y . The c o n t r o l s o f n a t i v e a n t i b o d y examined by E L I S A were c o n s i s t a n t w i t h t h e amount of a n t i b o d y bound a c c o r d i n g t o t h e f l u o r e s c a m i n e a s s a y . The amounts o f a n t i b o d y bound were t o o s m a l l t o e s t i m a t e a c c u r a t e l y w i t h t h e f l u o r e s c a m i n e a s s a y , i n t h e o r d e r of 0.2ng or l e s s , b u t an i n d i c a t i o n o f a s i g n i f i c a n t l o s s o f b i n d i n g a b i l i t y was a p p a r e n t . The i n a c t i v a t i o n o f t h e a n t i b o d y was most l i k e l y due t o s t e r i c i n t e r a c t i o n s c a u s e d by t h e b u l k o f t h e PEG r a t h e r t h a n t h e b l o c k a g e of t h e amine g r o u p s . T h i s i s s u g g e s t e d by t h e f a c t t h a t m o d i f i c a t i o n w i t h PEG 200 had l i t t l e e f f e c t on r a b b i t a n t i - h u m a n e r y t h r o c y t e a g g l u t i n a t i n g a b i l i t y , whereas 94 CO z =5 £ 0.021-O CO LU CC O 3 0 1 F i g . 2 3 . R e s u l t s of a b i n d i n g a s s a y f o r P E G - m o d i f i e d m o n o c l o n a l IgG t o r a b b i t IgG. A m i c r o E L I S A p l a t e was c o a t e d a t 1 ug/ml and t h e amount of n a t i v e and m o d i f i e d p r o t e i n bound t o t h e r a b b i t IgG was m e a s u r e d by t h e f l u o r e s c a m i n e a s s a y . A l l c o n c e n t r a t i o n s a r e t h e amount of m o n o c l o n a l a n t i b o d y r e a c t e d w i t h t h e bound r a b b i t IgG. 1. PEG 1900 m o d i f i e d m o n o c l o n a l a n t i b o d y (0.8mg/ml) 2. PEG 1900 m o d i f i e d m o n o c l o n a l a n t i b o d y (0.4mg/ml) 3. N a t i v e m o n o c l o n a l a n t i b o d y (0.9mg/ml) 4. N a t i v e m o n o c l o n a l a n t i b o d y (0.6mg/ml) f l u o r e s c e n t u n i t s E L I S A a b s o r b a n c e 95 PEG 5000 d e s t r o y e d a g g l u t i n a t i n g a b i l i t y a t t h e same d e g r e e o f m o d i f i c a t i o n ( 2 0 ) . However b e c a u s e t h e p a r t i t i o n c o e f f i c i e n t d e p ends e x p o n e n t i a l l y on t h e amount o f l i g a n d bound, even s m a l l amounts o f bound l i g a n d c a n have a s i g n i f i c a n t e f f e c t . 9. PARTITIONING OF NATIVE AND MODIFIED PROTEINS P a r t i t i o n i n g was p e r f o r m e d as d e s c r i b e d u s i n g a 5,3.4 s y s t e m . The p a r t i t i o n c o e f f i c i e n t s o f t h e m o n o c l o n a l a n t i b o d y a n d r a b b i t IgG, n a t i v e o r PEG m o d i f i e d shown a r e i n T a b l e 3. The p a r t i t i o n of t h e r a b b i t IgG was i n c r e a s e d f r o m 1.0 t o 1.94 by a d d i t i o n o f 29% PEG 1900 m o d i f i e d m o n o c l o n a l IgG but no s i g n i f i c a n t i n c r e a s e i n t h e p a r t i t i o n o f r a b b i t IgG was o b s e r v e d on a d d i t i o n o f 25% m o d i f i e d m o n o c l o n a l IgG. However, t h e 25% m o d i f i e d m o n o c l o n a l a n t i b o d y i t s e l f had a l o w e r p a r t i t i o n c o e f f i c i e n t t h e t h e 29% m o d i f i e d m o n o c l o n a l a n t i b o d y a n d would be e x p e c t e d t o have a l e s s e r e f f e c t on t h e p a r t i t i o n c o e f f i c i e n t o f t h e r a b b i t IgG. T h i s r e f l e c t s t h e l o s s i n a c t i v i t y o f t h e m o d i f i e d m o n o c l o n a l a n t i b o d y . The r e s u l t s i n e x p e r i m e n t s 1 a n d 2 show t h a t t h e m o d i f i e d m o n o c l o n a l a n t i b o d y i t s e l f has a p a r t i t i o n c o e f f i c i e n t 5 t o 10 t i m e s t h a t o f t h e n a t i v e a n t i b o d y , y e t t h e p a r t i t i o n o f t h e r a b b i t IgG i s i n c r e a s e d 2 t i m e s d e m o n s t r a t i n g t h a t t h e b i n d i n g i s n o t a s g r e a t a s i n i t i a l l y hoped f o r . Table 3. Partition of Modified and Native Antibodies detected by fluorescamine Expt. Cone, of 25% PCC-m/c in the Phase j System (ug/ml) Cone, of 29% PCC-m/c in the Phase , Sys tern (ug/ml) Cone, of Native m/c in the Phase . System (ug/ml) Cone, of rabbit IgG in the Phase , System (ug/ml) Borate Phosphate Partition Partition % of total buffer added' (ul) buffer added' (ul) Coefficient of PCC-m/c or m/c Coefficient of rabbit IgG protein in the upper Phase 1 2 3 4 5 6 7 8 13 13 13 13 13 13 20 20 20 20 75 75 35 35 5.3 ±0.15 10.0 ±0.1 5.3 3 103 1.0 ±0.1 1.0 ±o.i 4 84 91 1.1 ±0.1 66 1.9 ±0.2 75 1.0 ±0.05 50 51 1.0 ±0.1 50 1. Detection was by O.D. 2. Borate buffer (0.1M sodium borate, pH 9.0) Phosphate buffer (0.01M sodium phosphate, pH 8.0) 3. Values assumed from expts. 1 & 2 4. Values assumed from expts. 5 & 6 The degree of modification of the monoclonal antibody with PEG 1900 was assayed by fluorescamine. PCC-m/c = monoclonal antibody modified by attachment of PEG 1900 m/c = monoclonal antibody 97 10. E F F E C T I V E ATTACHED PEG 1900 As d e f i n e d e a r l i e r ( p . 1 6 ) , t h e p a r t i t i o n c o e f f i c i e n t of a m a c r o m o l e c u l e bound t o an a f f i n i t y l i g a n d , a s s u m i n g r e v e r s i b l e b i n d i n g , c a n be e x p r e s s e d by eqn [ 1 3 ] . I n t h e c a s e of t h e P E G - m o d i f i e d m o n o c l o n a l a n t i b o d y , t h e l i g a n d i s c o v a l e n t l y bound PEG 1900. T h e r e f o r e , t h e a s s o c i a t i o n T B c o n s t a n t s , K and K , a r e v e r y l a r g e and t h e same i n e i t h e r a a p h a s e . Eqn [ 1 3 ] , under t h e s e c o n d i t i o n s , s i m p l i f i e s t o , Km = K ° K 1 Or, i f n l i g a n d m o l e c u l e s w i t h p a r t i t i o n c o e f f i c i e n t a r e c o v a l e n t l y l i n k e d t o a p r o t e i n w i t h p a r t i t i o n c o e f f i c i e n t Kp, i f e a c h l i g a n d m o l e c u l e i s f u l l y e x p o s e d t o t h e s y s t e m and i f i t i s assumed t h a t a l l components e x h i b i t i d e a l b e h a v i o u r , t h e n t h e p a r t i t i o n c o e f f i c i e n t o f t h e m o d i f i e d p r o t e i n , K p j r c a n D e e x p r e s s e d by ( 1 4 ) , K , = K K n p i p i The K1 f o r r a d i o l a b e l e d PEG 1900 was measured a s 1.88±0.05. I f K i s t a k e n a s 1.0 ( T a b l e 3) and t h e a p p r o p i a t e P e x p e r i m e n t a l p a r t i t i o n c o e f f i c i e n t i s u s e d f o r K p j » t h e n t h e number o f e f f e c t i v e a t t a c h e d l i g a n d s c a n be c a l c u l a t e d a s 98 shown i n T a b l e 4. P C C t l y s i n e P a r t i t i o n A t t a c h e d PEG E f f e c t i v e m o l a r r a t i o c o e f f i c i e n t mol/mol a t t a c h e d PEG mol/mol 0 1.0 1 5.0 22-23 2-3 3 10.0 26-27 3-4 T a b l e 4. C a l c u l a t i o n o f t h e E f f e c t i v e Amount o f PEG 1900 A t t a c h e d t o t h e M o n o c l o n a l A n t i b o d y f r o m t h e P a r t i t i o n C o e f f i c i e n t . The e f f e c t i v e amount o f a t t a c h e d PEG 1900 was f o u n d t o be s e v e n t o e i g h t t i m e s s m a l l e r t h a n t h e a c t u a l v a l u e s measured by f l u o r e s c a m i n e . A p o s s i b l e r e a s o n f o r t h i s d i s c r e p a n c y i s t h a t i n c r e a s i n g amounts o f bound PEG 1900 may have l e s s e f f e c t as t h e y a r e i n c r e a s i n g l y h i d d e n f r o m t h e p h a s e s y s t e m by a l r e a d y a t t a c h e d PEG. The t h e o r y o u t l i n e d e a r l i e r assumes i d e a l thermodynamic b e h a v i o u r , w h i c h a p p a r e n t l y d o e s not a p p l y when more t h a n one l i g a n d i s bound p e r m a c r o m o l e c u l e . The e x p r e s s i o n s h o u l d a l s o i n c l u d e an a c t i v i t y c o e f f i c i e n t t o a l l o w f o r t h e a p p a r e n t s h i e l d i n g o f t h e i n n e r PEG m o l e c u l e s from t h e phase s y s t e m . T h i s h y p o t h e s i s i s s u p p o r t e d by t h e c h a r g e s h i e l d i n g e f f e c t s d e m o n s t r a t e d by DEAE c e l l u l o s e i o n e x c h a n g e c h r o m a t o g r a p h y o f P E G - m o d i f i e d IgG a t pH 9 ( d a t a n o t shown). The p i o f IgG i s 8, so a t pH 9 i t i s n e g a t i v e l y c h a r g e d and 99 b i n d s t o t h e column i n low s a l t . The m o d i f i e d IgG's w i t h l e s s l y s i n e s would be e x p e c t e d t o b i n d more s t r o n g l y . However, o n l y a s m a l l amount o f PEG 1900 bound t o t h e column u n d e r t h e s e c o n d i t i o n s and most was e l u t e d i n t h e v o i d v o lume. The n a t i v e IgG a c t u a l l y bound t o t h e column a t a g r e a t e r i o n i c s t r e n g t h t h a n t h e m o d i f i e d IgG. From t h i s e v i d e n c e i t seems l i k e l y t h a t t h e bound PEG r e d u c e s t h e m a g n i t u d e of t h e i n t e r a c t i o n between t h e p h a s e s y s t e m and t h e i n d i v i d u a l bound PEG m o l e c u l e . I t a l s o seems p o s s i b l e t h a t t h e same s t e r i c s h i e l d i n g p r e v e n t s t h e a n t i b o d y from c l o s e l y a s s o c i a t i n g w i t h t h e r a b b i t IgG, a c c o u n t i n g f o r t h e l o s s i n a c t i v i t y . The c a l c u l a t i o n o f t h e amount o f m o d i f i e d m o n o c l o n a l a n t i b o d y bound t o t h e r a b b i t IgG f r o m t h e p a r t i t i o n c o e f f i c i e n t i n a s i m i l a r way, r e q u i r e s s e v e r a l more a s s u m p t i o n s . S t a r t i n g w i t h t h e e x p r e s s i o n , K m = K o K j U ^ / K g 5 ) " eqn [17] T B and a s s u m i n g t h a t K =K , t h e n a v a l u e f o r n c a n be 3 3 c a l c u l a t e d f r o m K , = K K n p i p i However, t h e e x c l u s i o n o f t h e l i g a n d f r o m one of t h e p h a s e s i m p l i e s t h a t t h e s t a n d a r d s t a t e c h e m i c a l p o t e n t i a l of t h e l i g a n d i s d i f f e r e n t i n t h e two p h a s e s s i n c e t h e s t a n d a r d s t a t e c h e m i c a l p o t e n t i a l d e p ends on t h e t y p e and m a g n i t u d e of t h e i n t e r a c t i o n between t h e s o l u t e and s o l v e n t . The s t a n d a r d s t a t e c h e m i c a l p o t e n t i a l i s l i k e l y t o be l o w e r i n t h e p h a s e i n w h i c h i t i s most c o n c e n t r a t e d s i n c e t h e c h e m i c a l p o t e n t i a l i n e a c h p h a s e must be e q u a l and t h e 1 00 c h e m i c a l p o t e n t i a l i s d e p e n d e n t on t h e sum o f t h e s t a n d a r d s t a t e c h e m i c a l p o t e n t i a l and. t h e n a t u r a l l o g o r i t h m o f t h e s o l u t e a c t i v i t y . T h i s means t h a t K i s l i k e l y t o be g r e a t e r i n t h e b o t t o m p h a s e t h a n t h e t o p p h a s e , t h u s r e d u c i n g t h e T B r a t i o , K /K , s u g g e s t i n g t h a t t h e c a l c u l a t e d v a l u e f o r n i s 3 3 l i k e l y t o be s m a l l e r t h a n t h e a c t u a l v a l u e . U s i n g t h e e q u a t i o n ' K p i = K p K i > t n e e f f e c t i v e amount of a t t a c h e d m o d i f i e d m o n o c l o n a l a n t i b o d y i s a p p r o x i m a t e l y 0.3 mole p e r mole o f r a b b i t IgG. A l t h o u g h t h i s i s l i k e l y t o be a low e s t i m a t e , i t a g a i n r e f l e c t s t h e l o s s i n b i n d i n g a c t i v i t y o f t h e m o d i f i e d m o n o c l o n a l a n t i b o d y . No i n c r e a s e i n t h e p a r t i t i o n c o e f f i c i e n t of t h e r a b b i t IgG was o b s e r v e d on a d d i n g n a t i v e m o n o c l o n a l IgG. 11. PARTITION OF HUMAN ERYTHROCYTES IN THE ABSENCE OF  ANTIBODY B o t h f r e s h and f i x e d e r y t h r o c y t e s were u s e d i n i t i a l l y i n p a r t i t i o n i n g e x p e r i m e n t s . G l u t a r a l d e h y d e f i x a t i o n of e r y t h r o c y t e s i s due t o t h e r e a c t i o n of membrane and i n t r a c e l l u l a r p r o t e i n s , m o s t l y h a e m o g l o b i n , w i t h g l u t a r a l d e h y d e . T h i s m a i n t a i n s t h e c e l l s i n t h e i r o r i g i n a l c o n d i t i o n a s r e g a r d s s i z e , m o r p h o l o g y and s p a t i a l r e l a t i o n s h i p o f o r g a n e l l e s and m a c r o m o l e c u l e s f o r e x t e n d e d p e r i o d s o f t i m e ( 8 2 ) . F i x e d c e l l s were more c o n v e n i e n t t o use and p r o v i d e d a r e p r o d u c i b l e s u p p l y o f c e l l s , f o r t h e s e r e a s o n s t h e y were u s e d i n a l l t h e e x p e r i m e n t s r e p o r t e d i n t h i s t h e s i s . A n o t h e r a d v a n t a g e o f w o r k i n g w i t h f i x e d c e l l s 101 was t h e d e c r e a s e d t e n d e n c y o f f i x e d c e l l s t o a g g r e g a t e i n th e p h a s e s y s t e m compared t o f r e s h c e l l s . T h i s i s t h o u g h t t o be due t o t h e c e l l s d e c r e a s e d a b i l i t y t o d e f o r m . The a d d i t i o n of 0.01M p h o s p h a t e , pH 8 u s e d t o b u f f e r t h e p r o t e i n s had a s i g n i f i c a n t e f f e c t on t h e p a r t i t i o n of e r y t h r o c y t e s ( T a b l e 5 ) . F o r t h i s r e a s o n , i t was e s p e c i a l l y i m p o r t a n t t o run c o n t r o l s w i t h e v e r y e x p e r i m e n t . As c a n be seen from T a b l e 5 p h o s p h a t e had a f a r g r e a t e r e f f e c t t h a n N a C l . A l l t h e s y s t e m s had s i m i l a r t o t a l i o n i c s t r e n g t h s o f 84mM ±4mM. 0.1M 0.01M N a 2 B f l 0 7 , p H 9 p h o s p h a t e added added F r a c t i o n o f P a r t i t i o n c e l l s i n c o e f f i c i e n t u p p e r p h a s e 7 5 u l - 3% 0.03 3 5 u l 9% 0.10 7 5 u l 3 5 u l 11% 0.12 T a b l e 5. The E f f e c t of B u f f e r s on E r y t h r o c y t e P a r t i t i o n The i n c r e a s e i n e r y t h r o c y t e p a r t i t i o n i s due t o t h e p h o s p h a t e i o n s p a r t i t i o n i n g t o t h e l o w e r d e x t r a n r i c h phase c a u s i n g an e l e c t r o s t a t i c p o t e n t i a l between t h e two p h a s e s (83) and i n c r e a s i n g t h e s u r f a c e t e n s i o n . The N a C l and most l i k e l y t h e N a 2 B , 0 7 i o n s , s i n c e t h e y have a s m a l l e f f e c t on 1 02 e r y t h r o c y t e p a r t i t i o n , p a r t i t i o n e q u a l l y between t h e two p h a s e s and t h e s y s t e m w i t h o u t p h o s p h a t e i s e l e c t r i c a l l y n e u t r a l . The p h o s p h a t e c o n t a i n i n g s y s t e m (lOmM) i s ab o u t 2mV more p o s i t i v e i n t h e upper p h a s e ( 8 3 ) . Due t o t h i s p o t e n t i a l t h e p h o s p h a t e c o n t a i n i n g s y s t e m i s c h a r g e s e n s i t i v e and t h e n e g a t i v e l y c h a r g e d e r y t h r o c y t e w i l l p a r t i t i o n i n t o t h e upper p h a s e (83) . B e c a u s e t h e s y s t e m s u s e d were c l o s e t o t h e c r i t i c a l p o i n t , s m a l l u n a v o i d a b l e f l u c t u a t i o n s i n c o n d i t i o n s c a u s e d t h e p a r t i t i o n o f e r y t h r o c y t e s t o v a r y s l i g h t l y f r o m e x p e r i m e n t t o e x p e r i m e n t . To make t h e p a r t i t i o n r e p r o d u c i b l e , s u f f i c i e n t s t o c k s o l u t i o n o f PEG ( a p p r o x i m a t e l y 30%w/w) was add e d t o t h e c o n t r o l s and e x p e r i m e n t s i n e a c h r u n t o make t h e c o n t r o l p a r t i t i o n c o e f f i c i e n t a p p r o x i m a t e l y 10% so t h a t any i n c r e a s e i n p a r t i t i o n c o u l d be o b s e r v e d c l e a r l y . The e f f e c t s o f a n t i b o d i e s on e r y t h r o c y t e p a r t i t i o n a r e summarised i n T a b l e 6. I t seems t h a t o n l y t h e c o m b i n a t i o n o f r a b b i t IgG and P E G - m o d i f i e d m o n o c l o n a l a n t i b o d y i n c r e a s e d t h e p a r t i t i o n o f e r y t h r o c y t e s . A l l o t h e r c o m b i n a t i o n s of m o n o c l o n a l a n t i b o d y a n d / o r r a b b i t IgG had no e f f e c t o r d e c r e a s e d e r y t h r o c y t e p a r t i t i o n . The s m a l l d e c r e a s e i n e r y t h r o c y t e p a r t i t i o n c a u s e d by t h e n a t i v e r a b b i t IgG w i t h o r w i t h o u t m o n o c l o n a l a n t i b o d y was p r o b a b l y due t o t h e a g g r e g a t i o n o f e r y t h r o c y t e s by t h e r a b b i t IgG i n c r e a s i n g t h e r a t e of s e t t l i n g t o t h e i n t e r f a c e o r b o t t o m p h a s e . The d e c r e a s e i n p a r t i t i o n c a u s e d by t h e n a t i v e m o n o c l o n a l 103 a n t i b o d y i s s t a t i s t i c a l l y i n s i g n i f i c a n t . The m o d i f i e d m o n o c l o n a l a n t i b o d y had no e f f e c t i n t h e a b s e n c e o f r a b b i t IgG. A l t h o u g h t h i s i s a s m a l l i n c r e a s e i n p a r t i t i o n , i t i s s u f f i c i e n t t o p r o d u c e s e p a r a t i o n i n a c o u n t e r c u r r e n t d i s t r i b u t i o n a p p a r a t u s w i t h t w e n t y t r a n s f e r s . 104 12. THE EFFECT OF THE ANTIBODIES ON ERYTHROCYTE PARTITION E x p t . Amount of Amount of % c e l l s P.C. R a t i o o f a n t i b o d y b u f f e r i n upper p a r t i t i o n a d ded added p h a s e i n c r e a s e PCC-m/c r a b b i t IgG 31 ±4 0.45 3.75 3 4 5 PCC-m/c m/c r a b b i t IgG m/c r a b b i t IgG p h o s p h a t e 11 ±2 b o r a t e b o r a t e 11 ±2 p h o s p h a t e 7 ±1 b o r a t e 7 ±1 10 ±2 0.12 0.12 0.08 0.08 0.11 1 -0.7 -0.7 0.9 P.C. = P a r t i t i o n C o e f f i c i e n t PCC-m/c = PEG 1 9 0 0 - m o d i f i e d m o n o c l o n a l a n t i b o d y m/c = n a t i v e m o n o c l o n a l a n t i b o d y In a l l c a s e s t h e f o l l o w i n g amounts were u s e d : 91 u l p h o s p h a t e b u f f e r (0.01M, pH 7 . 2 ) , 73 u l b o r a t e b u f f e r (0.1M, pH 9.0), 0.37 mg n a t i v e o r c y a n u r i c c h l o r i d e m o d i f i e d m o n o c l o n a l a n t i b o d y a n d 0.48 mg r a b b i t IgG. The above a r e t h e a v e r a g e r e s u l t s t a k e n o v e r 10 e x p e r i m e n t s . T a b l e 6. The E f f e c t o f A n t i b o d i e s on E r y t h r o c y t e P a r t i t i o n 105 The t h e o r e t i c a l t r e a t m e n t a p p l i e d t o t h e e f f e c t of an a f f i n i t y l i g a n d on a s o l u t e c a n n o t be a p p l i e d t o p a r t i c l e a f f i n i t y p a r t i t i o n b e c a u s e t h e number o f b i n d i n g s i t e s i s much g r e a t e r and t h e c o n c e p t of c h e m i c a l p o t e n t i a l o f t h e p a r t i c l e d o e s not a p p l y . The change i n s u r f a c e f r e e e n e r g y of a p a r t i c l e on b i n d i n g a l i g a n d , i , i s o b t a i n e d i n s t e a d by i n t e g r a t i n g t h e G i b b s e q u a t i o n (84), d 7 = -Lr\ d * i i eqn[ 18] where r\ = s u r f a c e e x c e s s o f t h e i t h component. dn^ = t h e change i n c h e m i c a l p o t e n t i a l of t h e i t h component on b i n d i n g . To f i n d t h e d i f f e r e n c e i n s u r f a c e f r e e e n e r g y d i f f e r e n c e o f an e r y t h r o c y t e between t o p and b o t t o m p h a s e s on a d d i n g a l i g a n d , eqn [18] must be i n t e g r a t e d f r o m 0 t o t h e l i g a n d c o n c e n t r a t i o n and t h e s u r f a c e f r e e e n e r g y d i f f e r e n c e f o r an e r y t h r o c y t e i n t h e t o p phase s u b t r a c t e d f r o m t h a t i n t h e b o t t o m p h a s e . I f i t i s assumed t h a t t h e b i n d i n g of t h e l i g a n d d o e s n o t a f f e c t t h e p a r t i c l e s u r f a c e a r e a and o n l y t h e l i g a n d h a s a s i g n i f i c a n t e f f e c t on t h e p a r t i c l e s u r f a c e f r e e e n e r g y , t h e n t h i s g i v e s , d A 7 = d/ij - d ^ eqn [19] where 1 r e f e r s t o t h e l i g a n d . The c h e m i c a l p o t e n t i a l of t h e l i g a n d i n e i t h e r p h a s e i s g i v e n by n = M° + k T l n C . A s s u m i n g t h a t t h a t t h e s u r f a c e e x c e s s o f l i g a n d , 1^, o b e y s t h e L a n gmuir b i n d i n g i s o t h e r m (85) f o r n i n d e p e n d e n t b i n d i n g 106 s i t e s p e r u n i t a r e a ( c m 2 ) , i . e . n =(nC K ) / ( l + C K ) f o r t h e t o p p h a s e , t h e n t h e i n t e g r a t i o n and e v a l u a t i o n o f t h e l i m i t s g i v e s t h e f o l l o w i n g ( 8 4 ) , Ay^Ayo = nkT l n ( 1 + C B K B ) / ( 1 + C T K T ) eqn [ 2 0 ] where n = t h e s a t u r a t i o n v a l u e o f bound l i g a n d A7-^, A 7 o = s u r f a c e f r e e e n e r g y d i f f e r e n c e between t h e p h a s e s i n t h e p r e s e n c e , a b s e n c e o f a l i g a n d Knowing t h a t C T / C B = K 1 t h e n , A 7 1 - A 7 o = nkT l n l n V l / f n V R ^ eqn [21] T B I f i t i s assumed t h a t K =K and t h a t t h e l i g a n d i s an i r r e v e r s i b l y bound complex of m o d i f i e d m o n o c l o n a l a n t i b o d y and r a b b i t IgG ( t h e s e a s s u m p t i o n s were d i s c u s s e d e a r l i e r ) , t h e n t h e t o t a l number of bound l i g a n d s c a n be e s t i m a t e d from t h e e r y t h r o c y t e s u r f a c e f r e e e n e r g y change on b i n d i n g t h e l i g a n d . T h i s i s e s t i m a t e d f r o m t h e e r y t h r o c y t e p a r t i t i o n c o e f f i c i e n t s w i t h and w i t h o u t l i g a n d . T h e s e were m e a s u r e d e x p e r i m e n t a l l y and a r e l i s t e d i n T a b l e 6. The e q u a t i o n f o r p a r t i c l e p a r t i t i o n i s , K = n , / n 2 = e x p ( - A E / k T ) eqn [14] T h i s e x p r e s s i o n assumes t h a t c e l l p a r t i t i o n i s a t h e r m a l e q u i l i b r i u m p r o c e s s . In f a c t t h i s i s n o t t h e c a s e a s d i s c u s s e d on p.17-20. C h a r a c t e r i s t i c e r y t h r o c y t e p a r t i t i o n e n e r g i e s were f o u n d t o be l a r g e r t h a n p r e d i c t e d by t h i s e q u a t i o n , i n t h e o r d e r o f 2-20 x lO'kT p e r c e l l by K. S h a r p ( 2 6 ) . The v a l u e of 1 0 5 t h e r e f o r e i s u s e d t o s c a l e t h e B o l t z m a n n c o n s t a n t t h r o u g h o u t t h e c e l l s u r f a c e f r e e e n e r g y 107 c a l c u l a t i o n s . I t i s s t r a i g h t f o r w a r d t o show from thermodynamic c o n s i d e r a t i o n s t h a t ( 2 6 ) : AE = ( A T T B / 4 k T ) ( 1 - A 7 / 7 T B ) 2 B eqn [22] where A = p a r t i c l e s u r f a c e a r e a 7 T B = i n t e r f a c i a l t e n s i o n between t o p a n d b o t t o m phase A7 = d i f f e r e n c e i n c e l l s u r f a c e f r e e e n e r g y between t h e p h a s e s t h e r e f o r e l n K = ( A 7 T B / 4 b k T ) ( l - A 7 / 7 T B ) 2 eqn [23] where b d e n o t e s t h e e m p i r i c a l f a c t o r of 10 5 t o s c a l e t h e B o l t z m a n n c o n s t a n t t o a l l o w f o r t h e n o n - t h e r m a l d i s t r i b u t i o n o f e r y t h r o c y t e s . The s u r f a c e a r e a o f an e r y t h r o c y t e i s 1.4 x I 0 " 6 c m 2 and t h e i n t e r f a c i a l t e n s i o n o f t h e p h a s e s y s t e m u s e d was 4 x 10"* e r g / c m 2 . a. C a l c u l a t i o n o f A70, t h e e r y t h r o c y t e s u r f a c e f r e e e n e r g y d i f f e r e n c e w i t h o u t l i g a n d . The v a l u e f o r K 0 i s 0.12 f r o m e x p t . 2, T a b l e 6. S u b s t i t u t i n g t h e s e v a l u e s i n t o eqn [23] g i v e s , l n 0.12 = ( 1 . 4X 1 0 " 6 x 4X 1 0 " * ) / ( 4 x 10 s x 1.38 x 1 0 " 1 6 x 2 9 8 ) { 1 - A 7 § / ( 1 . 4 X 1 0 " 6 ) 2 } A 7 0 = 1 .11x10~ 5 e r g / c m 2 b. C a l c u l a t i o n o f A7^, t h e e r y t h r o c y t e s u r f a c e f r e e e n e r g y w i t h t h e maximum bound l i g a n d . The v a l u e f o r i s t a k e n f r o m e x p t . 1, T a b l e 6. l n 0.45 = O . 4 X 1 0 " 6 x 4X 1 0 " * ) / ( 4 x 10 5 x 1 . 3 8X 1 0 " 1 6 x 2 9 8 ) { 1 - A 7 ? / ( 1 . 4 X 1 0 " 6 ) 2 } 108 by1 = 0 . 6 8X 1 0 " 5 e r g / c m 2 By s u b s t i t u t i n g t h e s e v a l u e s f o r A7 0 and i n t o eqn [21] T B and a s s u m i n g t h a t K =K and t h e number of bound l i g a n d s i s a a T B above s a t u r a t i o n , i . e . n =n , t h e n n, t h e e f f e c t i v e number of l i g a n d s bound, can be e s t i m a t e d . The v a l u e o f i s t a k e n f r o m e x p t . 4, T a b l e 3. The c o r r e c t i o n f a c t o r f o r k i s no l o n g e r r e q u i r e d b e c a u s e t h e l i g a n d i s i n s o l u t i o n and t h e p r i n c i p l e s o f t h e r m a l e q u i l i b r i u m c a n be a p p l i e d . ( 0 . 6 8 - 1 . 1 1 ) x 1 0 - 5 = n x 1 . 3 8 X 1 0 ' 1 6 x 298 x In 1/1.9 n = 1.63x10 B cm" 2 As t h e s u r f a c e a r e a of an e r y t h r o c y t e i s 1 . 4X 1 0 ~ 6 cm" 2, t h e n t h e e f f e c t i v e number of l i g a n d s bound p e r c e l l = 1.63x10 s x 1.4x10" 6 = 228 l i g a n d s bound p e r c e l l The s a t u r a t i o n l e v e l o f t h e e r y t h r o c y t e f o r r a b b i t IgG was f o u n d t o be 8 x 1 0 5 m o l e c u l e s p e r c e l l i n PBS ( p e r s o n a l c o m m u n i c a t i o n - J . J a n z e n ) . A l t h o u g h a d i r e c t c o m p a r i s o n between K f o r t h e r a b b i t IgG i n PBS a n d p h a s e s y s t e m c a n n o t be made, t h i s s u g g e s t s t h a t many r a b b i t IgG m o l e c u l e s a r e bound t o t h e e r y t h r o c y t e and a g r e a t e r i n c r e a s e i n p a r t i t i o n c o u l d r e s u l t by i n c r e a s i n g K a f o r t h e m o d i f i e d m o n o c l o n a l a n t i b o d y . 109 13. THE EFFECT OF ALTERING THE RATIOS OF ANTIBODIES PCC-m/c R a b b i t P.C. C o n t r o l R a t i o o f E f f e c t i v e a dded IgG added P.C. p a r t i t i o n a t t a c h e d ( u l ) ( u l ) i n c r e a s e PEG (mol/mol) 35 65 0.25 0.1 2.5 1 .5 65 35 0.59 0.12 4.9 2.5 70 30 0.42 0.12 3.5 2.0 80 20 0.18 0.08 2.25 1 .3 P.C. = P a r t i t i o n C o e f f i c i e n t The a n t i b o d y c o n c e n t r a t i o n s u s e d were a s f o l l o w s : 0.37mg/ml PCC-m/c i n 0.1M b o r a t e (pH 9.0) 0.48mg/ml r a b b i t IgG i n 0.01M p h o s p h a t e (pH 7.2) C o n t r o l s c o n t a i n e d o n l y b u f f e r i n t h e same amounts as t h e e x p e r i m e n t . The a b o v e r e s u l t s a r e c o m b i n e d f r o m two e x p e r i m e n t s done i n d u p l i c a t e . T a b l e 7. The E f f e c t o f A l t e r i n g t h e R e l a t i v e Amounts o f A n t i b o d i e s on t h e P a r t i t i o n of Human E r y t h r o c y t e s The r e s u l t s i n T a b l e 7 d e m o n s t r a t e t h a t t h e p a r t i t i o n of e r y t h r o c y t e s i n c r e a s e s w i t h i n c r e a s i n g P E G - m o d i f i e d m o n o c l o n a l IgG c o n c e n t r a t i o n up t o a maximum r a t i o o f 65:35 m o d i f i e d m o n o c l o n a l I g G : r a b b i t IgG. T h i s was i n t e r p r e t e d as i n d i c a t i n g t h a t t h e d e c r e a s i n g r a b b i t IgG c o n c e n t r a t i o n 1 10 l i m i t e d t h e number o f b i n d i n g s i t e s f o r t h e m o d i f i e d m o n o c l o n a l a n t i b o d y . The optimum r a t i o (w/w) f o u n d w i t h i n t h i s r a n g e o f m o d i f i e d m o n o c l o n a l IgG t o r a b b i t IgG was 2. The p o l y c l o n a l a n t i b o d y was f o u n d t o be 8% a c t i v e by a f f i n i t y c h r o m a t o g r a p h y . T h i s i s a c o n s e q u e n c e o f i t ' s p o l y c l o n a l o r i g i n s . A s i m i l a r d e g r e e o f a c t i v i t y was f o u n d by b i n d i n g s t u d i e s w i t h e r y t h r o c y t e s ( r e s u l t s n o t shown). T h i s s u g g e s t s t h a t , f o r t h e p r e p a r a t i o n s u s e d , t h e optimum r a t i o w i t h i n t h i s r a n g e o f m o d i f i e d m o n o c l o n a l a n t i b o d y : a c t i v e r a b b i t IgG i s a c t u a l l y c l o s e r t o 65:3 o r 17:1(w/w). I f t h e m o d i f i e d a n t i b o d y were more a c t i v e , t h e n t h i s r a t i o w o u l d d e c r e a s e as more m o d i f i e d m o n o c l o n a l a n t i b o d y bound t o r a b b i t IgG. P r e i n c u b a t i o n o f t h e two a n t i b o d i e s a t 37°C, w i t h o r w i t h o u t e r y t h r o c y t e s , d i d n o t a l t e r t h e e r y t h r o c y t e p a r t i t i o n , t h e i n c r e a s e was seen when a l l components were p r e s e n t . T h i s i s a t r u e e q u i l i b r i u m as t h e r e s u l t was i n d e p e n d e n t o f t h e o r d e r i n w h i c h t h e r e a g e n t s were add e d . 111 14. THE EFFECT OF THE ANTIBODIES ON RABBIT ERYTHROCYTE PARTITION PCC-m/c R a b b i t IgG E r y t h r o c y t e % o f c e l l s P a r t i t i o n a d ded ( u l ) added ( u l ) s p e c i e s i n t h e c o e f f i c i e n t u pper p h a s e 65 35 human 35 0.53 human 10 0.11 65 35 r a b b i t 12 0.14 r a b b i t 12 0.14 A l l a n t i b o d y c o n c e n t r a t i o n s and c o n t r o l s a r e a s d e f i n e d f o r T a b l e 7. T a b l e 8. The E f f e c t o f A n t i b o d i e s on Human and R a b b i t E r y t h r o c y t e P a r t i t i o n The c o m b i n a t i o n o f m o d i f i e d m o n o c l o n a l a n t i b o d y and r a b b i t IgG had no e f f e c t on t h e p a r t i t i o n of r a b b i t e r y t h r o c y t e s , whereas t h e p a r t i t i o n o f human e r y t h r o c y t e s was t r i p l e d under t h e same c o n d i t i o n s a s shown i n T a b l e 7. The same r e s u l t was o b t a i n e d u s i n g a m i x t u r e o f r a d i o l a b e l l e d human e r y t h r o c y t e s and r a b b i t e r y t h r o c y t e s . I n t h i s c a s e t h e upper phase became e n r i c h e d w i t h human c e l l s . T h i s v e r i f i e s t h e s p e c i f i c i t y o f t h e t e c h n i q u e and c o n f i r m s t h a t t h e p a r t i t i o n i n c r e a s e o f human e r y t h r o c y t e s by m o d i f i e d m o n o c l o n a l 1 12 a n t i b o d y and r a b b i t IgG i s not due t o n o n - s p e c i f i c b i n d i n g . 1 1 3 15. SUMMARY The p a r t i t i o n o f e r y t h r o c y t e s was i n c r e a s e d s i g n i f i c a n t l y by a c o m b i n a t i o n o f PEG 1 9 0 0 - m o d i f i e d m o n o c l o n a l IgG and a n t i - e r y t h r o c y t e r a b b i t IgG. However, t h i s p a r t i t i o n i n c r e a s e was s i m i l a r i n m a g n i t u d e t o t h a t o b t a i n e d by d i r e c t m o d i f i c a t i o n o f t h e r a b b i t IgG ( 2 0 ) . A l t h o u g h t h e use o f t h e m o n o c l o n a l a n t i b o d y h a s s e v e r a l i n t r i n s i c a d v a n t a g e s , most i m p o r t a n t l y i t ' s v e r s a t i l i t y and h o m o g e n e i t y , i t was a l s o hoped t h a t t h e s e c o n d a n t i b o d y t e c h n i q u e m i g h t r e s u l t i n a g r e a t e r p a r t i t i o n i n c r e a s e . The m o n o c l o n a l a n t i b o d y was d e m o n s t r a t e d t o u n d e r g o a s i g n i f i c a n t l o s s i n b i n d i n g a c t i v i t y on m o d i f i c a t i o n w i t h a c t i v a t e d PEG 1900. As t h e p a r t i t i o n c o e f f i c i e n t i s d e p e n d e n t on t h e e x p o n e n t o f t h e number of bound l i g a n d s , any i n c r e a s e i n t h e b i n d i n g a c t i v i t y o f t h e m o d i f i e d m o n o c l o n a l a n t i b o d y s h o u l d r e s u l t i n a l a r g e i n c r e a s e of t h e p a r t i t i o n c o e f f i c i e n t . The power law dependence of p a r t i t i o n a l s o means t h a t t h e g r e a t e s t i n c r e a s e i n p a r t i t i o n would be o b s e r v e d i f t h e c o n t r o l p a r t i t i o n was c l o s e t o 1. I n t h i s c a s e , ( e x p ) - A E / k T t e n d s t o 1 t h e r e f o r e -AE/kT t e n d s t o 0 and t h e s y s t e m w i l l be s e n s i t i v e t o a s m a l l change i n c e l l s u r f a c e f r e e e n e r g y on b i n d i n g t h e l i g a n d . I f t h e c o n t r o l p a r t i t i o n s had been m a n i p u l a t e d t o a p p r o x i m a t e l y 1 i n s t e a d o f 0.1, p o s s i b l y a g r e a t e r p a r t i t i o n i n c r e a s e w o u l d have been o b s e r v e d . T h e r e a r e a t l e a s t two p o s s i b l e a p p r o a c h e s by w h i c h t h e p a r t i t i o n c o e f f i c i e n t may be i n c r e a s e d . One i s t o p r o t e c t 1 1 4 t h e a n t i b o d y b i n d i n g s i t e d u r i n g t h e m o d i f i c a t i o n r e a c t i o n o r t o e n r i c h t h e p r o p o r t i o n o f a c t i v e m o l e c u l e s r e m a i n i n g a f t e r m o d i f i c a t i o n . The o t h e r a p p r o a c h i s t o l o o k a t a l t e r n a t i v e c h e m i s t r y t o c y a n u r i c c h l o r i d e a t t a c h m e n t o f t h e PEG. O t h e r methods o f m o d i f y i n g p r o t e i n s have been e x t e n s i v e l y documented and t h e r e i s e v i d e n c e i n some c a s e s , f o r i n s t a n c e enzyme m o d i f i c a t i o n , t h a t some methods a l l o w a h i g h e r r e t e n t i o n o f enzyme a c t i v i t y t h a n c y a n u r i c c h l o r i d e a t t a c h m e n t . Such methods i n c l u d e : 1. F o r m a t i o n o f a 1 , 1 ' c a r b o n y l d i i m i d a z o l e a c t i v a t e d PEG s u b j e c t t o n u c l e o p h i l i c a t t a c k by p r o t e i n p r i m a r y amines ( 2 3 ) . 2. F o r m a t i o n o f a PEG e s t e r w i t h s u c c i n a m i d e f o l l o w e d by a n u c l e o p h i l i c s u b s t i t u t i o n o f t h e PEG s u c c i n a t e w i t h p r o t e i n p r i m a r y amines ( 7 8 ) . 3. R e a c t i o n o f a PEG s a l t w i t h d i t h i o c a r b o n a t e , f o l l o w e d by n u c l e o p h i l i c d i s p l a c e m e n t o f a C-S bond by p r o t e i n p r i m a r y amines ( 2 2 ) . 4. A d i r e c t r o u t e v i a r e d u c t i v e a m i n a t i o n o f PEG a l d e h y d e s by p r o t e i n p r i m a r y a m i n e s ( 8 6 ) . A l l o f t h e s e methods i n v o l v e m o d i f i c a t i o n o f t h e p r i m a r y a m i n e s . G l a s s has d e v e l o p e d a method u s i n g c y s t e i n e s u l p h y d r y l g r o u p s (87) and P o l l a k has r e a c t e d a PEG amine and p r o t e i n i n t h e p r e s c e n c e o f a c a r b o d i i m i d e h y d r o c h l o r i d e t o y i e l d amide l i n k a g e s ( 8 8 ) . I f t h e c o u p l i n g c h e m i s t r y i s c r u c i a l t o r e t e n t i o n of a c t i v i t y , i t i m p l i e s t h a t t h e c h e m i c a l n a t u r e o f t h e g r o u p 1 1 5 m o d i f i e d d e t e r m i n e s t h e e f f e c t on p r o t e i n a c t i v i t y . I f t h e l o s s o f b i n d i n g a c t i v i t y i s due i n s t e a d t o s t e r i c i n t e r f e r e n c e between bound PEG and t h e a n t i g e n , a s s u g g e s t e d by t h e e v i d e n c e q u o t e d i n t h e t h e s i s , i t i s u n l i k e l y t h a t t h e c o u p l i n g c h e m i s t r y i s t h e most i m p o r t a n t f a c t o r i n m a i n t a i n i n g a c t i v i t y . Hence, a l t h o u g h i n v e s t i g a t i o n i n t o a l t e r n a t i v e ways of m o d i f y i n g t h e a n t i b o d y may p r o v e f r u i t f u l , i t seems t h a t t h e s t e r i c i n t e r f e r e n c e w i t h b i n d i n g i s t h e r o o t o f t h e p r o b l e m . F o r t h i s r e a s o n I would s u g g e s t t h a t p r o t e c t i o n o f t h e a c t i v e s i t e d u r i n g m o d i f i c a t i o n o r e n r i c h m e n t o f a c t i v e m o l e c u l e s i s t h e d i r e c t i o n t h a t f u t u r e r e s e a r c h s h o u l d t a k e . One method by w h i c h t h i s m i g h t be done would be t o m o d i f y t h e whole complex o f m o n o c l o n a l and r a b b i t IgG i n s o l u t i o n , t h e n d i s s o c i a t e t h e bond and s e p a r a t e t h e a n t i b o d i e s by c h r o m a t o g r a p h y o r g e l f i l t r a t i o n . The p r o b l e m w i t h t h i s method i s t h a t an i o n exchange column c a n n o t be r u n under d i s s o c i a t i n g c o n d i t i o n s and t h e two m o l e c u l e s a r e t o o s i m i l a r i n s i z e t o be s e p a r a t e d by g e l f i l t r a t i o n . The F c f r a g m e n t a l o n e i s n o t p u r e , o n l y e n r i c h e d so i f t h i s were u s e d t h e sample w o u l d s t i l l be c o n t a m i n a t e d w i t h r a b b i t IgG and pose t h e same p r o b l e m , a l t h o u g h g e l f i l t r a t i o n c h r o m a t o g r a p h y may r e s o l v e t h e f r a g m e n t s . A more p r o d u c t i v e a p p r o a c h m i g h t be t o i m m o b i l i z e t h e a n t i g e n on a bead, b i n d t h e a n t i b o d y , c o u p l e t h e PEG, t h e n d i s s o c i a t e t h e a n t i g e n - a n t i b o d y bond and c o l l e c t t h e m o d i f i e d a n t i b o d y . CONCLUSION I t was d e m o n s t r a t e d t h a t a m o n o c l o n a l a n t i b o d y r a i s e d a g a i n s t t h e F c f r a g m e n t o f r a b b i t IgG and m o d i f i e d w i t h c y a n u r i c c h l o r i d e a t t a c h m e n t o f PEG 1900 i n c o m b i n a t i o n w i t h r a b b i t IgG r a i s e d a g a i n s t NN g l y c o p h o r i n A c o u l d i n c r e a s e t h e p a r t i t i o n o f human e r y t h r o c y t e s i n a two p h a s e aqueous p o l y m e r s y s t e m c o n t a i n i n g PEG and d e x t r a n . The p a r t i t i o n was n o t a l t e r e d by any of t h e components a l o n e nor was t h e p a r t i t i o n o f r a b b i t e r y t h r o c y t e s a f f e c t e d by t h e c o m b i n a t i o n o f a l l t h r e e components. I t seems t h a t m o n o c l o n a l a n t i b o d i e s and two phase aqueous p o l y m e r s y s t e m s a r e p o t e n t i a l l y u s e f u l f o r b i o l o g i c a l l y s p e c i f i c c e l l s e p a r a t i o n s . 1 16 GLOSSARY OF TERMS A n t i g e n i c i t y - an i n d i c a t i o n o f t h e r e a c t i v i t y o f an a n t i g e n w i t h an a n t i b o d y . B C e l l s - l y m p h o c y t e s l o c a l i s e d i n l y m p h o i d o r g a n s and t h e s p l e e n . They b i n d a n t i g e n s by means of s u r f a c e a n t i b o d i e s w h i c h s t i m u l a t e s t h e B c e l l t o d i v i d e and d i f f e r e n t i a t e . I n i t i a l s t i m u l a t i o n c a u s e s IgM s e c r e t i o n , f o l l o w e d by o t h e r i m m u n o g l o b u l i n s . B l a s t t r a n s f o r m a t i o n - s t i m u l a t i o n o f t h e l y m p h o c y t e p r o v o k i n g t r a n s f o r m a t i o n t o a r a p i d l y d i v i d i n g s t a t e . C a t a b o l i c r e g u l a t i o n - c o n t r o l o f d e g r a d a t i v e m e t a b o l i c p r o c e s s e s . Complement f i x a t i o n - a c h a i n r e a c t i o n i n i t i a t e d by C, component o f complement b i n d i n g t o t h e F c f r a g m e n t o f bound a n t i b o d i e s . The complement s y s t e m r e s u l t s i n c y t o l y s i s o f f o r e i g n p a r t i c l e s . D e x t r a n - p o l y a ( 1 , 6 ) D g l u c o s e . F i c o l l - a s y n t h e t i c c o p o l y m e r o f s u c r o s e and e p i c h l o r o h y d r i n . 1 17 1 1 8 H y b r i d o m a - a c o n t i n u o u s l y g r o w i n g c e l l l i n e formed by f u s i o n o f a m a l i g n a n t and n o r m a l c e l l . K l i g h t c h a i n - one of two t y p e s o f IgG l i g h t c h a i n , t h e o t h e r i s X. The c h a i n s d i f f e r c o n s i d e r a b l y i n amino a c i d s e q u e n c e and a n t i g e n i c i t y . I m m u n o g e n i c i t y - a measure o f t h e e f f e c t i v e n e s s o f a f o r e i g n s u b s t a n c e t o p r o v o k e an immune r e s p o n s e . I m m u n o s u p r e s s i o n - i n h i b i t i o n o f t h e immune r e s p o n s e Macrophage f i x a t i o n - s t i m u l a t i o n of macrophage p r o d u c t i o n . M a c r o p h a g e s d e s t r o y f o r e i g n p a r t i c l e s , p r o c e s s t h e a n t i g e n f o r t h e immune r e s p o n s e and a c t a s s e c r e t o r y c e l l s . Membrane t r a n s m i s s i o n - f o r m a t i o n of h o l e s i n t h e c e l l membrane of t h e a n t i g e n by a membrane-attack complex formed as a r e s u l t o f complement f i x a t i o n . Lysozyme c o m p l e t e s t h e a n t i g e n d e s t r u c t i o n by r u p t u r i n g t h e i n n e r c e l l membrane. Myeloma - a tumor c o n s i s t i n g o f a m a l i g n a n t f o r m of pl a s m a c e l l . N e u r a m i n i c a c i d -5 - a m i n o - 3 , 5 - d i d e o x y - D - g l y c e r o - D - g a l a c t o n o u l o s o n i c a c i d . 119 PEG - p o l y ( e t h y l e n e g l y c o l ) P l a s m a c y t o m a - s e e myeloma. S i a l o g l y c o p r o t e i n - p r o t e i n bound t o c a r b o h y d r a t e and s i a l i c a c i d . S i a l i c a c i d s a r e t h e N and 0 a c e t y l d e r i v a t i v e s o f n e u r a m i n i c a c i d . GLOSSARY OF SYMBOLS AND ABBREVIATIONS A p a r t i c l e s u r f a c e a r e a F F a r a d a y c o n s t a n t f m o l a l a c t i v i t y c o e f f i c i e n t K p a r t i t i o n c o e f f i c i e n t K a s s o c i a t i o n c o n s t a n t a k B o l t z m a n n ' s c o n s t a n t , 1 . 3 6 x 1 0 " 1 6 e r g / m o l e c u l e . K ° M n number a v e r a g e m o l e c u l a r w e i g h t M^ w e i g h t a v e r a g e m o l e c u l a r w e i g h t N A A v o g a d r o s number P number of p o l y m e r segments ( t h e r a t i o o f p o l y m e r m o l e c u l a r volume t o s o l v e n t m o l e c u l a r volume) PEG X p o l y ( e t h y l e n e g l y c o l ) , m o l . wt. a p p r o x . X g/mole R M o l a r gas c o n s t a n t , 8.314X10 7 e r g / m o l e . K ° z l a t t i c e c o o r d i n a t i o n number X F l o r y i n t e r a c t i o n p a r a m e t e r r \ s u r f a c e e x c e s s o f t h e i t h component 7 s u r f a c e f r e e e n e r g y 7 T B i n t e r f a c i a l t e n s i o n between t h e p h a s e s A E T I f r e e e n e r g y of p a r t i c l e / i n t e r f a c e a t t a c h m e n t AE,pB f r e e e n e r g y of p a r t i c l e t r a n s f e r between p h a s e s A G m f r e e e n e r g y of m i x i n g A H m e n t h a l p y o f m i x i n g A S m e n t r o p y o f m i x i n g A7 s u r f a c e f r e e e n e r g y d i f f e r e n c e between p h a s e s A7^,A7o s u r f a c e f r e e e n e r g y d i f f e r e n c e between p h a s e s i n t h e p r e s e n c e , a b s e n c e of a l i g a n d 120 121 A / i ? , j d i f f e r e n c e i n s t a n d a r d s t a t e c h e m i c a l p o t e n t i a l s between t h e p h a s e s n° ,u s t a n d a r d s t a t e c h e m i c a l p o t e n t i a l , c h e m i c a l p o t e n t i a l 0 volume f r a c t i o n \//,A\// G a l v a n i , or i n n e r p o t e n t i a l , p o t e n t i a l d i f f e r e n c e between t h e p h a s e s BIBLIOGRAPHY (1) H a n n i g , K . In "Methods i n M i c r o b i o l o g y " , v o l . 5 B , p.513. E d . by N o r r i s , J . R . and Ribbons,D.W. Academic P r e s s , New Y o r k . ( 1 9 7 1 ) (2) S t r i c k l e r , A . and S a c h s , T . Ann. N. Y. A c a d . S c i . 209, 497.(1973) (3) Melamed,M.R., M u l l a n e y , P . F . and Mendelsohn,M.L. (Ed.) Flow C y t o m e t r y and C e l l S o r t i n g , p.307. W i l e y , New Y o r k . ( 1 9 7 7 ) (4) Salzman,G.C., C r o w e l l , J . M . , Good,C.A., Hansen,K.M., H i e b e r t ,R.D. , Labauve , P.M. , M a r t i n , J . C , Ingram,M.L. and M u l l a n e y , P . F . C l i n . Chem. 21, 1297.(1975) (5) G h e t i e , V . , Mota,G. and S j o q u i s t , J . J . Immunol. M e t h o d s . 21, 133.(1978) (6) M a r s h a k - R o t h s t e i n , D . , F i n k , P . , G r i d l e y , T . , R a u l e t , D . H . , B a v a n , J . and G o f t e r , M . L . J . Immunol. 122, 2491.(1979) (7) Le Bien,T.W., Sepan,D.E., Bartholomew,R.M., Stong,R.C. and A n d e r s o n , J . M . B l o o d 65, 945.(1985) (8) A l b e r t s s o n , P - A . P a r t i t i o n of C e l l P a r t i c l e s and M a c r o m o l e c u l e s (2nd e d . ) . W i l e y , New Y o r k . ( 1 9 7 1 ) (9) W a l t e r , H . In "Methods of C e l l S e p a r a t i o n " , v o l . 1 , p.307. E d . by C a t s i u m p o o l a s , D . N . W i l e y , New Y o r k . ( 1 9 7 7 ) (10) A l b e r t s s o n , P - A . Biochem. e t B i o p h y s . A c t a . 27, 378.(1958) (11) P a u l i , W . and Rona,F. H o f m e i s t e r s B e i t r . 2, 1.(1902) (12) F l o r y , P . J . P r i n c i p l e s o f Polymer C h e m i s t r y . C o r n e l l U n i v e r s i t y P r e s s , New Y o r k . ( 1 9 5 3 ) (13) Huggins,M.L. J . Chem. P h y s . 9, 440.(1941) (14) B r o o k s , D . E . and Sharp,K.A. In " P a r t i t i o n i n g i n Aqueous Two P h a s e S y s t e m s " Chap.2. Ed . by W a l t e r , H . S . , B r o o k s , D . E . and F i s h e r , D . A cademic P r e s s , New Y o r k . ( 1 9 8 6 ) (15) B r o o k s , D . E . , Sharp,K.A., Tamblyn,C.H., Seaman,G.V.F. and W a l t e r , H . J . C o l l o i d I n t e r f a c e S c i . 102, 1.(1984) (16) E r i k s s o n , E . , A l b e r t s s o n , P - A . and J o h a n s s o n , G . M o l e c u l a r and C e l l u l a r Biochem. 10, 123.(1976) (17) Van A l s t i n e , J . M . and B r o o k s , D . E . C l i n . Chem. 30, 122 123 (18 (19 (20 (21 (22 (23 (24 (25 (26 (27 (28 (29 (30 (31 (32 (33 (34 (35 (36 (37 441(1986) F l a n a g a n , S . D . , B a r o n d e s , S . H . a n d T a y l o r , P . J . B i o l . Chem. 251, 858.(1976) L i n g , T . G . I . , Ramstorp,M. and M a t t i a s o n , B . A n a l . B i ochem. 122, 26.(1982) Sharp,K.A., Y a l p a n i , M . , Howard,S.J. and B r o o k s , D . E . A c c e p t e d f o r p u b l i c a t i o n . A n a l . Biochem. (1986) K i n g , T . P . and W e i n e r , C . I n t . J . P e p t i d e P r o t e i n R es. 16, 147.(1980) Beauchamp,C.O., G o n i a s , S . L . , Menapace,D.P., and P i z z o , S . V . A n a l . B iochem. 131, 25.(1983) B e n d i c h , A . , K a f k e w i t z , D . , Abuchowski,A. and D a v i s , F . F . C l i n . Exp. Immunol. 48, 273.(1982) A l b e r t s s o n , P - A . and B a i r d , G . D . Exp. C e l l R e s . 28, 296.(1962) E v a n s , E . A . B i o p h y s . J . 30,265.(1980) Sharp,K.A. T h e o r e t i c a l and E x p e r i m e n t a l S t u d i e s on E r y t h r o c y t e P a r t i t i o n i n Aqueous Polymer Two Phase S y s t e m s . Ph.D. T h e s i s . U n i v e r s i t y o f B r i t i s h C o l u m b i a . ( 1 9 8 5 ) E a s t , I . J . , H u r r e l l , J . G . R . , Todd,P.E.E. and L e a c h , S . J . J . B i o l . Chem. 257, 3199.(1982) B e r z o f s k y , J . A . , Buckmeyer,G.K., H i c k s , G . , Gurd,F.R.N., F e l d m a n n , R . J . and M i n n a , J . J . B i o l . Chem. 257, 3189.(1982) B l u m e n f i e l d , 0 . 0 . and Adamany,A.M. P r o c . N a t . A c a d . S c i . U.S.A. 75,2727.(1978) F u r t h m a y r , H. J . S u p r a m o l . S t r u c t u r e . 9, 79.(1978) A n s t e e , D . J . S e m i n a r s i n H e m a t o l o g y . 18, 1, 13.(1981) Cotton,R.G.H. and M i l s t e i n , C . N a t u r e . 224, 42.(1973) N o s s a l , G . J . V . and L e d e r b e r g , J . N a t u r e . 181, 1419.(1958) Okada,Y. Exp. C e l l R es. 26, 98.(1962) L i t t l e f i e l d , J . W . S c i e n c e . 145, 709.(1964) P o t t e r , M . and Boyce,C.R. N a t u r e . 193, 1086.(1962) H o r i b a t a , K . and H a r r i s , A . W . E x p . C e l l R es. 60, 1 (38 (39 (40 (41 (42 (43 (44 (45 (46 (47 (48 (49 (50 (51 (52 (53 (54 (55 (56 (57 (58 6 1.(1970) K i n g , T . P . and W e i n e r , C . I n t . J . P e p t i d e P r o t e i n Res. 16, 147.(1980) P o t t e r , M . Adv. Immunol. 25, 141.(1977) P o t t e r , M . P h y s i o l . Rev. 52, 631.(1972) Blow,A.M.J., Botham,G.M., F i s h e r , D . , G o o d a l l , A . H . , T i l c o c k , C . P . S . and L u c y , J . A . FEBS L e t t . 94, 305.(1978) R i n g e r t z , N . R . and Savage,R.E. C e l l H y b r i d s . A c a d e m i c P r e s s , New Y o r k . ( 1 9 7 6 ) L e r n h a r d t , W . , A n d e r s o n , J . , C o u t i n h o , A . and M e l c h e r s , F Exp. C e l l Res. 111, 309.(1978) G a l f r e , G . and M i l s t " e i n , C . M e t h . E n z y m o l . 73, 1 .(1978) H e n g a r t n e r , H . , L u z z a t i , A . L . and S c h r e i e r , M . C u r r . T o p i c s M i c r o b i o l . Immunol. 81, 92.(1978) L e f k o v i t s , I and Waldman,H. L i m i t i n g D i l u t i o n A n a l y s i s of C e l l s i n t h e Immune S y s t e m . C a m e b r i d g e U n i v e r s i t y P r e s s , C a m e b r i d g e . ( 1 9 7 9 ) A n d e r s s o n , J . and M e l c h e r s , F . C u r r . T o p i c s M i c r o b i o l . Immunol. 81, 130.(1978) E s h h a r , Z . , B l a t t , C , Bergman,Y. and H a i m o v i t c h , J . J . Immunol. 122, 240.(1979) K o h l e r , G . and M i l s t e i n , C . N a t u r e . 256, 495.(1975) G a l f r e , G . and M i l s t e i n , C . Meth. E n z y m o l . 73, 3.(1981) M a r c h e s i , V . T . and Andrews,E.P. S c i e n c e . 174, 1247.(1971) P o r t e r , R . R . Biochem. J . 73, 119.(1959) U t s u m i , S . Biochem. J . 112, 343.(1969) Keogh, R. and S t a n w o r t h , D.R. In "Handbook o f Immunology" 3 r d E d . p.6.17. E d . by W i e r . (1978) D a v i s , B . J . N.Y. A c a d . A n n u a l s . 121, 404.(1964) O r n s t e i n , L . N.Y. A c a d . A n n u a l s . 121, 321.(1964) F a i r b a n k s , G . , S t e c k , T . L . and W a l l a c h , D . F . H . B i o c h e m . 10, 2606.(1971) C a l a b r e s i , P . and P a r k s , R . E . In "The P h a r m a c o l o g i c a l 125 (59 (60 (61 (62 (63 (64 (65 (66 (67 (68 (69 (70 (71 (72 (73 (74 (75 (76 (77 B a s i s o f T h e r a p u t i c s " 5 t h Ed., P1254-1309. E d . by Goodman,L.S. and G i l m a n , A . M a c M i l l a n , New Y o r k . ( 1 9 7 5 ) Burnette,W.N. A n a l . Biochem. 112, 195.(1981) Abuchowski,A., Van E s , T . , P a l c z u k , N . C . and D a v i s , F . F . J . B i o l . Chem. 252, 3578.(1977) Edelman,G.M., Cunningham,B.A., G e l l , W . E . , G o t t l i e b , P . D . , R u t i s h a u s e r , U . and Waxdal,M.J. P r o c . N a t l . A c a d . S c i . 63, 78.(1969) Habeeb,A.F.S.A. A n a l . B iochem. 14, 328.(1966) B 6 h l e n , P . , S t e i n , S . , Dairman,W. and U d e n f r i e n d , S . A r c h . B i o c h e m . B i o p h y s . 155, 213.(1973) U d e n f r i e n d , S . , S t e i n , S . , B 6 h l e n , S . , Dairman,W., Leimgruber,W. and W e i g e l e , M . S c i e n c e 178, 871.(1972) Okuyama,T. and S a t a k e , K . J . J . B i o c h e m . 47, 454.(1960) Samejima,K., Dairman,W. and U d e n f r i e n d , S . A n a l . Biochem. 42, 222.(1971) A n d e r s s o n , K . , Benyamin,Y., Douzou,P. and B a l n y , C . J . Immunol. Methods 23, 17.(1978) O i , V . T . and H e r z e n b e r g , L.A. M o l e c . Immunol. 16, 1005.(1979) C l e a r d i n , P . , M c G r e g o r , J . L . , Manach,M., B o u k e r c h e , H . and Dechavanne,M. J . C h r o m a t o g r . 319, 67.(1985) Putnam,F.W. Plasma P r o t e i n s 2nd E d . , v o l 1. p.1-55, Academic P r e s s , New Y o r k . ( 1 9 7 5 ) Houslay,M.D. and S t a n l e y , K . K . In "Dynamics o f B i o l o g i c a l Membranes" p.141, W i l e y , New Y o r k . ( 1 9 8 2 ) Ooms,A.J.J. N a t u r e . 190, 533.(1961) F a h r n e y , D . E . and Gold,A.M. J.A.C.S. 85, 997.(1963) B r u c k , C , P o r t e l l e , D . , G l i n e u r , C . and B o l t o n , A . J . Immunol. M e t h o d s . 53, 313.(1982) M c G r e g o r , J . L . , B r o c h i e r , J . , W i l d , F . , F o l l e a , G . , T r z e c i a k , M . C . , James,E., Dechavanne,M., M c G r e g o r , L . and C l e m e t s o n , K . J . E u r . J . B i o c h e m . 131, 427.(1983) B o c c u , E . , V e l o , G . P . and V e r o n e s e , F . M . Pharm. Res. Comm. 14, 2, 113.(1982) K i n g , T . P . , Kochoumian,L. and C h i o r a z z i , N . J . E x p . Med. 126 149, 424.(1979) (78) B o c c u , E . , L a r g a j o l l i , R . and V e r o n e s e , F . M . Z. N a t u r f o r s c h . 38c, 94.(1983) (79) G l a z e r , A . N . , D e l a n g e , R . J . and Sigman,D.S. In " C h e m i c a l M o d i f i c a t i o n of P r o t e i n s " p.77, L a b o r a t o r y T e c h n i q u e s i n B i o c h e m i c a l and M o l e c u l a r B i o l o g y , v o l . 4 . E d . by Work,T.S. and Work,E. A m e r i c a n E l s e v i e r P u b l i s h i n g Co., New Y o r k . ( 1 9 7 6 ) (80) W i e g e l e , M . , D e d e r n a r d o , S . L . , T e n g i , J . P . and Leimgruber,W. J.A.C.S. 94:16, 5927.(1972) (81) H a r r i s , J . M . P e r s o n a l C o m m u n i c a t i o n and U n p u b l i s h e d O b s e r v a t i o n . ( 1 9 8 4 ) (82) V a s s a r , P . S . , H a r d s , J . M . , B r o o k s , D . E . , H a g e n b e r g e r , B . and Seaman,G.V.F. J . C e l l B i o l . 53', 809.( 1972) (83) Bamberger,S., Seaman,G.F.V., Brown,J.A. and B r o o k s , D . E . J . C o l l o i d I n t e r f a c e S c i . 99, 1, 187.(1984) (84) Sharp,K.A. and B r o o k s , D . E . S u b m i t t e d f o r p u b l i c a t i o n (1986) (85) Shaw,D.J. I n t r o d u c t i o n t o C o l l o i d and S u r f a c e C h e m i s t r y 2nd E d . p108-111, B u t t e r w o r t h & Co., Fakenham.(1970) (86) H a r r i s , J . M . , Y a l p a n i , M . , Van A l s t i n e , J . M . , S t r u c k , E . C . , Case,M.G., P a l e y , M . S . and B r o o k s , D . E . J . P o l y . S c i . P o l y . Chem. Ed. 22, 341.(1984) (87) G l a s s , J . D . , S i l v e r , L . , S o n d h e i m e r , J . , Pande,C.S. a n d C o d e r r e , J . B i o p o l y m e r s . 18, 383.(1979) (88) P o l l a k , A . and W h i t e s i d e s , G . M . J.A.C.S. 98, 289.(1976) 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0059392/manifest

Comment

Related Items