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Characterization of monoclonal antibodies to rubella virus structural proteins Brush, Bradley Alan 1989

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CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO RUBELLA. VIRUS STRUCTURAL PROTEINS by BRADLEY ALAN BRUSH B.M.L.Sc, The University of British Columbia, 1985 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PATHOLOGY UNIVERSITY OF BRITISH COLUMBIA We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1989 0BRADLEY ALAN BRUSH, 1989 MASTER OF SCIENCE in In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of 0frD-rt>LO 6~V The University of British Columbia Vancouver, Canada DE-6 (2/88) I . ABSTRACT M o n o c l o n a l a n t i b o d i e s (MAbs ) , due t o t h e i r i n t r i n s i c s p e c i f i c i t y t o s i n g l e d e t e r m i n a n t s , a r e c a p a b l e o f d e f i n i n g t h e a n t i g e n i c and s t r u c t u r a l p r o p e r t i e s o f v i r u s e s . A p a n e l o f t w e n t y - f i v e m o n o c l o n a l a n t i b o d i e s , t h e r e s u l t o f f u s i o n s be tween s p l e n o c y t e s f r om B a l b / c m i c e immun i zed w i t h RA 27 /3 v a c c i n e s t r a i n r u b e l l a v i r u s and myeloma c e l l l i n e N S l X 6 3 A g 8 . 6 5 3 , were c h a r a c t e r i z e d b y r a d i o i m m u n o p r e c i p i t a t i o n o f r u b e l l a v i r u s s t r u c t u r a l p r o t e i n s , IgG s u b - c l a s s i f i c a t i o n , h a e m a g g l u t i n a t i o n i n h i b i t i o n (HI ) and v i r u s n e u t r a l i z a t i o n (N t ) o f r u b e l l a v i r u s ( R V ) . The a n t i g e n s p e c i f i c i t i e s o f t h i r t e e n o f i t w e n t y - f i v e MAbs were i d e n t i f i e d 35 b y i m m u n o p r e c i p i t a t i o n o f [ S ] - m e t h i o n i n e l a b e l l e d v i r u s . A l l showed r e a c t i v i t y t o t h e E l e n v e l o p e g l y c o p r o t e i n o f e i t h e r M33 w i l d t y p e , o r RA 27 /3 v a c c i n e s t r a i n v i r u s , o r b o t h . The r e m a i n d e r p r e c i p i t a t e d m u l t i p l e v i r a l p r o t e i n s o r none a t a l l . These t r i a l s r e q u i r e d numerous a l t e r a t i o n s i n t h e c o m p o s i t i o n o f b u f f e r s t o a c h i e v e unambiguous r e s u l t s . IgG s u b c l a s s i f i c a t i o n was p e r f o r m e d on h y b r i d o m a c e l l c u l t u r e s u p e r n a t a n t s o r p u r i f i e d a s c i t e s f l u i d b y t h e O u c h t e r l o n y d o u b l e i m m u n o d i f f u s i o n t e c h n i q u e , w i t h a l l i m m u n o g l o b u l i n s t y p e d as I g G - , IgG„ , o r IgG„ , , b u t w i t h no J . Z. cL Z. D c a s e s o f IgG^ o r IgM s e c r e t o r s . I n h i b i t i o n o f r u b e l l a v i r u s a g g l u t i n a t i o n o f c h i c k e r y t h r o c y t e s b y n i n e o f t h e m o n o c l o n a l a n t i b o d i e s gave h a e m a g g l u t i n a t i o n i n h i b i t i o n t i t r e s o f g r e a t e r t h a n 8 w i t h t h e h i g h e s t t i t r e a t 16384 . The p a n e l o f m o n o c l o n a l a n t i b o d i e s was t e s t e d f o r i t s c a p a c i t y t o n e u t r a l i z e b o t h M33 and RA 27 /3 s t r a i n s o f r u b e l l a v i r u s , w i t h and w i t h o u t t h e p r e s e n c e o f comp lement . To d e t e c t r u b e l l a p l a q u e f o r m a t i o n e x c l u s i v e l y , an immunoc y t o c hem i c a l method o f d e t e c t i n g r u b e l l a v i r u s p r o t e i n s on c e l l s u r f a c e s was d e v e l o p e d . P l a q u e r e d u c t i o n e x p e r i m e n t s showed t h a t t e n o f t h e MAbs i n h i b i t e d the cytopathic effect of rubella virus to some extent. Five MAbs neutralized M33 RV without complement present, one showing a four-fold increase i n t i t r e upon i t s addition, and one MAb, negative i n the absence of complement displayed a pos i t i v e t i t r e i n i t s presence. Three MAbs showed ne u t r a l i z i n g a c t i v i t y toward RA 27/3 with no observed effect upon the addition of complement; two MAbs showed a s h i f t from negativity to n e u t r a l i z i n g t i t r e s . Of the ten MAbs that were active, four reacted with both ru b e l l a s t r a i n s . The capacity of the monoclonal antibodies to neutralize rubella virus was found to be independent of th e i r a b i l i t y to i n h i b i t the haemagglutinin of the v i r u s . Therefore the epitopes for virus n e u t r a l i z a t i o n and haemagglutination appear to be d i f f e r e n t . I I . TABLE OF CONTENTS I . ABSTRACT i i I I . TABLE OF CONTENTS i v I I I . L I S T OF TABLES v i I V . L I S T OF FIGURES v i i V . ABBREVIATIONS v i i i V I . ACKNOWLEDGMENTS i x V I I . INTRODUCTION 1 A . M o n o c l o n a l A n t i b o d i e s 1 B. C l i n i c a l R u b e l l a 2 C. The R u b e l l a V i r u s 2 D. C h r o n i c l e o f I m m u n o l o g i c a l I n v e s t i g a t i o n s o f R u b e l l a V i r u s 4 E. E x p e r i m e n t a l R a t i o n a l e 6 V I I I . MATERIALS AND METHODS 8 A . R u b e l l a V i r u s S t r a i n s 8 B. B a c k g r o u n d on O r i g i n o f M o n o c l o n a l A n t i b o d i e s 8 C. P u r i f i c a t i o n and S t a n d a r d i z a t i o n o f M o n o c l o n a l A n t i b o d i e s 9 D. V e r i f i c a t i o n o f S p e c i f i c i t y o f M o n o c l o n a l A n t i b o d i e s 9 E. I m m u n o p r e c i p i t a t i o ^ 9 1. P r e p a r a t i o n o f [ S ] - m e t h i o n i n e - l a b e l l e d R u b e l l a V i r u s 9 2 . P r e t r e a t m e n t o f S t a p h y l o c o c c u s a u r e u s 10 3 . P r e t r e a t m e n t o f R u b e l l a V i r u s 10 4 . A d s o r p t i o n o f N o n - s p e c i f i c P r o t e i n s f r om V i r u s L y s a t e s 11 5 . P r e c i p i t a t i o n o f R u b e l l a V i r u s P r o t e i n s 11 F. IgG C l a s s i f i c a t i o n 12 G. H a e m a g g l u t i n a t i o n I n h i b i t i o n 12 1. R u b e l l a V i r u s P r e p a r a t i o n 12 2 . D e t e r m i n a t i o n o f R u b e l l a A n t i g e n A c t i v i t y 13 3 . P r e t r e a t m e n t o f Serum t o Remove N o n - S p e c i f i c I n h i b i t o r s 13 4 . D e t e r m i n a t i o n o f HI A n t i b o d i e s i n Serum 13 H. R u b e l l a V i r u s N e u t r a l i z a t i o n 14 1. R u b e l l a V i r u s - Complement C o m p a t i b i l i t y 14 2 . V i r u s N e u t r a l i z a t i o n 14 3 . P l a q u e D e t e c t i o n by Immunoperox idase Me thod 15 I X . RESULTS 16 A . P u r i f i c a t i o n and S t a n d a r d i z a t i o n o f M o n o c l o n a l A n t i b o d i e s 16 B. V e r i f i c a t i o n o f R u b e l l a V i r u s M o n o c l o n a l A n t i b o d y S p e c i f i c i t y . . . . 17 C. I m m u n o p r e c i p i t a t i o n 18 D. IgG C l a s s i f i c a t i o n 27 E. H a e m a g g l u t i n a t i o n I n h i b i t i o n 29 F. R u b e l l a V i r u s N e u t r a l i z a t i o n 32 1. R u b e l l a V i r u s - Complement C o m p a t i b i l i t y 32 2 . V i r u s N e u t r a l i z a t i o n 33 - i v -X . DISCUSSION 37 X I . SUMMARY AND CONCLUSIONS 45 X I I . REFERENCES 48 X I I I . BIOGRAPHICAL INFORMATION 52 -v-I I I . L I ST OF TABLES Page T a b l e I . V e r i f i c a t i o n o f s p e c i f i c i t y o f m o n o c l o n a l a n t i b o d i e s 18 T a b l e I I . S p e c i f i c i t y o f M o n o c l o n a l A n t i b o d i e s t o I n d i v i d u a l R u b e l l a V i r u s S t r u c t u r a l P r o t e i n s 24 T a b l e I I I . I m m u n o g l o b u l i n c l a s s i f i c a t i o n o f m o n o c l o n a l a n t i b o d i e s . . . . 28 T a b l e I V . M o n o c l o n a l A n t i b o d y H a e m a g g l u t i n a t i o n I n h i b i t i o n (H I ) T i t r e s 30 T a b l e V . P l a q u e f o r m i n g u n i t ( P F U ) / w e l l a s s a y w i t h comp lement 32 T a b l e V I . R u b e l l a V i r u s N e u t r a l i z a t i o n T i t r e s 33 T a b l e V I I . Summary o f r e s u l t s o f m o n o c l o n a l 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 RA 27 /3 v a c c i n e and M33 w i l d s t r a i n r u b e l l a v i r u s 46 - v i -I V . L I S T OF FIGURES Page F i g . 1 SDS-PAGE a n a l y s i s o f p u r i f i e d and n o n - p u r i f i e d a s c i t e s f l u i d 17 F i g . 2 I m m u n o p r e c i p i t a t i o n o f r u b e l l a v i r a l p r o t e i n s d e m o n s t r a t i n g c o - p r e c i p i t a t i o n o f E2 and NC p r o t e i n s w i t h E l 20 F i g . 3 I m m u n o p r e c i p i t a t i o n w i t h v a r i a t i o n i n t h e N a C l c o n c e n t r a t i o n o f wash b u f f e r s 21 F i g . 4 I m m u n o p r e c i p i t a t i o n w i t h i n c l u s i o n o f 2 - m e r c a p t o e t h a n o l (2-ME) 22 F i g . 5 S p e c i f i c i t y o f m o n o c l o n a l a n t i b o d i e s t o M33 s t r a i n r u b e l l a v i r u s s t r u c t u r a l p r o t e i n s 25 F i g . 6 S p e c i f i c i t y o f m o n o c l o n a l a n t i b o d i e s t o RA 27 /3 s t r a i n r u b e l l a v i r u s s t r u c t u r a l p r o t e i n s 26 F i g . 7a O u c h t e r l o n y d o u b l e i m m u n o d i f f u s i o n s u b c l a s s i f i c a t i o n o f h y b r i d o m a c e l l c u l t u r e s u p e r n a t a n t s 28 F i g . 7 b / c O u c h t e r l o n y d o u b l e i m m u n o d i f f u s i o n s u b c l a s s i f i c a t i o n o f d i l u t i o n s o f p u r i f i e d a s c i t e s f l u i d and g o a t a n t i - m o u s e se rum . . . 29 F i g . 8 H a e m a g g l u t i n a t i o n i n h i b i t i o n (HI ) t i t r a t i o n o f m o n o c l o n a l and p o l y c l o n a l serum w i t h s u s p e n s i o n s o f c h i c k e r y t h r o c y t e s 31 F i g . 9 I n h i b i t i o n o f t h e c y t o p a t h i c e f f e c t o f r u b e l l a v i r u s by complement 32 F i g . 10 R u b e l l a v i r u s n e u t r a l i z a t i o n by p l a q u e r e d u c t i o n 34 F i g . 11 I m m u n o p r e c i p i t a t i o n o f M33 r u b e l l a v i r u s p r o t e i n s d e m o n s t r a t i n g c r o s s r e a c t i v i t y o f MAb 21D10B w i t h V e r o c e l l p r o t e i n s 42 - v i i -V . ABBREVIATIONS BSA CPE E1/E2 (VP1/VP2) EDTA EL ISA FCS HAT Hepes HA HI HPV/77 DE/5 HRP HSAG IgG IgM LWB M199 M33 MAb MEM M N6 (VP3) NP40 N t PAGE PBS PEG PMSF PS PFU RA 27 /3 RK RPMI RV SDS T r i s - H C l V e r o b o v i n e serum a l b u m i n c y t o p a t h i c e f f e c t r u b e l l a v i r u s s t r u c t u r a l e n v e l o p e g l y c o p r o t e i n s e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d enzyme l i n k e d immunosorbent a s s a y f e t a l c a l f se rum h y p o x a n t h i n e - a m i n o p t e r i n - t h y m i d i n e med i a N- 2 - h y d r o x y e t h y l p i p e r a z i n e - N - 2 - e t h a n e s u l f o n i c a c i d h a e m a g g l u t i n i n H a e m a g g l u t i n i n I n h i b i t i o n v a c c i n e s t r a i n r u b e l l a v i r u s h o r s e - r a d i s h p e r o x i d a s e H e p e s - s a l i n e - a l b u m i n - g l y c i n e i m m u n o g l o b u l i n G i m m u n o g l o b u l i n M l y s a t e wash b u f f e r medium 199 w i l d s t r a i n r u b e l l a v i r u s m o n o c l o n a l a n t i b o d y m i n i m a l e s s e n t i a l medium r e l a t i v e m o l e c u l a r w e i g h t r u b e l l a v i r u s c a p s i d p r o t e i n N o n i d e t - P 4 0 v i r u s n e u t r a l i z a t i o n 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 p ho spha t e b u f f e r e d s a l i n e p o l y e t h y l e n e g l y c o l p h e n y l m e t h y l s u l f o n y l f l u o r i d e p e n i c i l l i n - s t r e p t o m y c i n p l a q u e f o r m i n g u n i t V a c c i n e s t r a i n r u b e l l a v i r u s r a b b i t k i d n e y c e l l s anagram o f R o s w e l l P a r k M e m o r i a l I n s t i t u t e ( c u l t u r e medium) r u b e l l a v i r u s sod i um d o d e c y l s u l p h a t e 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 C 1 A f r i c a n g r e e n monkey k i d n e y - v i i i -V I . ACKNOWLEDGMENTS I w o u l d l i k e t o e x p r e s s my g r a t i t u d e t o my s u p e r v i s o r , D r . S h i r l e y G i l l a m who gave me t h e chance t o p r o v e m y s e l f and s u p p o r t e d me f a r b e y o n d wha t c o u l d e v e r be e x p e c t e d . My t h a n k s t o t h e members o f my s u p e r v i s o r y c ommi t t ee f o r t h e i r s u g g e s t i o n s and g u i d a n c e i n t h i s e ndea vou r . I w o u l d l i k e a l s o t o a cknow ledge two f r i e n d s . A l l a n H u s t a d and E l i z a b e t h Donna ch i e have g i v e n me t h e i r s t e a d f a s t f r i e n d s h i p and g e n e r o s i t y . I c a n o n l y r e p a y them w i t h my a b i d i n g l o y a l t y . F u n d i n g f o r t h i s r e s e a r c h was p a r t i a l l y p r o v i d e d f o r by a g r a d u a t e s t u d e n t s h i p f r o m t h e B r i t i s h C o l u m b i a C h i l d r e n ' s H o s p i t a l R e s e a r c h D i v i s i o n . - i x -V I I . INTRODUCTION A . M o n o c l o n a l A n t i b o d i e s I m m u n o l o g i c a l s t u d y o f a n t i g e n s p r i o r t o t h e i n t r o d u c t i o n o f m o n o c l o n a l a n t i b o d i e s ha s b e en o b s c u r e d by t h e p r e s e n c e o f a s p e c t r u m o f i m m u n o g l o b u l i n s o f d i f f e r e n t c l a s s e s and s p e c i f i c i t i e s d e s p i t e r i g o r o u s p u r i f i c a t i o n o f a n t i g e n s and i m m u n i z a t i o n . I t was known t h a t myeloma c e l l l i n e s c o u l d s e c r e t e l a r g e q u a n t i t i e s o f h i g h l y s p e c i f i c a n t i b o d i e s , and t h a t t h e s e myeloma tumors c o u l d be i n d u c e d t h r o u g h c h e m i c a l o r v i r a l s t i m u l a t i o n . A t t e m p t s were f i r s t made t o immun ize a n i m a l s w i t h a p a r t i c u l a r a n t i g e n and t h e n c r e a t e a n t i g e n - s p e c i f i c myelomas f r om t h e i r s p l e e n c e l l s . However , d e s p i t e c o m p r e h e n s i v e s c r e e n i n g programmes, i t d i d n o t p r o v e f e a s i b l e t o p r o d u c e a n t i b o d i e s t o a s p e c i f i c a n t i g e n b y t h i s t e c h n i q u e ( G o d i n g , 1 9 8 3 ) . C o t t o n and M i l s t e i n i n 1973 f o rmed mouse-mouse and m o u s e - r a t c e l l h y b r i d s b y u s i n g i n a c t i v a t e d S e n d a i v i r u s as a f u s i o n a i d . They f o u n d t h a t i m m u n o g l o b u l i n e x p r e s s i o n was p r e s e r v e d and c o - d o m i n a n t . E x p a n d i n g on t h e s e c o n c e p t s , K o h l e r and M i l s t e i n ( 1 9 7 5 ) , f u s e d n o n - i m m u n o g l o b u l i n s e c r e t i n g myeloma c e l l l i n e s w i t h s p l e e n c e l l s f r om m i ce immun i zed w i t h sheep r e d b l o o d c e l l s . T h i s p r o d u c e d h y b r i d s w h i c h s e c r e t e d a n t i - s h e e p i m m u n o g l o b u l i n s . S e l e c t i o n o f h y b r i d s was f a c i l i t a t e d b y t h e i r a b i l i t y t o s u r v i v e i n HAT ( 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 ) medium, due t o s u p p l e m e n t a t i o n o f t h e d e f e c t s p r e s e n t i n b o t h p a r e n t a l c e l l l i n e s ( L i t t l e f i e l d , 1 9 6 4 ) . L a t e r e x p e r i m e n t s , s u b s t i t u t i n g p o l y e t h y l e n e g l y c o l ( D a v i d s o n and G e r a l d , 1976) f o r S e n d a i v i r u s , i n c r e a s e d t h e c e l l f u s i o n f r e q u e n c y . S p l e e n c e l l - m y e l o m a h y b r i d c e l l l i n e s a r e c a p a b l e o f p r o d u c i n g a v i r t u a l l y u n l i m i t e d amounts o f p u r e , w e l l d e f i n e d a n t i b o d i e s w h i l e n o t r e q u i r i n g e x t e n s i v e p u r i f i c a t i o n o f a n t i g e n s . The c h a r a c t e r i s t i c s o f s i n g l e - d e t e r m i n a n t s p e c i f i c i t y , and r e t e n t i o n o f a f r a c t i o n o f t h e a c t i v i t i e s o f h e t e r o l o g o u s - 1 -se rum e s t a b l i s h e s m o n o c l o n a l a n t i b o d y t e c h n o l o g y as an i m p o r t a n t t o o l f o r t h e i m m u n o l o g i c a l a n a l y s i s o f v i r u s e s . B. C l i n i c a l R u b e l l a R u b e l l a (German m e a s l e s ) i s u s u a l l y a m i l d d i s e a s e a f f e c t i n g c h i l d r e n and young a d u l t s w i t h o u t s i g n i f i c a n t c o n s e q u e n c e s . A l t h o u g h i t s i n c i d e n c e ha s b e en d r a m a t i c a l l y r e d u c e d due t o t h e i n t r o d u c t i o n o f a r e l a t i v e l y s u c c e s s f u l v a c c i n e i n 1966 (Pa r kman , 1 9 6 6 ) , i t s t i l l r ema i n s an i m p o r t a n t human p a t h o g e n . C o m p l i c a t i o n s o f r u b e l l a i n b o t h c h i l d r e n and a d u l t s i n c l u d e a r t h r i t i s ( P h i l l i p s , 1965) and e n c e p h a l i t i s (Sherman, 1 9 7 5 ) . The most d r a m a t i c e f f e c t s howeve r , o c c u r as a r e s u l t o f c o n g e n i t a l r u b e l l a syndrome; t h e m a n i f e s t a t i o n s o f w h i c h i n c l u d e c a t a r a c t s , c a r d i a c d e f e c t s , d e a f n e s s , m e n t a l and g r ow th r e t a r d a t i o n , m i c r o c e p h a l y , bone d e f e c t s and s t i l l b i r t h s . I m m u n i z a t i o n ha s a l s o b e en shown t o c au se s i d e e f f e c t s i n c h i l d r e n and a d u l t r e c i p i e n t s , p r i m a r i l y o c c u r r i n g as a t r a n s i t o r y o r c h r o n i c a r t h r a l g i a a n d / o r a r t h r i t i s ( T i n g l e , 1979; D e i n a r d , 1973; L e o n i d a , 1973 ; S w a r t z , 1 9 7 1 ) . The r e i s a l s o a r a t e o f f a i l e d i m m u n i z a t i o n s o f up t o 5% o f t h o s e immun i zed w i t h t h e c u r r e n t a t t e n u a t e d v a c c i n e s ( T i n g l e , 1985; W i l k i n s , 1972; Dudgeon, 1 9 6 9 ) . C. The R u b e l l a V i r u s R u b e l l a v i r u s , t h e s o l e member o f t h e genus R u b i v i r u s i n t h e f a m i l y o f T o g a v i r i d a e , i s a s p h e r i c a l e n v e l o p e d v i r u s , 60 nm i n d i a m e t e r , c o n t a i n i n g a s i n g l e s t r a n d RNA o f p o s i t i v e p o l a r i t y , a p p r o x i m a t e l y 11 , 000 n u c l e o t i d e r e s i d u e s . The e n v e l o p e c o n s i s t s o f a h o s t l i p i d b i l a y e r and two g l y c o s y l a t e d v i r u s - s p e c i f i e d s t r u c t u r a l p r o t e i n s , E l and E2 , w h i c h e n c l o s e a n o n - g l y c o s y l a t e d n u c l e o c a p s i d p r o t e i n C, w h i c h i s a s s o c i a t e d w i t h t h e genomic RNA. The E l and E2 p o l y p e p t i d e s were f o u n d t o be g l y c o s y l a t e d b y b o r o h y d r i d e r a d i o l a b e l l i n g o f c a r b o h y d r a t e s and t u n i c a m y c i n b l o c k a g e o f g l y c o s y l a t i o n - 2 -( O k e r - B l o m , 1 9 8 3 ) . The E2 g l y c o p r o t e i n a ppea r s as two d i s t i n c t s p e c i e s , E_ and E^^ i n 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 . However , t h e r e s u l t s f r om a m i n o - t e r m i n a l s equence a n a l y s i s b y Edman d e g r a d a t i o n f o l l o w e d b y c a r b o x y l - t e r m i n a l a n a l y s i s b y c a r b o x y p e p t i d a s e d i g e s t i o n and q u a n t i t a t i v e amino a c i d c o m p o s i t i o n i n d i c a t e t h a t E2a and E2b a r e t h e r e s u l t o f h e t e r o g e n e o u s p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s o f t h e same p r o t e i n ba ckbone ( K a l k k i n e n , 1 9 8 4 ) . I n a d d i t i o n t o t h e genomic 40S RNA, v i r u s - i n f e c t e d c e l l s a l s o c o n t a i n a subgenomic 24S mRNA w h i c h i s d e r i v e d f r om t h e 3 ' o n e - t h i r d o f t h e genomic 40S RNA ( O k e r - B l o m e t a l 1 9 8 4 ) . The subgenomic 24S mRNA i s t r a n s l a t e d i n t o a p r e c u r s o r t o t h e s t r u c t u r a l p r o t e i n s and i s p r o t e o l y t i c a l l y p r o c e s s e d t o g i v e t h e s t r u c t u r a l p r o t e i n s C, E2 and E l ( K a l k k i n e n e t a l 1984; O k e r - B l o m , 1 9 8 4 ) . The r e i s a l a c k o f c on s en su s on t h e m o l e c u l a r w e i g h t s (M^) o f t h e s t r u c t u r a l p r o t e i n s . E s t i m a t e s o f t h e E l M^ r ange f r om 55 , 000 ( H o - T e r r y , 1984) t o 62 , 000 (Waxham, 1 983 ) ; t h e E2 w h i c h a ppea r s as a c omp l e x on p o l y a c r y l a m i d e g e l s r a n g e s f r om 4 7 , 0 0 0 - 4 2 , 0 0 0 ( O k e r - B l o m , 1983) t o 5 4 , 0 0 0 - 4 7 , 0 0 0 (Waxham, 1983) and t h e n u c l e o p r o t e i n NC M r r a n g e s f r o m 26 , 000 (Chong , 1983) t o 38 , 000 (Waxham, 1 9 8 3 ) . I n o u r l a b o r a t o r y , hype r - immune r a b b i t 35 serum r o u t i n e l y p r e c i p i t a t e d f o u r p r o t e i n s f r om [ S ] - l a b e l l e d v i r u s h a v i n g r e l a t i v e m o l e c u l a r w e i g h t s o f 5 8 , 000 ( E l ) , 4 5 , 0 0 0 ( E 2 a ) , 4 2 , 0 0 0 ( E 2 b ) , and 30 , 000 (NC ) , w h i c h a r e c l o s e t o t h e v a l u e s o b t a i n e d b y O k e r - B l o m ( 1 9 8 3 ) . Meager y i e l d s o f r u b e l l a v i r u s f r e e o f h o s t c e l l p r o t e i n s have hampered p r o g r e s s i n b i o c h e m i c a l l y c h a r a c t e r i z i n g t h e i n d i v i d u a l s t r u c t u r a l v i r a l g l y c o p r o t e i n s . - 3 -D. C h r o n i c l e o f I m m u n o l o g i c a l I n v e s t i g a t i o n s o f R u b e l l a V i r u s M o n o c l o n a l a n t i b o d i e s , b e c au s e o f t h e i r a b i l i t y t o d i s c r i m i n a t e a n t i g e n i c d i f f e r e n c e s , a r e u s e f u l t o o l s f o r d e f i n i n g t h e i m p o r t a n t a n t i g e n i c r e g i o n s o f v i r u s s t r u c t u r a l p o l y p e p t i d e s . I t h a s b e en r e a l i z e d t h a t t h e i m m u n i z i n g a c t i v i t y o f mos t v i r u s e s c a n be p o s s e s s e d by one s p e c i e s o f p r o t e i n o r p r o t e i n s u b - u n i t (B rown , 1985; Wunner, 1 9 8 3 ) . F o r r u b e l l a , t h i s i s s u s p e c t e d t o be t h e s t r u c t u r a l p r o t e i n E l ( V P l ) (Ka tow, 1985 , Waxham, 1985 , Ho, 1 9 8 4 ) . I n p r e v i o u s s t u d i e s , m o n o c l o n a l a n t i b o d i e s (MAbs) d i r e c t e d t o w a r d d i s s o c i a t e d E l s u b u n i t s h a d v i r u s - n e u t r a l i z i n g a c t i v i t y (N t ) a n d / o r h a e m a g g l u t i n i n i n h i b i t i o n (H I ) a c t i v i t y (Umino, 1 9 8 5 ) . The r e i s m o u n t i n g e v i d e n c e t o s u g g e s t t h a t t h e E l p o l y p e p t i d e p l a y s a m a j o r r o l e i n immune r e s p o n s e ( H o - T e r r y , 1 9 8 4 ) . However , t h e HI and N t ( v i r u s n e u t r a l i z a t i o n ) a c t i v i t i e s o f MAbs have b e en shown t o n o t a l w a y s c o r r e l a t e (G r e en , 1 9 8 6 ) . Up t o s i x s p a t i a l l y s e p a r a t e e p i t o p e s , w i t h d i v e r g e n t HI and N t r e a c t i o n p a t t e r n s , have been mapped w i t h MAbs (Waxham, 1 9 8 5 ) . G r e en e t a l ( 1 9 8 6 ) , have r e c e n t l y i s o l a t e d a MAb c a p a b l e o f n e u t r a l i z i n g r u b e l l a v i r u s w h i c h i s E 2 - s p e c i f i c . Waxham e t a l ( 1 9 8 2 ) , p o s t u l a t e d t h a t t h e E l and E2 e n v e l o p e g l y c o p r o t e i n s e x i s t i n t h e i n t a c t v i r i o n as m i x e d d ime r s w h i c h a r e a s s o c i a t e d v i a d i s u l p h i d e l i n k a g e s . T h i s i s s u p p o r t e d b y t h e p r e s e n c e o f bands o f m o l e c u l a r w e i g h t 105 , 000 and 95 , 000 i n 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 u n d e r u n r e d u c e d c o n d i t i o n s . R e d u c t i o n and r a d i o l a b e l l i n g by c a r b o x y m e t h y l a t i o n o f t h e two e n v e l o p e g l y c o p r o t e i n s ( H o - T e r r y , 1981) a l s o s u p p o r t s t h e i d e a o f m i x e d d i m e r s . D o r s e t t e t a l ( 1 9 8 5 ) , have shown i n t h e i r s t u d y o f t h e i n t e r a c t i o n s o f t h e v i r a l s t r u c t u r a l components o f r u b e l l a v i r u s s t r a i n HPV -77 , t h a t t h e r e were E l : E l , NC:NC and E1 :E2 d ime r s p r e s e n t i n p u r i f i e d p r e p a r a t i o n s o f v i r u s . - 4 -H o - T e r r y e t a l ( 1 9 8 5 ) , w h i l e a t t e m p t i n g t o s e p a r a t e E l and E2 s t r u c t u r a l p r o t e i n s u s i n g T w e e n - e t h e r e x t r a c t i o n and t r y p s i n d e g r a d a t i o n , f o u n d t h a t e ven t hough t h e E l g l y c o p r o t e i n was c l e a v e d i n t o s e p a r a t e ( 4 3 , 0 0 0 and 13 , 000 MW) e n t i t i e s , t h e l a r g e r c l e a v a g e p r o d u c t f a i l e d t o s e p a r a t e f r om E2 . Chong e t a l (1985) f o u n d d i f f i c u l t y i n i s o l a t i n g i n d i v i d u a l r u b e l l a s t r u c t u r a l p r o t e i n s u s i n g MAbs i m m o b i l i z e d t o C N B r - a c t i v a t e d Sepha ro se 4B . H o - T e r r y (1981) showed t h a t p r i o r t o i m m u n o p r e c i p i t a t i o n , t r e a t m e n t o f t h e p r o t e i n s b y SDS (2%) and / 3 -me r c ap t oe t hano l (2%) r e s u l t e d i n a r e d u c t i o n o f p o l y v a l e n t a n t i b o d y b i n d i n g c a p a c i t y . I n 1984 , t h e y f o u n d t h a t i n c u b a t i o n o f t h e r u b e l l a v i r u s w i t h SDS and m e r c a p t o e t h a n o l a l t e r e d t h e a n t i g e n i c p r o p e r t i e s o f t h e v i r u s and " m a r k e d l y r e d u c e d t h e i r i m m u n o r e a c t i v i t y " . When g e l p u r i f i e d p r o t e i n s were u s e d t o immun i ze r a b b i t s , t h e serum o b t a i n e d r e a c t e d o n l y a t l o w t i t r e s i n HI and N t t e s t s w i t h r u b e l l a v i r u s ( H o - T e r r y ) . I n 1986 , H o - T e r r y f o u n d t h a t r u b e l l a E l and E2 p r o t e i n s c o u l d o n l y be i s o l a t e d b y u s i n g p o w e r f u l d e n a t u r i n g and r e d u c i n g a g e n t s (SDS and m e r c a p t o e t h a n o l ) , r e s u l t i n g i n a l o s s o f " a l l b i o l o g i c a l a c t i v i t y and a l m o s t a l l i m m u n o l o g i c a l a c t i v i t y " . T h i s may be t h e s o u r c e o f t h e d i f f i c u l t y t h a t Loo e t a l (1986) h a d i n i s o l a t i n g t h e two e n v e l o p e p r o t e i n s w h i l e m a i n t a i n i n g t h e i r n a t i v e f u n c t i o n a l and i m m u n o l o g i c a l c h a r a c t e r . G r e e n e t a l (1986) u s i n g W e s t e r n b l o t a n a l y s i s , f o u n d t h a t two E l s p e c i f i c MAbs r e a c t e d w i t h t h e E l o f i n t a c t v i r u s ; one MAb l o s t i t s r e a c t i v i t y upon v i r a l p r o t e i n s o l u b i l i z a t i o n w i t h 0.15% SDS b u t r e g a i n e d i t when E l was a b s o r b e d t o t h e s o l i d phase o f t h e W e s t e r n b l o t . The o t h e r MAb l o s t r e a c t i v i t y when t h e a n t i g e n s were r e d u c e d b y m e r c a p t o e t h a n o l p r i o r t o e l e c t r o p h o r e s i s . D o r s e t t e t a l ( 1 9 8 5 ) , howeve r , h ad some s u c c e s s i n r a i s i n g p o l y v a l e n t serum i n r a b b i t s a g a i n s t p u r i f i e d p o l y p e p t i d e s d e r i v e d f r om p r e p a r a t i v e g e l - 5 -s l i c e s unde r r e d u c i n g c o n d i t i o n s . The MAbs r e a c t e d s p e c i f i c a l l y w i t h t h e a p p r o p r i a t e p r o t e i n s i n W e s t e r n b l o t s w i t h o u t e x p e r i e n c i n g c r o s s - r e a c t i v i t y w i t h t h e o t h e r v i r a l p o l y p e p t i d e s . E. E x p e r i m e n t a l R a t i o n a l e I m m u n o l o g i c a l t e c h n i q u e s may be a b l e , t h r o u g h t h e c o n s t r u c t i o n o f i m m u n o a f f i n i t y c o l umns , t o s u p p l y r e l a t i v e l y l a r g e amounts o f p u r i f i e d r u b e l l a v i r u s p r o t e i n s f o r f u r t h e r a n a l y s i s . The t e c h n i q u e s c o u l d a l s o be u s e d t o t h e c h a r a c t e r i z e h o s t immune r e s p o n s e s t o t h e i n d i v i d u a l p o l y p e p t i d e s w h i c h a r e t h o u g h t t o be a l t e r e d i n c o n g e n i t a l and p e r s i s t e n t r u b e l l a i n f e c t i o n . R u b e l l a v i r u s i s v e r y d i f f i c u l t t o p r o du c e i n l a r g e q u a n t i t i e s . C u r r e n t l y o u r l a b o r a t o r y i s a t t e m p t i n g , t h r o u g h cDNA t e c h n o l o g y , t o g e n e r a t e s e c r e t a b l e v i r a l p r o t e i n s f o r a n a l y s i s and p o s s i b l e s u b - u n i t v a c c i n e a p p l i c a t i o n s . A b a t t e r y o f c h a r a c t e r i z e d m o n o c l o n a l a n t i b o d i e s w i l l be needed f o r v i r a l p r o t e i n p u r i f i c a t i o n , e p i t o p e mapp i ng , s t u d y o f t h e i r b i o l o g i c a l f u n c t i o n s , and f o r t h e s t u d y o f t h o s e v i r a l p r o t e i n s a l t e r e d by i n v i t r o s i t e - s p e c i f i c m u t a g e n e s i s . The m o n o c l o n a l a n t i b o d i e s may a l s o be u s e d t o c h a r a c t e r i z e n a t u r a l a n t i g e n i c v a r i a n t s o f r u b e l l a v i r u s and t o examine a n t i g e n i c d e t e r m i n a n t s f o r v a c c i n e d e s i g n . I n t h i s t h e s i s , I have c h a r a c t e r i z e d a p a n e l o f m o n o c l o n a l a n t i b o d i e s p r o d u c e d by t h e f u s i o n s be tween a mu r i n e myeloma c e l l l i n e and s p l e e n c e l l s o b t a i n e d f r om m i c e w h i c h have b e en immun i zed b y v a c c i n e s t r a i n v i r u s . The m o n o c l o n a l a n t i b o d i e s have b e en c h a r a c t e r i z e d by i m m u n o g l o b u l i n s u b - c l a s s t y p i n g , h a e m a g g l u t i n i n i n h i b i t i o n a s s a y ( H I ) , r a d i o i m m u n o p r e c i p i t a t i o n and v i r u s n e u t r a l i z i n g a c t i v i t y . I m m u n o g l o b u l i n s u b - c l a s s t y p i n g i s e s s e n t i a l t o t h e s t r a t e g y o f p u r i f i c a t i o n o f a n t i b o d i e s and t o t h e i r u s e i n i m m u n o a f f i n i t y t e c h n i q u e s , i n c l u d i n g i m m u n o p r e c i p i t a t i o n . B i n d i n g and e l u t i o n c h a r a c t e r i s t i c s o f t h e -6-a n t i b o d i e s b y t h e i r Fc r e g i o n t o S t a p h y l o c o c c a l p r o t e i n A v a r i e s w i t h e a ch i m m u n o g l o b u l i n s u b - c l a s s , w i t h mu r i n e IgG^ show i ng r e l a t i v e l y p o o r a f f i n i t y ( G o d i n g , 1 9 8 3 ) . The r e a r e i n d i c a t i o n s t h a t t h e p r e s e n c e o f h a e m a g g l u t i n i n a c t i v i t y (HA) o f p r o t e i n E l i s a s i g n i f i c a n t f a c t o r i n t h e immune r e s p o n s e . V i r u s i n d u c e d c e l l m e d i a t e d c y t o t o x i c i t y ha s b e en b l o c k e d i n v i t r o b y m o n o c l o n a l a n t i b o d i e s t o t h e h a e m a g g l u t i n i n o f v i r u s e s ( J u s t e w i c z , 1 9 8 4 ) . The HA o f r u b e l l a v i r u s may a l s o f a c i l i t a t e t h e d i s e a s e p r o c e s s , as a t t a c h m e n t o f v i r u s t o c e l l s i s t h o u g h t t o be a f a c t o r i n i n f e c t i v i t y . H a e m a g g l u t i n i n i n h i b i t i n g (H I ) a b i l i t i e s may a l s o s e r v e t o d i f f e r e n t i a t e two m o n o c l o n a l c e l l l i n e s t h a t , o t h e r w i s e , have c l o s e l y r e l a t e d a c t i v i t i e s . However , HI i s s u s c e p t i b l e t o t h e e f f e c t s o f n o n - s p e c i f i c i n h i b i t o r s . R a d i o i m m u n o p r e c i p i t a t i o n was u s e d , n o t o n l y t o d e l i n e a t e t h e a n t i b o d y s p e c i f i c i t i e s o f t h e MAb, b u t t o s t u d y t h e a n t i g e n i c d i f f e r e n c e s be tween w i l d and v a c c i n e s t r a i n v i r u s e s . T h i s s t u d y was a t t e m p t e d t o s o l v e p r e v i o u s w o r k e r s ' d i f f i c u l t i e s i n d i s s o c i a t i n g t h e E l and E2 g l y c o p r o t e i n s f r o m ea ch o t h e r w h i l e m a i n t a i n i n g t h e i m m u n o l o g i c a l i n t e g r i t y o f t h e g l y c o p r o t e i n s w i t h r e s p e c t t o i n d i v i d u a l m o n o c l o n a l a n t i b o d i e s . R u b e l l a v i r u s n e u t r a l i z a t i o n ha s p r e v i o u s l y b een p e r f o r m e d on i n d i v i d u a l s t r a i n s i n t h e p r e s e n c e o f a n t i - i m m u n o g l o b u l i n s (Ge r n a , 1987; Umino , 1985) and complement ( R a w l s , 1 9 6 7 ) . T h i s s t u d y , howeve r , compared t h e n e u t r a l i z a t i o n p a t t e r n s o f M33 ( w i l d ) and RA 27 /3 ( v a c c i n e ) v i r u s s t r a i n s b y m o n o c l o n a l a n t i b o d i e s w i t h and w i t h o u t t h e p r e s e n c e o f comp lement , as d e t e c t e d b y p l a q u e r e d u c t i o n a s s a y . An o v e r v i e w o f t h e above r e s u l t s , a c o m p a r i s o n o f i m m u n o p r e c i p i t a t i o n and n e u t r a l i z a t i o n p a t t e r n s be tween w i l d and v a c c i n e s t r a i n v i r u s , and HI v a l u e s o b t a i n e d a g a i n s t w i l d s t r a i n v i r u s u s i n g m o n o c l o n a l s r a i s e d a g a i n s t t h e v a c c i n e s t r a i n , w i l l be d e t a i l e d and may f u r t h e r c l a r i f y t h e n a t u r e o f t h e r u b e l l a v i r u s . V I I I . 1 M A T E R I A L S AND METHODS A . R u b e l l a V i r u s S t r a i n s W i l d t y p e s t r a i n (M33) r u b e l l a v i r u s ha s commonly b e e n a c q u i r e d f r om newborns o r a b o r t e d f e t u s e s o f women who have b e en d i a g n o s e d as h a v i n g r u b e l l a v i r u s i n f e c t i o n d u r i n g p r e g n a n c y . The RA 27 /3 v a c c i n e s t r a i n h a s b e e n p r o d u c e d b y p a s s age o f w i l d t y p e s t r a i n i n human emb r yon i c k i d n e y and human d i p l o i d WI-38 c e l l l i n e s , and t h e HPV-77/DE5 s t r a i n b y p a s s age t h r o u g h v e r v e t monkey k i d n e y c e l l s and duck embryo f i b r o b l a s t s . B. B a c k g r o u n d on O r i g i n o f M o n o c l o n a l A n t i b o d i e s The h y b r i d o m a c e l l l i n e s p r o d u c i n g a n t i b o d i e s a g a i n s t r u b e l l a v i r u s have b e en i s o l a t e d i n o u r l a b o r a t o r y b y I v y H u i and T i p Loo and m u r i n e a s c i t e s f l u i d was u s e d f o r t h i s p r o j e c t . B r i e f l y , s p l e n o c y t e s h a r v e s t e d f r o m a B a l b / c mouse immun i zed w i t h RA 27 /3 v a c c i n e s t r a i n r u b e l l a v i r u s were f u s e d w i t h a myeloma c e l l l i n e (NS l X63Ag8 . 653 ) i n t h e p r e s e n c e o f f u s i o n medium c o n s i s t i n g o f PEG, RPM1 1640 and Hepes ( G o d i n g , 1 9 8 3 ) . H y b r i d c e l l l i n e s were s e l e c t i v e l y c u l t u r e d i n HAT medium u s i n g mouse t h ymocy t e s as a f e e d e r l a y e r i n 96 w e l l p l a t e s . S u p e r n a t a n t s were s c r e e n e d f o r t h e p r e s e n c e o f r u b e l l a - s p e c i f i c a n t i b o d i e s b y E L ISA ( E n g v a l l , 1971) and c e l l s f r om p o s i t i v e w e l l s were d i l u t e d f o r s i n g l e - c e l l c l o n i n g and a s s a y e d a g a i n f o r s p e c i f i c a n t i b o d y . Hyb r i doma 7 8 c e l l l i n e s (10 t o 10 c e l l s / m o u s e ) were i n j e c t e d i n t o t h e p e r i t o n e a l c a v i t i e s o f p r i s t a n e - p r i m e d B a l b / c m i c e and i m m u n o g l o b u l i n b e a r i n g a s c i t e s f l u i d was removed b y p a r a c e n t e s i s . The c e l l l i n e s were a l s o grown i n t i s s u e c u l t u r e medium c o n t a i n i n g RPMI 1640 , 10% FCS and 1% PS (100 u n i t s / m l p e n i c i l l i n , 0 . 1 mg/ml s t r e p t o m y c i n ) . The m o n o c l o n a l s u p e r n a t a n t s were removed and p r o t e i n s p r e c i p i t a t e d u s i n g 41% w/v ammonium s u l p h a t e i n PBS . The p r e c i p i t a t e s were r e s u s p e n d e d i n 1^0, and d i a l y z e d a g a i n s t 0 .15M N a C l . C. P u r i f i c a t i o n and S t a n d a r d i z a t i o n o f M o n o c l o n a l A n t i b o d i e s A s c i t e s f l u i d f r om mouse h y b r i d o m a tumors was p u r i f i e d b y t h e B i o r a d P r o t e i n A Mapps I I me thod . A c h r oma t og r aphy co l umn was p a c k e d w i t h a s l u r r y o f A f f i - G e l P r o t e i n A e q u i l i b r a t e d w i t h 5 vo l umes o f b i n d i n g b u f f e r (10 mM PBS, 1% sod i um a z i d e , pH 9 . 0 ) . A s c i t e s f l u i d was d i l u t e d one t o one w i t h b i n d i n g b u f f e r , a p p l i e d t o t h e co l umn and washed w i t h 15 b e d vo l umes o f b i n d i n g b u f f e r . The IgG f r / c t i o n was e l u t e d w i t h 5 b e d vo l umes o f e l u t i o n b u f f e r (0 .1M a c e t i c a c i d i n s a l i n e , pH 3 . 0 ) , n e u t r a l i z e d w i t h 1M T r i s - H C l pH 9 . 0 and d i a l y z e d a g a i n s t PBS, pH 7 . 4 a t 4°C o v e r n i g h t . The e l u a t e s were s t a n d a r d i z e d t o 1 mg p e r mL as measu r ed b y t h e B i o r a d Coomass i e B r i l l i a n t B l u e G-250 P r o t e i n A s s a y and s amp l e s were a n a l y z e d by PAGE and s t a i n e d w i t h Coomass i e b l u e t o a s s e s s p u r i t y . D. V e r i f i c a t i o n o f S p e c i f i c i t y o f M o n o c l o n a l A n t i b o d i e s A l t h o u g h c u l t u r e s u p e r n a t a n t s were p r e v i o u s l y s c r e e n e d f o r r u b e l l a s p e c i f i c a n t i b o d i e s b y E L ISA t e c h n i q u e , t h e s p e c i f i c i t y o f m o n o c l o n a l a n t i b o d i e s was d e t e r m i n e d a g a i n u s i n g p u r i f i e d MAbs on r u b e l l a v i r u s (M33 and RA 27 /3 ) i n f e c t e d RK c e l l s ( u s i n g goa t a n t i - m o u s e IgG c o n j u g a t e d w i t h HRP as a s e c o n d a n t i b o d y f o r v i s u a l i z a t i o n ; see immunope rox i da se method o f p l a q u e d e t e c t i o n i n v i r u s n e u t r a l i z a t i o n s e c t i o n ) . E. I m m u n o p r e c i p i t a t i o n 35 1. P r e p a r a t i o n o f \ — S I - m e t h i o n i n e - l a b e l l e d R u b e l l a V i r u s 35 P r e p a r a t i o n o f [ S ] - m e t h i o n i n e - l a b e l l e d M33 and RA 27 /3 r u b e l l a v i r u s was c a r r i e d o u t b y i n c u b a t i n g a s u b c o n f l u e n t m o n o l a y e r o f V e r o c e l l s , ( a t 24 - 9 -h o u r s p o s t - i n f e c t i o n ) w i t h MEM (5% FCS, 1% PS) d e f i c i e n t i n m e t h i o n i n e , b u t 35 c o n t a i n i n g [ S ] - m e t h i o n i n e (10 / i C i / m l ) o v e r n i g h t . The c e l l c u l t u r e s u p e r n a t a n t was h a r v e s t e d 24 h o u r s l a t e r b e f o r e t h e a d v e n t o f c e l l d e a t h , and c e n t r i f u g e d t o remove c e l l d e b r i s . N a C l and PEG 8000 were added t o t h e s u p e r n a t a n t t o a c o n c e n t r a t i o n o f 0 .5M and 10% r e s p e c t i v e l y , m i x e d and a l l o w e d t o s t a n d f o r 2 h o u r s a t 20°C, t h e n c e n t r i f u g e d a t 1 0 , 000 rpm i n a S o r v a l c e n t r i f u g e u s i n g an SS34 r o t o r f o r t e n m i n u t e s . The s u p e r n a t a n t was d i s c a r d e d , and t h e p e l l e t was d i s s o l v e d i n 500 fil o f TBS b u f f e r ( 0 . 1M N a C l , 20 mM T r i s - H C l , 1 mM EDTA, pH 7 . 4 ) . As c o n t r o l s , m o c k - i n f e c t e d V e r o c e l l s t h a t were d e t e r i o r a t i n g due t o age (7 d a y s ) , were u s e d and t r e a t e d as i n f e c t e d c e l l s . 2. P r e t r e a t m e n t o f S t a p h y l o c o c c u s au r eu s P a n s o r b i n S t a p h y l o c o c c u s au r eu s c e l l s (10% w/v i n f o r m a l i n , b i n d i n g c a p a c i t y 2 . 4 mg o f I gG /m l c e l l s u s p e n s i o n ) were p e l l e t e d , s u spended i n p r e t r e a t m e n t b u f f e r ( 0 . 5M N a C l , 20 mM T r i s - H C l , pH 7 . 4 , 2 % T r i t o n X - 1 0 0 , 0 .2M i o d o a c e t a m i d e , 2 . 0 mM EDTA, 1 % sod i um d e o x y c h o l a t e , 10% SDS and 5% m e r c a p t o e t h a n o l ) , and b o i l e d f o r 30 m i n . The S. a u r eu s c e l l s were t h e n p e l l e t e d and t h i s p r o c e s s was r e p e a t e d i n o r d e r t o r educe , b a c t e r i a l s u r f a c e p r o t e i n s h e d d i n g t h a t o c c u r s w i t h f o r m a l i n f i x e d S. a u r eu s c e l l s w i t h a g i n g . The p e l l e t was washed t h r e e t i m e s i n PBS, c o n t a i n i n g 1% N P ^ , r e s u s p e n d e d i n PBS, c o n t a i n i n g 1% NP^ Q and 5% BSA, a l l o w e d t o s t a n d a t 4°C f o r 1 h o u r , washed t w i c e and r e s u s p e n d e d t o o r i g i n a l vo lume i n PBS-1% N P ^ Q . 3 . P r e t r e a t m e n t o f R u b e l l a V i r u s 35 [ S ] - m e t h i o n i n e - l a b e l l e d M33 o r RA 27 /3 s t r a i n v i r u s was p e l l e t e d i n TBS b u f f e r . ( 0 . 1M N a C l , 20 mM T r i s - H C l , 1 mM EDTA, pH 7 . 4 ) , was d i s r u p t e d i n l a r g e vo l umes o f immune l y s a t e b u f f e r (10 mM T r i s - H C l , 1% T r i t o n X - 1 0 0 , 1.0 mM EDTA, 0.5% sod i um d e o x y c h o l a t e , 2 .5 mM p h e n y l m e t h y l s u l f o n y l f l u o r i d e (PMSF) , pH 7 . 4 w i t h v a r i a t i o n o f N a C l a t c o n c e n t r a t i o n s o f 0 . 1 t o 0 . 5 M, w i t h o r w i t h o u t - 1 0 -0.1% SDS, 0 .02M i o d o a c e t a m i d e , o r 0.25% BSA) f o r 30 m i n u t e s a t 4 °C . 4 . A d s o r p t i o n o f N o n - s p e c i f i c P r o t e i n s f r om V i r u s L y s a t e s 0 . 1 i L n o r m a l r a b b i t serum was added t o 1 mL o f d i s r u p t e d r u b e l l a v i r u s , a l l o w e d t o s t a n d on i c e f o r t e n m i n u t e s , t h e n 0 . 5 mL o f p r e t r e a t e d S. a u r eu s was added and t h e i n c u b a t i o n c o n t i n u e d f o r 30 m i n u t e s . The s u p e r n a t a n t c o n t a i n i n g a d s o r b e d r u b e l l a v i r u s p r o t e i n s was o b t a i n e d b y c e n t r i f u g a t i o n . 5 . P r e c i p i t a t i o n o f R u b e l l a V i r u s P r o t e i n s I n s i l i c o n i z e d E p p e n d o r f t u b e s , 10 /xl S. a u r eu s and 10 pi o f r a b b i t serum ( r u b e l l a p o s i t i v e o r n e g a t i v e ) o r 25 pi p u r i f i e d MAb ( 1 . 0 mg/ml) were i n c u b a t e d b y i n v e r s i o n a t 4°C f o r two h o u r s . Immunoadsorbents were washed t h r e e t i m e s w i t h l y s a t e wash b u f f e r ( 0 . 5M N a C l , 10 mM T r i s - H C l , 1% T r i t o n X - 1 0 0 , 0.5% sod i um d e o x y c h o l a t e , pH 7 . 4 ) and p e l l e t e d . D i s r u p t e d v i r a l p r o t e i n s were added , and i n v e r t e d f o r two h o u r s a t 4 °C . The i m m u n o p r e c i p i t a n t s were washed 3 t i m e s u s i n g LWB w i t h 1M N a C l , and t h e n 3 t i m e s w i t h LWB c o n t a i n i n g 0 .5M N a C l . The p e l l e t was washed t w i c e w i t h 0 .06M T r i s - H C l , pH 6 .8 and l e f t d r y . 50 pi o f r e d u c t i o n m i x t u r e (0 .0625M T r i s - H C l , 2% SDS, 10% g l y c e r o l , 5% m e r c a p t o e t h a n o l and 0.002% b romopheno l b l u e ) was added , m i x e d and b o i l e d f o r 5 m i n u t e s , a l l o w e d t o c o o l , and c e n t r i f u g e d f o r 10 m i n u t e s . R a d i o a c t i v i t y i n s u p e r n a t a n t s was d e t e r m i n e d and samp les were a p p l i e d t o a p o l y a c r y l a m i d e g e l c o n s i s t i n g o f a 4% s t a c k i n g and 10% r u n n i n g g e l ( Laemml i 1 9 7 0 ) , a l o n g w i t h s t a n d a r d v i r u s s amp le s and (BRL) h i g h r ange m o l e c u l a r w e i g h t m a r k e r s ( m y o s i n 2 0 0 , 0 0 0 ; p h o s p h o r y l a s e B 9 2 , 5 0 0 ; BSA 6 8 , 0 0 0 ; o v a l b u m i n 4 3 , 0 0 0 ; a - c h y m o t r y p s i n o g e n 2 5 , 7 0 0 ; / 3 - l a c t o g l o b u l i n 1 8 , 4 0 0 ; c y t o ch r ome C 1 2 , 3 0 0 ) . E l e c t r o p h o r e s i s was p e r f o r m e d a t c o n s t a n t c u r r e n t , and t h e g e l f i x e d w i t h 10% a c e t i c a c i d , i m p r e g n a t e d w i t h A m p l i f y (Amersham), d r i e d and f l u o r o g r a p h e d . M o l e c u l a r w e i g h t s o f v i r a l p r o t e i n s were d e t e r m i n e d b y c o m p a r i s o n w i t h t h e - 1 1 -d i s t a n c e s m i g r a t e d b y m o l e c u l a r w e i g h t m a r k e r s . F. IgG C l a s s i f i c a t i o n The O u c h t e r l o n y d o u b l e i m m u n o d i f f u s i o n t e c h n i q u e ( O u c h t e r l o n y , 1 9 4 9 ) , u s i n g 2% a g a r o s e was emp loyed f o r IgG c l a s s i f i c a t i o n . A g a r o s e was m e l t e d b y h e a t i n g i n b a r b i t o n e b u f f e r ( s od i um b a r b i t o l [12 g i n 800 mL H 2 0 ] and b a r b i t o l [ 4 . 4 g i n 150 mL H 2 0 a t 9 5 ° C ] , were c omb ined , t h e pH a d j u s t e d t o 8 . 2 , 0 . 1 5 g a z i d e added , and t h e vo lume a d j u s t e d t o 1 L ) , p o u r e d o n t o p l a s t i c s h e e t s , c o o l e d and w e l l s were punched 3 mm a p a r t . MAb ( h yb r i d oma c e l l c u l t u r e s u p e r n a t a n t p u r i f i e d and . c o n c e n t r a t e d b y s a t u r a t e d ammonium s u l p h a t e ) and a n t i - m o u s e serum (Goa t a n t i - m o u s e i m m u n o g l o b u l i n t o mouse IgM, I gG , IgG^, I g G 2 a > IgG2^, IgG^ f r om M e l o y , S p r i n g f i e l d , VA o r Tago , B u r l i n g a m e , CA) were added t o o p p o s i n g w e l l s and i n c u b a t e d i n a h u m i d i f i e d chamber a t 37°C o v e r n i g h t . G e l s were removed f r om s h e e t s , s o a k e d i n PBS f o r 24 t o 48 h o u r s t o remove e x c e s s and n o n - c o m p l e x e d p r o t e i n s , p l a c e d on p l a s t i c s h e e t s , c o v e r e d w i t h f i l t e r p a p e r and a i r d r i e d . The g e l s were t h e n s t a i n e d w i t h 1% amido b l a c k i n PBS, d e s t a i n e d w i t h 7% a c e t i c a c i d , r i n s e d i n ^ 0 and a i r d r i e d . A l t e r n a t i v e l y , p u r i f i e d mu r i n e a s c i t e s f l u i d was u s e d i n s t e a d o f c e l l c u l t u r e s u p e r n a t a n t , s e q u e n t i a l l y d i l u t e d w i t h PBS u n t i l o n l y one unamb iguous p r e c i p i t i n p a t t e r n was a p p a r e n t . D i l u t i o n o f t h e a n t i - m o u s e serum was a l s o p e r f o r m e d t o d e t e r m i n e c l o n a l i m m u n o g l o b u l i n t y p e . G. H a e m a g g l u t i n a t i o n I n h i b i t i o n 1. R u b e l l a V i r u s P r e p a r a t i o n R u b e l l a v i r u s s u i t a b l e f o r HA t e s t i n g was p r e p a r e d b y p r e c i p i t a t i n g v i r u s i n f e c t e d c u l t u r e s u p e r n a t a n t s w i t h 10% w/v PEG 6000 and p u r i f i e d b y a s u c c e s s i o n o f d i s c o n t i n u o u s and l i n e a r s u c r o s e g r a d i e n t c e n t r i f u g a t i o n s ( T r u d e l and Payment , 1 9 8 0 ) . - 1 2 -2. D e t e r m i n a t i o n o f R u b e l l a A n t i g e n A c t i v i t y A h epa r i n /manganou s c h l o r i d e t e c h n i q u e ( L i e b h a b e r , 1970) was u s e d f o r HI a s s a y s . I n a 96 w e l l r o unded b o t t o m m i c r o t i t r a t i o n p l a t e , 25 fil o f HSAG b u f f e r ( 0 .025M Hepes , 0 .14M N a C l , 0 .025M C a C l 2 . 2 H 2 0 , 1% BSA, 0 . 025 mg g e l a t i n / L , pH 6 . 5 ) was added t o 10 w e l l s and 50 fil ( c e l l c o n t r o l ) i n t o a s e p a r a t e w e l l , f o l l o w e d by 25 p i o f r e c o n s t i t u t e d r u b e l l a HA a n t i g e n i n t o t h e f i r s t w e l l , mak i ng s e r i a l d o u b l i n g d i l u t i o n s o f t h e a n t i g e n . 25 fil o f HSAG b u f f e r was p l a c e d i n e a c h w e l l c o n t a i n i n g a n t i g e n and c h i l l e d a t 4 °C . 50 fil o f t h e 0.25% ( v / v ) day o l d c h i c k e r y t h r o c y t e s u s p e n s i o n i n HSAG b u f f e r , was added t o e a ch w e l l , a g i t a t e d , c o v e r e d and l e f t u n d i s t u r b e d a t 4°C f o r 1.5 t o 2 h o u r s . The h i g h e s t d i l u t i o n e x h i b i t i n g a g g l u t i n a t i o n was d e s i g n a t e d as h a v i n g 1 HA u n i t . The a n t i g e n d i l u t i o n c o n t a i n i n g 4 HA u n i t s was u s e d i n a l l s u b s equen t t e s t s . 3 . P r e t r e a t m e n t o f Serum t o Remove N o n - S p e c i f i c I n h i b i t o r s N o n - s p e c i f i c se rum i n h i b i t o r s o f r u b e l l a v i r u s h a e m a g g l u t i n a t i o n a r e known t o be m o s t l y se rum l i p o p r o t e i n s , n o t a b l y f3-lipoproteins ( S h o r t r i d g e , 1 9 7 4 ) , w h i c h c a n be removed by h e p a r i n t r e a t m e n t (Kawano, 1 9 8 7 ) . 20 pi o f se rum, and 30 fil HSAG b u f f e r were m i x e d w i t h 20 fil o f h e p a r i n - M n C ^ s o l u t i o n (1M M n C l 2 , 2500 I U / m l h e p a r i n ) and i n c u b a t e d a t 4°C f o r 15 m i n u t e s f o l l o w e d b y 20 fil o f a 50% c h i c k e r y t h r o c y t e s u s p e n s i o n i n HSAG b u f f e r , a g i t a t e d and i n c u b a t e d a t 4°C f o r one h o u r . 80 fil HSAG b u f f e r was added , and t h e e r y t h r o c y t e s were p e l l e t e d by c e n t r i f u g a t i o n f o r 15 m i n u t e s a t 1500 rpm (600 g ) , r e s u l t i n g i n a s u p e r n a t a n t d i l u t e d 1 :8 . 4 . D e t e r m i n a t i o n o f HI A n t i b o d i e s i n S e r a 25 ftl o f r u b e l l a a n t i g e n (4 HA u n i t s ) was added t o e a ch o f t h e r o u n d b o t t o m e d w e l l s c o n t a i n i n g 25 fil o f p r e t r e a t e d serum t h a t h a d b e en s e r i a l l y -13-d o u b l y d i l u t e d i n HSAG b u f f e r , and a l s o t o a w e l l c o n t a i n i n g o n l y 25 fil o f HSAG b u f f e r ( se rum c o n t r o l ) , and i n c u b a t e d f o r one h o u r a t 4 °C . 50 fil o f t h e 0.25% ( v / v ) day o l d c h i c k e r y t h r o c y t e s u s p e n s i o n was added t o a l l w e l l s i n c l u d i n g two c o n t a i n i n g o n l y 50 fil HSAG b u f f e r ( c e l l c o n t r o l ) , f o l l o w e d b y i n c u b a t i o n a t 4°C f o r one h o u r , t h e n room t e m p e r a t u r e f o r 15 m i n u t e s . The h i g h e s t d i l u t i o n o f serum s how i ng no a g g l u t i n a t i o n was d e s i g n a t e d as h a v i n g one HI u n i t , and i t s r e c i p r o c a l was t h e HI t i t r e o f t h e se rum. H. R u b e l l a V i r u s N e u t r a l i z a t i o n 1. R u b e l l a V i r u s - Complement C o m p a t i b i l i t y Complement when a t h i g h c o n c e n t r a t i o n s , i s known t o i n a c t i v a t e v i r u s e s a l o n e , e i t h e r b y a c t i v a t i o n o f a l t e r n a t e pa thways o r s i m p l e s u r f a c e a c c u m u l a t i o n . P l a q u e f o r m a t i o n s t u d i e s a t v a r y i n g c o n c e n t r a t i o n s o f complement were p e r f o r m e d i n o r d e r t o a v o i d i n a c t i v a t i o n o f v i r u s s o l e l y b y comp lement . E q u a l v o l umes o f M33 o r RA 27 /3 r u b e l l a v i r u s (2 PFU / / i l i n M199, 2% FCS/1% PS) and r a b b i t complement s e r i a l l y d o u b l y d i l u t e d f r om 1 i n 5 p a r t s i n s i m i l a r medium were i n c u b a t e d f o r 1 h o u r a t 37°C and t h e n 50 fil was a p p l i e d t o m o n o l a y e r s o f RK c e l l s , f o u r w e l l s p e r d i l u t i o n . The s e c o n d d i l u t i o n o f complement t h a t f a i l e d t o i n h i b i t t h e CPE o f r u b e l l a was u s e d as t h e f i n a l c o n c e n t r a t i o n i n n e u t r a l i z a t i o n t e s t s e m p l o y i n g comp lement . 2. V i r u s N e u t r a l i z a t i o n 40 fil o f p u r i f i e d a s c i t e s f l u i d o r c o n t r o l serum was h e a t e d a t 56°C f o r 20 m i n u t e s t o i n a c t i v a t e comp lement , d i l u t e d 1:5 i n M199 medium w i t h 2% FCS/1% PS, c e n t r i f u g e d f o r 10 m i n u t e s a t 10 , 000 rpm and s t e r i l i z e d b y f i l t r a t i o n t h r o u g h a 0 . 22 fi p o r e s i z e Ge lman f i l t e r . S e r i a l d o u b l i n g d i l u t i o n s o f t h e p u r i f i e d serum (1 mg I gG /m l ) were p e r f o r m e d i n M199 medium w i t h 2% FCS/1% PS t o w h i c h was added e q u a l amounts o f - 1 4 -M33 o r RA 27 /3 r u b e l l a v i r u s (2 PFU/>1, i n M199 medium, 2% FCS/1% P S ) , w i t h o r w i t h o u t 2.5% r a b b i t comp lement . The v i r u s - a n t i b o d y m i x t u r e was i n c u b a t e d a t 37°C f o r one h o u r , t h e n 50 fil was l a y e r e d on t o one day o l d RK c e l l mono l a y e r s i n 96 w e l l m i c r o t i t r e p l a t e s , m i x i n g f o r one h o u r a t 37°C . The v i r u s - a n t i b o d y m i x t u r e was removed and t h e mono l a y e r s were l a y e r e d i n M199 medium c o n t a i n i n g 5% FCS and 1% PS , i n c u b a t e d a t 35°C f o r 60 t o 72 h o u r s . 3 . P l a q u e D e t e c t i o n by Immunoperox idase Me thod RK c e l l m o n o l a y e r s were g e n t l y washed t w i c e w i t h 0 . 2 mL o f PBS, pH 7 . 4 , f i x e d w i t h 0 . 2 mL 2% n e u t r a l b u f f e r e d f o r m a l i n f o r 15 m i n u t e s a t room t e m p e r a t u r e , and t h e n washed t w i c e w i t h 0 . 2 mL PBS, pH 7 . 4 . M o n o l a y e r s were t h e n b r o u g h t t h r o u g h 70%, t h e n 95% m e t h a n o l , and endogenous p e r o x i d a s e was i n a c t i v a t e d w i t h 0 . 2 mL o f 0.5% ^2^2 * n a b s ° l u t e m e t h a n o l f o r 15 m i n u t e s a t room t e m p e r a t u r e . Samples were r e h y d r a t e d w i t h 95% t h e n 70% m e t h a n o l and washed t w i c e w i t h 0 . 2 mL PBS, pH 7 . 4 . The n o n - s e l e c t i v e i m m u n o g l o b u l i n b i n d i n g s i t e s o f t h e m o n o l a y e r s were b l o c k e d by i n c u b a t i o n f o r one h o u r a t 37°C w i t h 0 . 2 mL o f p re - immune r a b b i t se rum ( d i l u t e d 1:200 i n PBS/0.5% BSA) and washed t w i c e w i t h wash b u f f e r (PBS, 0.1% BSA, pH 7 . 4 ) . 0 . 2 mL o f r u b e l l a immune mouse serum ( a n t i - M33 d i l u t e d 1:200 i n PBS/0 .5% BSA ) , was added and i n c u b a t e d a t 37°C f o r 60 m i n u t e s f o l l o w e d b y t h r e e wash b u f f e r r i n s e s . P e r o x i d a s e - c o n j u g a t e d (HRP) r a b b i t i m m u n o g l o b u l i n t o mouse IgG ( D a k o - i m m u n o g l o b u l i n s a / s , G u l d b o r g v e j 22 , DK-2000 Copenhagen F . , Denmark, 1:500 i n PBS/0.5% BSA, 0 . 1 mL p e r w e l l ) was added and i n c u b a t e d f o r one h o u r a t 37°C and t h e n r i n s e d t h r e e t i m e s w i t h wash b u f f e r . 0 . 1 mL c o l d PBS c o n t a i n i n g 0.02% ^2^2 a n C * 3 , 3 ' d i a m i n o b e n z i d i n e t e t r a h y d r o c h l o r i d e ( 0 . 5 mg/m l , pH 7 . 4 ) was a p p l i e d and m o n o l a y e r s were i n c u b a t e d a t room t e m p e r a t u r e . When b rown d e p o s i t s d e v e l o p e d , r e a c t i o n s were - 1 5 -h a l t e d b y r e m o v i n g t h e s u b s t r a t e w i t h a PBS r i n s e and a d d i n g 10% n e u t r a l b u f f e r e d f o r m a l i n . P l a q u e s were enume ra t ed f r om t h r e e w e l l s p e r a n t i b o d y d i l u t i o n , and a v e r a g e d . The n e u t r a l i z a t i o n t i t r e (N t ) was t h e r e c i p r o c a l o f t h e d i l u t i o n t h a t d e m o n s t r a t e d a t l e a s t a 50% r e d u c t i o n i n p l a q u e f o r m a t i o n compared t o c o n t r o l w e l l s (50 t o 100 P F U / w e l l ) . I X . RESULTS A . P u r i f i c a t i o n and S t a n d a r d i z a t i o n o f M o n o c l o n a l A n t i b o d i e s Twenty f i v e MAb samp les i n t h e f o rm o f a s c i t e s f l u i d were a v a i l a b l e , r a n g i n g i n vo l ume f r o m 1 t o 18 m l . A l l were p u r i f i e d (maximum o f 10 m l / a s c i t e s samp le ) b y t h e B i o r a d P r o t e i n A Mapps c h r oma t og r aphy method and d i a l y z e d a g a i n s t PBS-1% sod ium a z i d e t o remove e l u t i o n b u f f e r . Vo lumes o f s amp le s r a n g e d f r o m 0 . 8 t o 2 .0 m l , h a v i n g a c o n c e n t r a t i o n r ange o f 0 . 5 t o 1 6 . 0 mg I g G / m l as d e t e r m i n e d b y a t o t a l p r o t e i n s t a n d a r d c u r v e u s i n g Coomass i e b l u e and b o v i n e IgG s t a n d a r d s . U n p r o c e s s e d and p u r i f i e d a s c i t e s f l u i d were s u b j e c t e d t o 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 and g e l s were s t a i n e d w i t h Coomass i e b l u e . A l l s amp l e s showed a marked o r t o t a l r e d u c t i o n i n n o n - i m m u n o g l o b u l i n ( p r o t e i n s o t h e r t h a n h e a v y and l i g h t c h a i n s o f IgG) bands when p u r i f i e d b y t h e P r o t e i n A Mapps me thod ( F i g . 1 ) . S e v e r a l f l o w t h r o u g h f r a c t i o n s o f a s c i t e s f l u i d i n b i n d i n g b u f f e r were r e - p u r i f i e d w i t h n e g l i g i b l e amounts o f IgG h e a v y o r l i g h t c h a i n s f o u n d i n t h e s e c o n d e l u t i o n , i n d i c a t i n g h i g h i n i t i a l r e c o v e r y o f IgG f r om u n p r o c e s s e d f l u i d . A l l p u r i f i e d a s c i t e s f l u i d s were a d j u s t e d t o a c o n c e n t r a t i o n o f 1 .0 mg I gG /m l w i t h PBS, 1% sod i um a z i d e . A l l t e s t i n g , e x c e p t f o r t h e IgG s u b c l a s s i f i c a t i o n o f some MAbs, was done a t t h i s c o n c e n t r a t i o n . - 1 6 -200,000-92,500-68,000-43,00 0-m w A a B b C c D d w E e F f F i g . 1 SDS-PAGE a n a l y s i s o f p u r i f i e d and n o n - p u r i f i e d a s c i t e s . C a p i t a l i z e d l e t t e r s : n o n - p u r i f i e d a s c i t e s , l o w e r c a se l e t t e r s : c o r r e s p o n d i n g p u r i f i e d a s c i t e s . MW, m o l e c u l a r w e i g h t m a r k e r s ; A, 4G6A; B, 14B1F; C, 15C11B; D, 16A10E; E, 21D9C; and F, 21D10B. B. V e r i f i c a t i o n o f R u b e l l a V i r u s M o n o c l o n a l A n t i b o d y S p e c i f i c i t y A l t h o u g h t he h y b r i d o m a s h a d been s c r e e n e d f o r r u b e l l a s p e c i f i c a n t i b o d i e s by E L I SA , t h e immunope rox i da se method was u s e d t o c o n f i r m t h e 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 i e s . A l l MAbs i n t e r a c t e d s p e c i f i c a l l y w i t h e i t h e r M33 o r RA 27/3 r u b e l l a v i r u s p l a q u e s i n RK c e l l c u l t u r e s w i t h v a r y i n g d e g r e e s o f i n t e n s i t y . Mos t MAbs r e a c t e d w i t h b o t h s t r a i n s ; t h r e e MAbs i n t e r a c t e d w i t h o n l y one s t r a i n ( T a b l e I ) . One MAb (21D10B) was f o u n d t o have e x t e n s i v e c r o s s - r e a c t i v i t y t o RK c e l l s . I have more c o n f i d e n c e i n t h i s t e c h n i q u e t h a n i n t he EL ISA s c r e e n i n g t e c h n i q u e as t h e MAb r e a c t i v i t y a g a i n s t r u b e l l a v i r u s - i n i t i a t e d CPE was e a s i l y d i f f e r e n t i a t e d f r om t h a t o f h o s t c e l l p r o t e i n s t h a t may be p r e s e n t i n EL ISA s c r e e n i n g s . P l a q u e s f r om u n s t a i n e d and n e u t r a l r e d s t a i n e d m o n o l a y e r s (Nawa, 1979) (see - 1 7 -f i g u r e s l l a / b , page 4 5 ) , c o u l d n o t be r e l i a b l y d i f f e r e n t i a t e d f r o m b a c k g r o u n d - c e l l a n o m a l i e s , s u c h as n a t u r a l c e l l d e a t h and c e l l b u n c h i n g due t o o v e r g r o w t h . The r u b e l l a immunope rox i dase s t a i n i n g t e c h n i q u e u s e d aRA 27 /3 hype r - immune mouse serum as t h e p r i m a r y a n t i b o d y . S t a i n i n g o f RK c e l l m o n o l a y e r s was l i m i t e d t o a r e a s o f CPE b e l i e v e d t o be r u b e l l a p l a q u e s , e i t h e r M33 o r RA 27 /3 s t r a i n i n o r i g i n ( F i g s . 10 d / e / f ) . P re - immune se rum f a i l e d t o s t a i n p l a q u e s ( F i g . 1 0 c ) . T a b l e I . V e r i f i c a t i o n o f s p e c i f i c i t y o f m o n o c l o n a l a n t i b o d i e s . MAb v s M33 RA 27 /3 MAb v s M33 RA 27 /3 2F4B + + 15C3E + + 2F12F + + 15C11B + Neg 3D5D + + 16A3C + . + 3D9F + + 16A10E + + 4G6A + Neg 16B2C + + 4G12E + Neg 16B8D + + 12B2D + + 16D9D + + 12B3G + + 16D11C + + 13A1F + + 21B8H + + 13A4H + + 21B9H • + + 13G6H Neg + 21D9C + + 14B1F + + 21D10B + * + * 14B3D + + MAb r e a c t i v i t y t o M33 o r RA 27 /3 r u b e l l a v i r u s p l a q u e s on RK c e l l m o n o l a y e r s as d e t e c t e d by immunope rox i da se t e c h n i q u e . * s i g n i f i c a n t c r o s s - r e a c t i v i t y o f MAb a g a i n s t c e l l u l a r p r o t e i n s . C. I m m u n o p r e c i p i t a t i o n B a s e d on t h e p r o c e d u r e s d e s c r i b e d b y Waxham e t a l ( 1 9 8 5 ) , [ v i r u s d i s r u p t e d i n immune l y s a t e b u f f e r (10 mM T r i s - H C l , 0 .5M N a C l , 0.1% SDS, 0 .1M i o d o a c e t a m i d e , 1% T r i t o n X - 1 0 0 , 1 mM EDTA, 0.5% sod i um d e o x y c h o l a t e , 2 .5 mM PMSF, pH 7 . 4 ) ] , I f o u n d t h a t a l l 25 m o n o c l o n a l a n t i b o d i e s f e l l i n t o two c o n t r a s t i n g g r oup s b a s e d on t h e i r i m m u n o p r e c i p i t a t i o n p a t t e r n s . One g roup d i s p l a y e d no p r e c i p i t a t i o n o f r u b e l l a s t r u c t u r a l p r o t e i n s w h i l e t h e o t h e r g roup p r o d u c e d i n t e n s e E l p r o t e i n p r e c i p i t a t i o n as w e l l as c o - p r e c i p i t a t i o n o f E2 and - 1 8 -NC ( n u c l e o c a p s i d ) p r o t e i n s t o some d e g r e e . These r e s u l t s c a s t u n c e r t a i n t y on t h e s p e c i f i c i t i e s o f t h e a n t i b o d i e s t o i n d i v i d u a l v i r a l p r o t e i n s ( F i g . 2 ) . C a p s i d p r o t e i n s have e v en a p p e a r e d i n p re - immune se rum l a n e s . S i n c e a l l MAbs i n t h e p a n e l r e a c t e d w i t h s i t e s on r u b e l l a v i r u s p l a q u e s on RK c e l l m o n o l a y e r s , t h e l a c k o f m o n o c l o n a l s d e m o n s t r a t i n g immune r e a c t i v i t y d u r i n g r a d i o i m m u n o p r e c i p i t a t i o n c o u l d o n l y be a t t r i b u t e d t o a l t e r e d c o n f o r m a t i o n o f s p e c i f i c e p i t o p e s b e c au s e o f i n s u f f i c i e n t o r e x c e s s i v e s o l u b i l i z a t i o n b y t h e d e t e r g e n t s , s u b - o p t i m a l N a C l c o n c e n t r a t i o n s , o r a l t e r a t i o n o f t h e a n t i b o d i e s i n v i r u s l y s a t e b u f f e r s . I n an a t t e m p t t o u n c o v e r e p i t o p e s t h a t may be i n a d e q u a t e l y e x p o s e d , I emp l oyed r e l a t i v e l y s t r i n g e n t s o l u b i l i z e r s s u c h as 0.5% SDS w i t h up t o 2% m e r c a p t o e t h a n o l i n t h e v i r u s l y s a t e b u f f e r ( s ee M a t e r i a l s and Me thods ) w i t h no improvement i n t h e p r e c i p i t a t i o n p a t t e r n o f v i r a l g l y c o p r o t e i n s . To overcome t h e p o s s i b i l i t y o f e x c e s s i v e e p i t o p e d e n a t u r a t i o n , t h e v i r u s was d i s r u p t e d i n m i l d e r s o l u t i o n s , s u c h as PBS c o n t a i n i n g 1% N P ^ Q . T r i t o n X - 1 0 0 , o r 0.5% sod i um d e o x y c h o l a t e . Comp lexes were washed i n e i t h e r t h e o r i g i n a l wash b u f f e r s o r r e l a t i v e l y m i l d e r s o l u t i o n s o f PBS, 1% N P ^ Q , 0.5% sod i um d e o x y c h o l a t e . The use o f a v i r a l l y s a t e b u f f e r l a c k i n g SDS and i o d o a c e t a m i d e b u t c o n t a i n i n g 1% T r i t o n X - 1 0 0 , 0.5% sod i um d e o x y c h o l a t e , 1 mM EDTA, 2 . 5 mM PMSF, 10 mM T r i s - H C l pH 7 .4 p r o d u c e d s p e c i f i c v i r a l p r e c i p i t a t i o n b y s e v e r a l m o n o c l o n a l a n t i b o d i e s , a l l t o t h e E l p r o t e i n . I n o r d e r t o o p t i m i z e i o n i c c o n d i t i o n s f o r e a ch e p i t o p e / a n t i b o d y c o m b i n a t i o n , I d u p l i c a t e d p r e c i p i t a t i o n s a t 0 .18M ( p h y s i o l o g i c a l l e v e l ) , 0 . 3 M, and 0 .5M N a C l c o n c e n t r a t i o n s and I f o u n d more s p e c i f i c p r e c i p i t a t i o n by o t h e r m o n o c l o n a l a n t i b o d i e s , dependen t on N a C l c o n c e n t r a t i o n . E x p e r i m e n t s were t h e n r e p e a t e d f o r t h e r e m a i n i n g u n d e t e r m i n e d MAbs u s i n g a s a l t g r a d i e n t ( 0 . 1M t o 0 .5M N a C l ) , r e s u l t i n g i n an o v e r a l l i n c r e a s e i n r u b e l l a s t r u c t u r a l p r o t e i n b a n d i n g . - 1 9 -A B C D E F G H NC E1 E2 92.5 K 68K 4 3 K F i g . 2 I m m u n o p r e c i p i t a t i o n o f r u b e l l a v i r a l p r o t e i n s d e m o n s t r a t i n g C j f ) - p r e c i p i t a t i o n o f E2 and NC p r o t e i n s w i t h E l . Lane A , S - m e t h i o n i n e - l a b e l l e d M33 r u b e l l a v i r u s ; l a n e B, i m m u n o p r e c i p i t a t e d w i t h r a b b i t a-M33 s e r a ; l a n e C, r a b b i t p re - immune s e r a , l a n e D, m o n o c l o n a l 2F4B; l a n e E, 2F12F; l a n e F, 3D5D; l a n e G, 4G6A, l a n e H, 14B1F . However , two and o c c a s i o n a l l y t h r e e p r o t e i n s were g e n e r a l l y p r e c i p i t a t e d , a l o n g w i t h n o n - v i r a l p r o t e i n s t h a t were d i f f i c u l t t o remove e ven w i t h t h e mos t h a r s h wash b u f f e r s ( 1 . 0M N a C l , 0.5% SDS, 2% m e r c a p t o e t h a n o l , 1% T r i t o n X - 1 0 0 , 1% sod ium d e o x y c h o l a t e , 0 .1M i o d o a c e t a m i d e , 1 mM EDTA, 10 mM T r i s - H C l , pH 7 . 4 ) . I n o r d e r t o r e s o l v e t h e s p e c i f i c i t i e s o f t h o s e m o n o c l o n a l a n t i b o d i e s t h a t showed m u l t i p l e p r o t e i n p r e c i p i t a t i o n p a t t e r n s w i t h t h e Waxham t e c h n i q u e , I f u r t h e r m o d i f i e d t h e c o n s t i t u e n t s o f b o t h t h e v i r u s l y s a t e and wash b u f f e r s . I f o u n d n u c l e o c a p s i d (NC) c o - p r e c i p i t a t i o n w i t h t h e E l g l y c o p r o t e i n was a f u n c t i o n o f n o n - s p e c i f i c a d s o r p t i o n t o t he s u r f a c e o f t h e S . a u r e u s c e l l s . T h i s was i n d i c a t e d b y c a p s i d band appea rance when S. a u r e u s a l o n e was i n c u b a t e d w i t h l a b e l l e d v i r u s , d e s p i t e e x t e n s i v e p r e - a d s o r p t i o n o f t h e l a b e l l e d v i r u s - 2 0 -w i t h S. a u r e u s . I n s e r t i o n o f a s e t o f t h r e e i m m u n o p r e c i p i t a n t washes w i t h l y s a t e wash b u f f e r c o n t a i n i n g 1.0M N a C l ( see m a t e r i a l s and me thods ) b e f o r e t h e 0.5M N a C l washes , and t h e e l i m i n a t i o n o f t h e u se o f i o d o a c e t a m i d e , s i g n i f i c a n t l y d e c r e a s e d o r removed c o - p r e c i p i t a t i o n o f t h e NC p r o t e i n s i n p re - immune serum o r m o n o c l o n a l t e s t s ( F i g . 3 ) . T h i s i n d i c a t e d t h a t t h e n o n - s p e c i f i c c a p s i d b i n d i n g was due t o s i m p l e i o n i c i n t e r a c t i o n s . The use o f more t h a n f i v e washes d i d n o t f u r t h e r v i s i b l y d e c r e a s e b a n d i n g p a t t e r n s o f any p r o t e i n s . M o n o c l o n a l a n t i b o d i e s t h a t have h i g h HI and N t t i t r e s have i n v a r i a b l y ( w i t h t he e x c e p t i o n o f one n e u t r a l i z i n g E2 MAb; G r e e n , 1986) b e e n d i r e c t e d t o t h e E l g l y c o p r o t e i n . These m o n o c l o n a l a n t i b o d i e s o f p r e s u m p t i v e E l s p e c i f i c i t y (due F i g . 3 I m m u n o p r e c i p i t a t i o n w i t h v a r i a t i o n i n t h e N a C l c o n c e n t r a t i o n o f wash b u f f e r s . Lane A , S - m e t h i o n i n e - l a b e l l e d M33 r u b e l l a v i r u s ; l a n e B, i m m u n o p r e c i p i t a t e d w i t h r a b b i t o-M33 s e r a ; l a n e C, r a b b i t p re - immune s e r a , l a n e D t o H, a l l m o n o c l o n a l 12B3G (a E l ) unde r v a r i e d wash c o n d i t i o n s ; l a n e D, f i v e 1.0M N a C l washes ; l a n e E, f i v e 1.0M and f i v e 0 .5M N a C l wa she s ; l a n e F, f i v e 1.0 M, f i v e 0 .5M and f i v e 0 .17M N a C l washes ; l a n e G, t e n 1.0M N a C l washe s ; l a n e H, t e n 1.0M and f i v e 0 .5M N a C l washe s . - 2 1 -t o h i g h HI and N t t i t r e s ) have a l s o shown c o - p r e c i p i t a t i o n o f c o m p a r a t i v e l y s m a l l ( b u t v i s i b l e ) amounts o f t h e E2 g l y c o p r o t e i n , a p r o b l e m t h a t h a s p l a g u e d many p r e v i o u s r e s e a r c h e r s and t h i s a u t h o r . I n my e x p e r i m e n t s , a l t e r a t i o n s were made i n N a C l c o n c e n t r a t i o n s o f t h e wash b u f f e r s r a n g i n g f r om b u f f e r s d e v o i d o f s a l t t o b u f f e r s c o n t a i n i n g 1M N a C l . As w e l l , t h e SDS c o n c e n t r a t i o n s were e l e v a t e d as h i g h as 1% SDS ( r i s k i n g d i s s o c i a t i o n o f t h e immune c o m p l e x e s ) , b u t t h e s e a l t e r a t i o n s f a i l e d t o r e s o l v e t h e s p e c i f i c i t i e s o f t h e MAbs. P r e v i o u s r e s e a r c h e r s had a t t e m p t e d d i s s o c i a t i o n o f E1 - E2 comp l exe s t h r o u g h m e r c a p t o e t h a n o l r e d u c t i o n o f h y p o t h e t i c a l d i s u l p h i d e l i n k a g e s , e i t h e r f o r r e a c t i o n w i t h , o r i n p r e p a r a t i o n o f v i r a l p e p t i d e s u s e d as immunogens f o r t h e A B C D E F G /H I J F i g ^ 4 I m m u n o p r e c i p i t a t i o n w i t h i n c l u s i o n o f 2 - m e r c a p t o e t h a n o l ( 2 - M E ) . Lane A , S - m e t h i o n i n e - l a b e l l e d M33 r u b e l l a v i r u s ; l a n e B, i m m u n o p r e c i p i t a t e d w i t h r a b b i t a-M33 serum w i t h no 2-ME; l a n e C, r a b b i t Q-M33 se rum w i t h 1% 2-ME i n l y s a t e wash b u f f e r (LWB); l a n e D, r a b b i t a-M33 serum w i t h 2% 2-ME i n i n c u b a t i o n b u f f e r and 1% 2-ME i n LWB; l a n e E, r a b b i t p re - immune s e r a ; l a n e F, 2F4B w i t h no 2-ME; l a n e G, 2F4B w i t h 1% 2-ME i n LWB; l a n e H, 14B1F w i t h no 2-ME; l a n e I , 14B1F w i t h 1% 2-ME i n LWB; l a n e J , 14B1F w i t h 2% 2-ME i n i n c u b a t i o n b u f f e r and 1% 2-ME i n LWB. - 2 2 -p r o d u c t i o n o f p o l y v a l e n t hype r - immune se rum. I i n c o r p o r a t e d m e r c a p t o e t h a n o l i n t o t h e wash b u f f e r s o f s e v e r a l i m m u n o p r e c i p i t a t i o n s i n v o l v i n g m o n o c l o n a l a n t i b o d i e s t h a t c o - p r e c i p i t a t e d E l and E2 p r o t e i n s . T h i s was done t o o b s e r v e t h e e f f e c t upon i n d i v i d u a l e p i t o p e b i n d i n g , and i n a t t e m p t t o e l i m i n a t e c o - p r e c i p i t a t i o n a f t e r t h e f o r m a t i o n o f immune c o m p l e x e s . I e x p e c t e d t h a t t h e a n t i b o d y - E l c omp l ex w o u l d r e m a i n i n t a c t a f t e r s e p a r a t i o n o f t h e E1 -E2 d i m e r , p o s s i b l y due t o some s t a b i l i z a t i o n o f t h e c o n f o r m a t i o n o f t h e e p i t o p e b y t h e a n t i g e n - a n t i b o d y c omp l e x . R u b e l l a p o l y v a l e n t hype r - immune se rum p r e c i p i t a t i o n o f t h e v i r a l g l y c o p r o t e i n s was n o t s i g n i f i c a n t l y a f f e c t e d , b u t f o r m o n o c l o n a l s e r a , an i n c l u s i o n o f 1% m e r c a p t o e t h a n o l i n w a s h i n g b u f f e r s a f t e r p r e c i p i t a t i o n c o n s i d e r a b l y r e d u c e d b o t h E l and E2 bands ( F i g . 4 ) . When 2% m e r c a p t o e t h a n o l was i n c o r p o r a t e d i n t o t h e l y s a t e i n c u b a t i o n b u f f e r p r i o r t o p r e c i p i t a t i o n , p r e c i p i t a t e s were m a r k e d l y r e d u c e d r e s u l t i n g i n a f a i n t b and o f E l . E2 c o - p r e c i p i t a t i o n i n t e n s i t y i s g e n e r a l l y l e s s i n c o m p a r i s o n t o E l , t h e r e f o r e i t c a n n o t be c o n c l u d e d t h a t i t was e l i m i n a t e d . The r e was no s e l e c t i v e r e m o v a l o f e i t h e r E l o r E2 p r o t e i n bands f r om t he p r e c i p i t a t e , i n s t e a d t h e r e was p r o g r e s s i v e and b r o a d b a s e d r e m o v a l o f a l l r u b e l l a s t r u c t u r a l p r o t e i n s , a s p r e d i c t e d i n r e s u l t s by H o - T e r r y ( 1 9 8 4 ) . T h i s i m p l i e s t h a t m e r c a p t o e t h a n o l c l e a v a g e o f d i s u l p h i d e bonds was s u f f i c i e n t t o a l t e r t h e c o n f o r m a t i o n o f t h e e p i t o p e i n q u e s t i o n , o r a l t e r t h e a n t i g e n b i n d i n g s i t e o f t h e MAb. The g r o s s s t r u c t u r e o f t h e a n t i b o d y was n o t a f f e c t e d as b o t h h e a v y and l i g h t c h a i n s o f IgG were f o u n d i n t a c t when s t a i n e d b y Coomass i e b l u e a f t e r e l e c t r o p h o r e s i s . Th rough a l t e r a t i o n s o f t h e c o n s t i t u e n t s o f l y s a t e and wash b u f f e r s , t h e a n t i g e n s p e c i f i c i t i e s o f t h i r t e e n o f t w e n t y - f i v e MAbs were d e t e r m i n e d by 35 i m m u n o p r e c i p i t a t i o n o f [ S ] - m e t h i o n i n e l a b e l l e d r u b e l l a v i r u s . The a p p l i c a b l e c o n d i t i o n s i n w h i c h t h e m o n o c l o n a l s r e a c t e d a r e shown i n T a b l e I I and F i g u r e s 5 and 6 . - 2 3 -T a b l e I I S p e c i f i c i t y o f M o n o c l o n a l A n t i b o d i e s t o I n d i v i d u a l R u b e l l a V i r u s S t r u c t u r a l P r o t e i n s M o n o c l o n a l P r o t e i n A n t i b o d v S p e c i f i c i t y M33 RA 2F4B E l a E 1 b 2F12F - E 1 b 3D9F E 1 b E l b 4G6A E l b -12B2D " k E la 12B3G E l b E l a 13A1F E l 3 "v. 13A4H E l b 14B1F E l b E 1 b 16A3C E 1 b E 1 b 16A10E E l b E l b 21B8H - E l a 21B9H - E l a a = l y s a t e b u f f e r c o n s i s t i n g o f 0 .5M N a C l , 1% T r i t o n X - 1 0 0 , 0 5% sod i um d e o x y c h o l a t e , 1 mM EDTA, 2 .5 mM PMSF, 10 mM T r i s - H C l pH 7 . 4 ; = l y s a t e b u f f e r w i t h i n c l u s i o n o f 0.1% SDS. ( - ) = ambiguous r e a c t i v i t y o r no r e a c t i v i t y . I f o u n d two o p t i m a l p r e c i p i t a t i n g c o n d i t i o n s , one o f a v i r a l l y s a t e b u f f e r o f 0 .5M N a C l , 1% T r i t o n X - 1 0 0 , 0.5% sod ium d e o x y c h o l a t e , 1 mM EDTA, 2 .5 mM PMSF, 10 mM T r i s - H C l , pH 7 . 4 , and t h e s e c ond o f t h e same l y s a t e b u f f e r w i t h t h e a d d i t i o n o f 0.1% SDS. A l t e r a t i o n s i n t h e N a C l c o n c e n t r a t i o n s , f r om 0.18M t o 0 . 5 M, were made i n o r d e r t o o p t i m i z e i o n i c c o n d i t i o n s f o r e a c h m o n o c l o n a l a n t i b o d y . T h i s imp r o v ed some p r e c i p i t a t i o n s , b u t n o t d r a m a t i c a l l y . Immunoadsorbents were washed t h r e e t i m e s w i t h l y s a t e wash b u f f e r c o n t a i n i n g 1.0M N a C l , 0.1% SDS, 1% T r i t o n X - 1 0 0 , 0.5% sod ium d e o x y c h o l a t e , 10 mM T r i s - H C l , pH 7 . 4 , and t h e n t h r e e t i m e s w i t h t h e same b u f f e r w i t h 0 .5M N a C l , f o l l o w e d b y two washes w i t h 0 .06M T r i s - H C l , pH 6 .8 i n o r d e r t o remove e x c e s s d e t e r g e n t p r i o r t o d i s s o c i a t i o n o f t h e i m m u n o p r e c i p i t a n t s w i t h t h e Laemml i r e d u c t i o n m i x t u r e ( L a e m m l i , 1 9 7 0 ) . - 2 4 -A B C D E F 68K 4 3 K F i g . 5a G H I J K L 68K - 4 3 K F i g . 5b F i g . 5 a /b S p e c i f i c i t y o f M o n o c l o n a l A n t i b o d i e s t o M33 S t r a i n R u b e l l a V i r u s S t r u c t u r a l P r o t e i n s . " a " - l y s a t e b u f f e r c o n s i s t i n g o f 0 .5M N a C l , 1% T r i t o n X - 1 0 0 , 0.5% sod i um d e o x y c h o l a t e , 1 mM EDTA, 2 .5 mM PMSF, 10 mM T r i s - H C l pH 7 . 4 ; " b " - l y s a t e b u f f e r w i t h i n c l u s i o n o f 0.1% SDS. A and G, r a b b i t a -RA 27 /3 s e r a ; B and H, r a b b i t p re - immune s e r a ; C, 2F4B; D, 3D9F; E, 13A1F; F, 16A3C; I , 4G6A; J , 12B3G; K, 14B1F; L, 16A10E. - 2 5 -A B C D E F G H F i g . 6a I J K L M N 0 F i g . 6b F i g . 6a /b S p e c i f i c i t y o f M o n o c l o n a l A n t i b o d i e s t o RA 27 /3 S t r a i n R u b e l l a V i r u s S t r u c t u r a l P r o t e i n s . " a "= l y s a t e b u f f e r c o n s i s t i n g o f 0 .5M N a C l , 1% T r i t o n X - 1 0 0 , 0.5% sod i um d e o x y c h o l a t e , 1 mM EDTA, 2 .5 mM PMSF, 10 mM T r i s - H C l pH 7 . 4 ; "b"= l y s a t e b u f f e r w i t h i n c l u s i o n o f 0.1% SDS. A and I , r a b b i t a -RA 27 /3 s e r a ; B and J , r a b b i t p re - immune s e r a ; C, 2F4B; D, 12B2D; E, 12B3G; F, 14B1F; G, 21B8H; H, 21B9H; K, 2F12F; L, 3D9F; M, 13A4H; N, 16A3C; 0 , 16A10E. - 2 6 -D. IgG C l a s s i f i c a t i o n C o n c e n t r a t e d c e l l c u l t u r e s u p e r n a t a n t s were u s e d t o t y p e f i f t e e n MAbs. P u r i f i e d a s c i t e s f l u i d were Used t o t y p e t h e r e m a i n i n g t e n MAbs, due t o l ow q u a n t i t i e s o f i m m u n o g l o b u l i n f o u n d i n t h e c u l t u r e s u p e r n a t a n t s . As shown i n F i g 7a and summar i z ed i n T a b l e I I I , a l l MAbs were c l a s s e d as IgG.. , IgG„ , o r IgG2^, and no IgG^ o r IgM s e c r e t o r s were f o u n d . T h i s i s n o t a t y p i c a l , as t h e l a c k o f IgM s e c r e t i n g h yb r i d omas i s c h a r a c t e r i s t i c o f a ma tu r e immune r e s p o n s e . The ab sence o f IgG^ s e c r e t o r s was e x p e c t e d as t h e r e a r e r e l a t i v e l y l ow l e v e l s o f IgG^ f o und i n mouse se rum ( G o d i n g , 1 9 8 3 ) . I n many c a s e s , b o t h p u r i f i e d a s c i t e s f l u i d and t h e g o a t a n t i - m o u s e se rum were d i l u t e d i n o r d e r t o o b t a i n an unambiguous r e s u l t . A c o n s i s t e n t c r o s s - r e a c t i v e p a t t e r n was a l s o e v i d e n t b e tween t h e c o m m e r c i a l m o n o s p e c i f i c g o a t a n t i s e r a d i r e c t e d a g a i n s t t o t a l mouse IgG and IgG^ and be tween IgG- and IgG„ ( F i g . 7 b / c ) . - 2 7 -Table III. Immunoglobulin classification of monoclonal antibodies. Monoclonal Ig Monoclonal Ig Antibody Class Antibody Class 2F4B 1 15C3E 2b 2F12F 2a 15C11B 1 3D5D 2b 16A3C 2b 3D9F 2b 16A10E 1 4G6A 1 16B2C 2b 4G12E 2a 16B8D 2b 12B2D 2a 16D9D 1 12B3G 2a 16D11C 1 13A1F 2b 21B8H 1 13A4H 2b 21B9H 1 13G6H 1 21D9C 2a 14B1F 1 21D10B 2a 14B3D 1 •I u I 6\ l o t 1> m 3 i I 0 TV 2* r 3 -lb Fig. 7a Ouchterlony double immunodiffusion subclassification of hybridoma cell culture supernatants. Pos = mouse serum with a l l subclasses; MAbs 4G6A, 14B1F, 15C11B, - IgG • 21D9C, 21D10B - IgG2 ; 16A10E - (IgG^ - not determined on this gel. T - goat anti-mouse Ig§, M - goat anti-mouse IgM, 1 = anti-IgG1, 2a - anti-IgG2a, 2b - anti-IgG2^, 3 = anti-IgG3. -28-F i g 7c 1 1 F i g . 7 b / c O u c h t e r l o n y d o u b l e i m m u n o d i f f u s i o n s u b c l a s s i f i c a t i o n o f d i l u t i o n s o f p u r i f i e d a s c i t e s and g o a t a n t i - m o u s e s e r a . Pos = mouse s e r a ; A = 13G6H, B -16A3C i n d o u b l i n g d i l u t i o n s o f PBS t o 1/64 o r 1 /128 . 13G6H - IgG^ ( n o t d e t e r m i n e d t h i s g e l ) ; 16A3C = IgG2^. E. H a e m a g g l u t i n a t i o n I n h i b i t i o n P o l y v a l e n t r u b e l l a immune mouse s e r a c o n s i s t e n t l y d e m o n s t r a t e d HI t i t r e s o f 9 11 512 t o 2048 (2 -2 ) and r u b e l l a n e g a t i v e mouse se rum d e m o n s t r a t e d a t i t r e l e s s t h a n 8 ( T a b l e I V , F i g . 8 ) . T h i s n a r r ow r ange o f c o n t r o l v a l u e s was c o n s i s t e n t w i t h d i f f e r e n t l o t s o f v i r u s and c h i c k e r y t h r o c y t e s , s u g g e s t i n g t h a t t h e r e i s no s i g n i f i c a n t v a r i a t i o n i n HA r e c e p t o r s on e r y t h r o c y t e s u r f a c e s . A l l MAbs were t e s t e d i n d u p l i c a t e i f t h e t i t r e was l e s s t h a n 8, o r i f p o s i t i v e , a t l e a s t i n t r i p l i c a t e w i t h a d i f f e r e n t l o t o f e r y t h r o c y t e s . - 2 9 -S e v e r a l o f t h e l ow d i l u t i o n w e l l s o f h i g h HI t i t r e MAbs showed n o n - s p e c i f i c a g g l u t i n a t i o n t h a t d i s a p p e a r e d w i t h d i l u t i o n o f s e r a ( F i g . 8b - 16B2C, 16B8D) , i n d i c a t i n g t h a t a d s o r p t i o n w i t h t h e 50% e r y t h r o c y t e m i x t u r e t o remove n o n - s p e c i f i c h a e m a g g l u t i n i n s was n o t e n t i r e l y s u c c e s s f u l . F i v e MAbs d e m o n s t r a t e d c o n s i d e r a b l e (4096 -16384) HI t i t r e s and f i v e showed m a r g i n a l ( 8 - 6 4 ) t i t r e s . T a b l e I V . M o n o c l o n a l A n t i b o d y H a e m a g g l u t i n a t i o n I n h i b i t i o n (H I ) T i t r e s A n t i b o d y HI T i t r e * A n t i b o d y HI T i t r e Pos 1024 14B3D < 8 Neg < 8 15C3E < 8 2F4B < 8 15C11B < 8 2F12F < 8 16A3C < 8 3D5D 8192 16A10E 32 3D9F > 16384 16B2C > 16384 4G6A < 8 16B8D > 16384 4G12E 32 16D9D 64 12B2D 4096 16D11C < 8 12B3G 8 21B8H < 8 13A1F 32 21B9H <. 8 13A4H < 8 21D9C < 8 13G6H < 8 21D10B < 8 14B1F < 8 * The r e c i p r o c a l o f t h e d i l u t i o n t h a t i n h i b i t s a g g l u t i n a t i o n . < 8 = h a e m a g g l u t i n a t i o n i n h i b i t i o n u n d e t e c t a b l e . - 3 0 -U A 1 F 1JG6H 2J 24 25 26 27 28 29 210 211 212 213 214 M l — ^ F i g 8a F i g 8b F i g 8c F i g . 8 H a e m a g g l u t i n a t i o n i n h i b i t i o n (HI ) t i t r a t i o n o f m o n o c l o n a l and p o l y c l o n a l serum w i t h s u s p e n s i o n s o f c h i c k e r y t h r o c y t e s . C e l l b u t t o n i n d i c a t e s i n h i b i t i o n o f h a e m a g g l u t i n a t i o n o f r u b e l l a v i r u s , smooth c e l l " c a r p e t " i n d i c a t e s h a e m a g g l u t i n a t i o n n o t i n h i b i t e d . -31-F. Rubella Virus Neutralization 1. Rubella Virus - Complement Compatibility As shown i n Fig 9, a five percent solution of complement was the f i r s t to f a i l to inhibit the CPE of rubella (Table V). Therefore, for the MAb neutralizations (Nt) with complement, a concentration of 2.5% complement was used and found to increase the Nt t i t r e of polyvalent rubella immune mouse serum by a factor of 8 (titres of 20 to 160). Table V. Plaque forming unit (PFU)/well assay with complement * Percent complement 50 10 5 2.5 1.25 0.625 PFU/well 162 3710 517 5113 579 556 * Average of four wells 1 SD Control value (no complement), average of 24 wells = 5511 PFU/well. 791 —— 1 69 Percent RaLbit Ctaplmnnt a p*rc«nt dilution + Control iv«r»o» a -1 SD control v*lu» ? +1 SO control vxUn Fig. 9 Inhibition of CPE of rubella virus by complement. -32-2. V i r u s N e u t r a l i z a t i o n Hyper - immune r a b b i t se rum h a d a N t t i t r e o f 20 w i t h M33 and 10 t o 20 w i t h RA 27 /3 s t r a i n s o f r u b e l l a v i r u s , w h i c h i n c r e a s e d t o a t i t r e o f 160 a g a i n s t b o t h s t r a i n s w i t h t h e a d d i t i o n o f complement ( T a b l e V I ) . A change i n N t t i t r e o f one d i l u t i o n ( i e t i t r e o f 5 t o 10) upon a d d i t i o n o f complement was n o t c o n s i d e r e d s i g n i f i c a n t . As shown i n T a b l e V I , t e n MAbs were f o u n d t o T a b l e V I . R u b e l l a V i r u s N e u t r a l i z a t i o n T i t r e s M33 ( w i l d s t r a i n ) w i t h o u t w i t h complement complement RA 27 /3 ( v a c c i n e s t r a i n ) w i t h o u t w i t h complement complement POS 20 160 POS 10 -20 160 NEG < < NEG < < 2F4B 10 5 2F4B 5 < 2F12F < < 2F12F < < 3D5D < < 3D5D < < 3D9F < < 3D9F < < 4G6A < < 4G6A < < 4G12E 5 < 4G12E < < 12B2D 5 20 12B2D NA 5 12B3G < < 12B3G 5 < 13A1F < < 13A1F < < 13A4H < < . 13A4H < 5 13G6H < < 13G6H < < 14B1F < < 14B1F < < 14B3D < < 14B 3D < < 15C3E < < 15C3E < < 15C11B < < 15C11B < < 16A3C 20 NA 16A3C 10 NA 16A10E 5 < 16A10E < < 16B2C < < 16B2C < < 16B8D < < 16B8D < < 16D9D < 10 16D9D < < 16D11C < < 16D11C < < 21B8H < < 21B8H < 10 21B9H < 5 21B9H < 20 21D9C < < 21D9C < < 21D10B < < 21D10B < < < = n e u t r a l i z a t i o n t i t r e l e s s t h a n 5; NA = n o t a v a i l a b l e . * r e c i p r o c a l o f t h e a n t i b o d y d i l u t i o n d e m o n s t r a t i n g a t l e a s t a 50% r e d u c t i o n i n P F U / w e l l . POS, NEG = r a b b i t p o l y c l o n a l s e rum. - 3 3 -F i g . 10 a F i g . 10 b F i g . 10 a , b R u b e l l a v i r u s n e u t r a l i z a t i o n b y p l a q u e r e d u c t i o n : a , r u b e l l a p l a q u e s ( l O O x ) ; b , p l a q u e s s t a i n e d by n e u t r a l r e d ( l O O x ) . ui -34-F i g . 10 c F i g . 10 d F i g . 10 c , d R u b e l l a v i r u s n e u t r a l i z a t i o n by p l a q u e r e d u c t i o n . C, s t a i n i n g w i t h p re - immune mouse se rum ( 4 0 0 x ) ; d , s t a i n i n g o f p l a q u e w i t h p o l y v a l e n t immune mouse serum and g o a t a n t i - m o u s e IgG l a b e l l e d w i t h HRP ( 4 0 0 x ) . - 3 5 -F i g . 10 e , f R u b e l l a v i r u s n e u t r a l i z a t i o n by p l a q u e r e d u c t i o n . E, MAb 16D9D w i t h RA 27 /3 v i r u s i n absence o f complement , f i r s t d i l u t i o n o f se rum ( l O O x ) ; f , MAb 16D9D w i t h RA 27 /3 v i r u s i n p r e s e n c e o f comp lement , f i r s t d i l u t i o n o f serum ( l O O x ) . - 3 6 -n e u t r a l i z e e i t h e r M33 o r RA 27 /3 s t r a i n s o r b o t h t o some e x t e n t . F i v e MAbs n e u t r a l i z e d M33 RV w i t h o u t comp lement , w i t h 12B2D show i ng a f o u r - f o l d i n c r e a s e i n t i t r e upon i t s a d d i t i o n , and one MAb, 16D9D, was n e g a t i v e i n t h e ab sence o f complement b u t d i s p l a y e d a t i t r e o f 10 i n i t s p r e s e n c e ( F i g s . l O e / f ) . I n t e s t i n g w i t h RA 27 /3 v a c c i n e s t r a i n v i r u s , t h r e e MAbs showed n e u t r a l i z i n g a b i l i t y w i t h no e l e v a t i o n when complement was a dded . However , two MAbs, 21B8H and 21B9H showed a change f r om n e g a t i v i t y t o t i t r e s o f 10 and 20 r e s p e c t i v e l y . Of t h e t e n MAbs t h a t h a d any r e a c t i v i t y t o e i t h e r s t r a i n , f o u r r e a c t e d commonly w i t h b o t h s t r a i n s . X . DISCUSSION I m m u n o p r e c i p i t a t i o n The a n t i g e n s p e c i f i c i t i e s o f h a l f (13 o f 25) o f t h e m o n o c l o n a l a n t i b o d i e s 35 were i d e n t i f i e d b y i m m u n o p r e c i p i t a t i o n o f [ S ] - m e t h i o n i n e l a b e l l e d v i r u s , r e q u i r i n g m u l t i p l e m o d i f i c a t i o n s i n t h e c h e m i c a l e n v i r o n m e n t o f t h e v i r u s t o a c h i e v e t h e s e r e s u l t s . I f o u n d t h e o p t i m a l p r e c i p i t a t i n g c o n d i t i o n s t o c o n s i s t o f a v i r a l l y s a t e b u f f e r o f 1% T r i t o n X - 1 0 0 , 0.5% sod i um d e o x y c h o l a t e , 1 mM EDTA, 2 . 5 mM PMSF, 10 mM T r i s - H C l , pH 7 . 4 , w i t h a l t e r a t i o n s i n t h e N a C l c o n c e n t r a t i o n s f r om 0 .18M t o 0 . 5 M, i n o r d e r t o o p t i m i z e i o n i c c o n d i t i o n s f o r e a ch m o n o c l o n a l a n t i b o d y . The u se o f 0.1% SDS i n t h e l y s a t e b u f f e r was f o u n d t o e l i m i n a t e r e a c t i v i t y i n some m o n o c l o n a l s and e s t a b l i s h i t i n o t h e r s . The i n c l u s i o n o f i o d o a c e t a m i d e c o i n c i d e d w i t h t h e n o n - s p e c i f i c p r e c i p i t a t i o n o f t h e n u c l e o c a p s i d p r o t e i n on t o t h e S. a u r e u s . I o d o a c e t a m i d e was e l i m i n a t e d f r om t he me thod . The opt imum immunoadsorbent wash t e c h n i q u e was f o u n d t o be t h r e e t i m e s w i t h a l y s a t e wash b u f f e r c o n t a i n i n g 1.0M N a C l , 0.1% SDS, 1% T r i t o n X - 1 0 0 , 0.5% sod i um d e o x y c h o l a t e , 10 mM T r i s - H C l , pH 7 . 4 , and t h e n t h r e e t i m e s w i t h t h e same - 3 7 -b u f f e r w i t h 0 .5M N a C l , f o l l o w e d by two washes w i t h 0 .06M T r i s - H C l , pH 6 .8 i n o r d e r t o remove any t r a c e o f N a C l and e x c e s s d e t e r g e n t s p r i o r t o d i s s o c i a t i o n o f t h e i m m u n o p r e c i p i t a n t s w i t h t h e Laemml i r e d u c t i o n m i x t u r e . U l t i m a t e l y , f o u r p a t t e r n s o f m o n o c l o n a l a n t i b o d y r e a c t i v i t y emerged , w i t h one g roup f a i l i n g t o p r e c i p i t a t e any o f t h e s t r u c t u r a l p r o t e i n s , w h i l e a s e c ond p r e c i p i t a t e d b o t h t h e E l and E2 g l y c o p r o t e i n s . A f u r t h e r g roup p r e c i p i t a t e d t h e E l p o l y p e p t i d e f r om b o t h M33 and RA 27 /3 s t r a i n b u t a n o t h e r o n l y p r e c i p i t a t e d t h e E l g l y c o p r o t e i n o f one v i r u s s t r a i n b u t n o t t h e o t h e r s t r a i n . Of t h e e l e v e n m o n o c l o n a l s ( g e n e r a t e d t o RA 27 /3 v i r u s ) t h a t p r e c i p i t a t e d E l , s i x a l s o p r e c i p i t a t e d E l o f M33. Of t h e e i g h t m o n o c l o n a l s t h a t p r e c i p i t a t e d E l o f M33, s i x o f t h o s e a l s o p r e c i p i t a t e d t h e E l o f RA 2 7 / 3 . The c a u s e s o f t h e s e d i f f e r e n t p a t t e r n s o f r e a c t i v i t y a r e l i k e l y t o be r e l a t e d t o t h e c o n s e r v a t i o n o f s i t e s c r i t i c a l t o a n t i g e n - a n t i b o d y r e a c t i o n s . Some o f t h e s e r e s u l t s may be e x p l a i n e d as a r e s u l t o f s t r a i n s p e c i f i c a n t i g e n i c v a r i a n c e s , as i n c a s e s where t h e m o n o c l o n a l s ( p r o d u c e d a g a i n s t RA 27 /3 v a c c i n e s t r a i n v i r u s ) u n a m b i g u o u s l y r e a c t e d w i t h RA 27 /3 b u t n o t M33. C e r t a i n a n t i b o d i e s may l a c k t h e a f f i n i t y ( t h e c l o s e n e s s o f f i t ) t o s e c u r e l y b i n d t h e p r o t e i n by t h e i r s p e c i f i c e p i t o p e t h r o u g h t he s e r i e s o f s t r i n g e n t w a s h i n g c o n d i t i o n s r e q u i r e d t o remove e x t r a n e o u s p r o t e i n s . Loo e t a l (1986) showed t h a t i m m u n o p r e c i p i t a t i o n c a n be a f f e c t e d by t h e p r e s e n c e o f h i g h i o n i c s t r e n g t h b u f f e r s , w h i c h a l t e r t h e " t y p e and r e l a t i v e amount o f e a ch r u b e l l a p o l y p e p t i d e p r e c i p i t a t e d b y a p a r t i c u l a r a n t i s e r u m " . I n my t r i a l s , I s u b j e c t e d m o n o c l o n a l a n t i b o d y - a n t i g e n m i x t u r e s t o a N a C l g r a d i e n t ( . 1 8 t o .50M N a C l ) b y a d d i n g c o n c e n t r a t e d N a C l s o l u t i o n s a t i n t e r v a l s d u r i n g t h e r e a c t i o n p e r i o d . MAbs t h a t h a d p r e v i o u s l y n o t shown r e a c t i v i t y a t any s i n g l e N a C l c o n c e n t r a t i o n , now d e m o n s t r a t e d r e a c t i v i t y t o e n v e l o p e g l y c o p r o t e i n s , p o s s i b l y due t o an - 3 8 -e x p o s u r e o f t h e s i t e o f t h e e p i t o p e w h i c h w o u l d a l l o w t h e u n i f i c a t i o n o f a n t i b o d y and a n t i g e n . B r i e f l y , a change o f p r o t e i n / a n t i b o d y r e a c t i v i t y c a n o c c u r w i t h v a r i a t i o n s i n N a C l o r d e t e r g e n t c o n c e n t r a t i o n s due e i t h e r t o f a i l u r e t o u n c o v e r c r i t i c a l s i t e s o r d e n a t u r a t i o n o f t h o s e s i t e s upon i s o l a t i o n o f t h o s e r u b e l l a p r o t e i n s . A l l o f t h e m o n o c l o n a l a n t i b o d i e s i n t h i s p a n e l t h a t have shown unambiguous p o l y p e p t i d e s p e c i f i c i t i e s have b e en d e d i c a t e d t o t h e E l p r o t e i n . P r e v i o u s r e s e a r c h e r s have f o u n d c o n s i s t e n t l y t h a t most h y b r i d o m a c e l l l i n e s g e n e r a t e d t o r u b e l l a v i r u s have b e e n g e n e r a t e d t o t h e E l p o l y p e p t i d e , w i t h r a r e ones g e n e r a t e d t o E2 o r t h e c a p s i d p r o t e i n . T h i s ha s l e d t h e E2 p r o t e i n t o be c o n s i d e r e d as l e s s immunogen i c , " h i d d e n " o r d e e p l y imbedded , o r n o t as p r o m i n e n t l y " p r omo t ed " b y t h e immune s y s t e m ' s MHC ( M a j o r H i s t o c o m p a t i b i l i t y Comp lex) as a f o r e i g n p r o t e i n as i s t h e E l p r o t e i n . D o r s e t t e t a l (1985) f o und t h a t h i g h e r t i t r e s o f E2 a n t i b o d y i n p o l y v a l e n t serum d e v e l o p e d when t h e r a b b i t s we re i n j e c t e d w i t h s o l u b i l i z e d , p u r i f i e d E2 r a t h e r t h a n w i t h an i n t a c t v i r u s i n o c u l u m . I t a ppea r s t h a t s u c c e s s f u l i m m u n i z a t i o n ( f o r m a t i o n o f n e u t r a l i z i n g a n t i b o d i e s ) w i t h i n t a c t v i r u s d i m i n i s h e s p r o d u c t i o n o f a n t i b o d i e s t o E 2 . T h i s may be r e l a t e d t o t h e f a c t t h a t p e o p l e w i t h c o n g e n i t a l o r p e r s i s t e n t r u b e l l a f r e q u e n t l y have an e l e v a t e d t i t r e o f a n t i b o d i e s t o t h e E2 p r o t e i n . An immune d e f e c t may l i m i t t h e i r a b i l i t y t o p r e s e n t , o r r e a c t t o t h e E l p r o t e i n , l e a d i n g t o a l a c k o f n e u t r a l i z i n g a n t i b o d i e s . A n o t h e r r e a s o n f o r t h e l a c k o f h y b r i d o m a s d i r e c t e d t o E2 and NC p r o t e i n s i s f o u n d i n e x p e r i m e n t s i n w h i c h a n t i b o d y t i t r e s t o E2 and NC d e c l i n e d f i r s t i n se rum samp l e s t a k e n o v e r t i m e f r om immun i zed r a b b i t s ( D o r s e t t , 1 9 8 5 ) . S ene s cence o f l y m p h o c y t e s s e c r e t i n g i m m u n o g l o b u l i n t o E2 o r NC may be a m a n i f e s t a t i o n o f a m a t u r i n g immune r e s p o n s e t o t h e r u b e l l a v i r u s . - 3 9 -A l l o f t h e m o n o c l o n a l s i n t h i s p a n e l have b e e n shown t o r e a c t w i t h r u b e l l a v i r u s p r o t e i n s embedded i n i n f e c t e d V e r o c e l l s , t h u s e s t a b l i s h i n g t h e i r g r o s s s p e c i f i c i t y t o r u b e l l a v i r u s , y e t o n l y 50% o f them c o u l d be f o u n d t o r e l i a b l y p r e c i p i t a t e a s t r u c t u r a l p r o t e i n . A n t i b o d i e s a r e g e n e r a t e d b y B c e l l s i n r e s p o n s e t o T c e l l a c t i v a t i o n c a u s e d b y p r e s e n t i o n o f f o r e i g n a n t i g e n s i n a s s o c i a t i o n w i t h t h e MHC o f a c c e s s o r y c e l l s . Thus , t h e s e a n t i b o d i e s may be g e n e r a t e d t o e p i t o p e s a r t i c u l a t e d i n a c o n f o r m a t i o n t h a t may n o t be f o u n d i n t h e i n t a c t v i r u s o r t h e i s o l a t e d p o l y p e p t i d e s . Some p r o t e i n d e g r a d a t i o n ha s b e en f o u n d t o be n e c e s s a r y f o r a n t i g e n e x p r e s s i o n on t h e s u r f a c e o f a c c e s s o r y c e l l s f o r T h e l p e r l y m p h o c y t e - m e d i a t e d immune r e s p o n s e ( S h i m o n k e v i t z , 1 9 8 3 ) . I t i s o n l y a m a t t e r o f e s t a b l i s h i n g a l a r g e enough b a t t e r y o f r u b e l l a s p e c i f i c h y b r i d o m a s t o f i n d a n t i b o d y p r o d u c t i o n c o n f i g u r e d f o r c e l l membrane bound a n t i g e n s t h a t w i l l a l s o r e a d i l y r e a c t w i t h p u r i f i e d p r o t e i n s . I n my e x p e r i m e n t s , t h e r e were v a r i o u s MAbs t h a t p r e c i p i t a t e d b o t h E l and E2 p r o t e i n s . T h i s may be due t o t h e p r o p o s e d e x i s t e n c e o f i n t e r - c h a i n d i s u l p h i d e l i n k a g e s be tween E l and E2 o r i t may be due t o some amino a c i d sequence homo logy be tween t h e two e n v e l o p e g l y c o p r o t e i n s . M e r c a p t o e t h a n o l r e d u c t i o n o f r u b e l l a v i r u s p r i o r t o p r e c i p i t a t i o n w i t h m o n o c l o n a l a n t i b o d i e s c a u s e d a g e n e r a l r e d u c t i o n i n t h e p r o t e i n p r e c i p i t a t e d , l e a v i n g MAb s p e c i f i c i t y u n r e s o l v e d . R e d u c i n g c o n d i t i o n s were a p p l i e d i n t h e wash s t e p s o f s e v e r a l i m m u n o p r e c i p i t a t i o n s a f t e r f o r m a t i o n o f t h e a n t i g e n - a n t i b o d y - S . a u r e u s c o m p l e x e s . T h i s was done t o a v o i d d e n a t u r a t i o n o f e i t h e r t h e e p i t o p e o r t h e a n t i b o d y b i n d i n g s i t e . T h i s a n t i c i p a t e s t h a t t h e a n t i b o d y w o u l d r e m a i n a t t a c h e d e ven i f t h e E l g l y c o p e p t i d e was d e n a t u r e d due t o t h e s e p a r a t i o n o f E l and E 2 . C o n c e i v a b l y t h e r e c o u l d be some " e n f o r c e m e n t " o f c o n f o r m a t i o n a l s t a b i l i t y o f t h e e p i t o p e b y t h e a n t i g e n - a n t i b o d y c omp l e x . However , once a g a i n , - 4 0 -t h e r e was a r e d u c t i o n i n b o t h E l and E2 p r e c i p i t a t i o n . D i s s o c i a t i o n o f t h e E l and E2 p r o t e i n s b y r e d u c t i o n o f e i t h e r i n t e r - o r i n t r a - m o l e c u l a r d i s u p h i d e bonds may s u f f i c i e n t l y a l t e r t h e e p i t o p e i n q u e s t i o n t o p r e v e n t o r remove b i n d i n g o f a m o n o c l o n a l a n t i b o d y . A c h a l l e n g e e x i s t s i n e x p o s i n g c r i t i c a l e p i t o p e s on r u b e l l a p r o t e i n s f o r a n t i b o d y b i n d i n g w i t h o u t d e n a t u r i n g them beyond u s e . D u r i n g t h e c o u r s e o f my e x p e r i m e n t s , a v a r i a t i o n was f o u n d i n t h e m o l e c u l a r w e i g h t o f t h e E l p r o t e i n be tween M33 ( 5 7 , 6 00 ) and RA 27 /3 ( 5 6 , 3 0 0 ) s t r a i n s by u s i n g t h e PAGE t e c h n i q u e . A l t h o u g h t h e s t a t i s t i c a l e r r o r i n v o l v e d i n t h i s method (SD = 3000) c a s t s doub t on i t s s i g n i f i c a n c e , r e p e a t e d s i d e - b y - s i d e l a n e c o m p a r i s o n s s u g g e s t t h a t t h e r e i s v a r i a b i l i t y i n t h e amino a c i d sequence o r d e l e t i o n i n t h e E l p r o t e i n be tween s t r a i n s . A l s o o f i n t e r e s t i s t h e number o f bands o c c a s i o n a l l y p r e c i p i t a t e d f r om i n d i v i d u a l h a r v e s t s o f r a d i o l a b e l l e d M33 o r RA 27 /3 r u b e l l a v i r u s w i t h p o l y v a l e n t a n t i s e r a r a i s e d a g a i n s t M33, RA 27 /3 and HPV/77 v i r u s s t r a i n s . P r e v i o u s w o r k e r s have p u b l i s h e d r e s u l t s r e m a r k i n g on t h e b r o a d band o f t h e E2 c omp l e x , r a t h e r t h a n r e s o l v a b l e bands o f E2a and E2b . I have f u r t h e r r e s o l v e d t h e s e m u l t i p l e bands i n t h e r e g i o n o f E2 and t h e NC p r o t e i n u s i n g a s e c o n d wash j u s t p r i o r t o e l u t i o n o f t h e immune comp lexes b y t h e d i s s o c i a t i n g samp le b u f f e r o f Laemml i ( 1 9 7 0 ) . O k e r - B l o m (1983) f o u n d f o u r m a j o r and f i v e m i n o r p o l y p e p t i d e b a n d s , and C h a n t l e r (1985) o b s e r v e d n i n e r u b e l l a v i r u s " i n d u c e d " p o l y p e p t i d e s u s i n g p o l y c l o n a l serum t o p r e c i p i t a t e r a d i o l a b e l l e d c e l l e x t r a c t f r om i n f e c t e d p e r i p h e r a l b l o o d mononuc l e a r c e l l s . O f t h e s e , o n l y f o u r were t e rmed ma tu r e v i r i o n p o l y p e p t i d e s w i t h t h e r e m a i n d e r t e rmed as p r e c u r s o r o r i n t e r m e d i a t e p o l y p e p t i d e s . I n a c o m p a r i s o n o f r a d i o l a b e l l e d RA 27 /3 r u b e l l a and m o c k - i n f e c t e d V e r o c e l l l y s a t e s , i t a p pea r s t h a t a t l e a s t f o u r o f t h e s e - 4 1 -bands i n t h e 30 , 000 t o 45 , 000 M r r ange a r e o f h o s t c e l l o r i g i n and n o t v i r a l as some have s p e c u l a t e d . T h i s c r o s s - r e a c t i v i t y w i t h c e l l u l a r p r o t e i n s c a n b e s t be a v o i d e d by s e v e r a l p a s s a g e s o f t h e v i r u s t o be u s e d as a n i m a l immunogen i n an u n r e l a t e d c e l l l i n e f r om t he one u s e d t o grow l a b e l l e d v i r u s , o r by i n f e c t i n g s u b - c o n f l u e n t and new l y s eeded c e l l l i n e s and h a r v e s t i n g c e l l c u l t u r e s u p e r n a t a n t s b e f o r e t h e c y t o p a t h i c e f f e c t s o f r u b e l l a f i r s t a p p e a r . I h ave a l s o f o u n d one m o n o c l o n a l , 21D10B ( F i g . 1 1 ) , t h a t c r o s s - r e a c t s h e a v i l y i n r a d i o i n m u n o p r e c i p i t a t i o n s w i t h V e r o c e l l p r o t e i n s and b i n d s t o u n i n f e c t e d RK c e l l s i n immunope rox idase t e s t s u s e d t o v e r i f y t h e s p e c i f i c i t y o f t he a n t i b o d i e s t o t h e r u b e l l a v i r u s . The m o n o c l o n a l may be d i r e c t e d t o a A B C D E F G H I J F i g . 11 I m m u n o p r e c i p i t a t i o n o f M33 r u b e l l a v i r u s p r o t e i n s d e m o n s t r a t i n g ^ r o s s - r e a c t i v i t y o f MAb 21D10B w i t h V e r o c e l l p r o t e i n s . Lane A , S - m e t h i o n i n e - l a b e l l e d M33 r u b e l l a v i r u s ; l a n e B, i m m u n o p r e c i p i t a t e d w i t h r a b b i t a-M33 s e r a ; l a n e C, r a b b i t p re- immune s e r a , l a n e D, MAb 3D5D; l a n e E, 4G6A (a E l ) ; l a n e F, 4G12E; l a n e G, 12B3G; l a n e H, 13G6H; l a n e I , 16A10E; l a n e J , 21D10B. - 4 2 -v i r a l e p i t o p e s t r u c t u r e common t o many c e l l membrane p r o t e i n s , o r a c e l l u l a r p r o t e i n , s t r i p p e d f r om t he c e l l s u r f a c e d u r i n g v i r u s p r o d u c t i o n , i n c o r p o r a t e d as an i n t e g r a l component o f t h e v i r u s s t r u c t u r e . IgG C l a s s i f i c a t i o n S e q u e n t i a l d i l u t i o n o f b o t h p u r i f i e d mu r i n e a s c i t e s f l u i d and g o a t a n t i - m o u s e se rum, s u b s t i t u t i n g f o r t h e l a c k o f c e l l c u l t u r e s u p e r n a t a n t s , a l l o w e d unamb iguous d e t e r m i n a t i o n o f t h e c l o n a l i m m u n o g l o b u l i n t y p e s o f t h e e n t i r e m o n o c l o n a l p a n e l b y t h e O u c h t e r l o n y d o u b l e i m m u n o d i f f u s i o n t e c h n i q u e . H a e m a g g l u t i n a t i o n I n h i b i t i o n The h e p a r i n manganous c h l o r i d e t e c h n i q u e ( L i e b h a b e r , 1970) upon r e p e a t e d t r i a l s w i t h m o n o c l o n a l a n t i b o d i e s and p o l y v a l e n t c o n t r o l se rum g e n e r a t e d n a r r o w r e p r o d u c i b l e r a n g e s o f r e s u l t s f o r e a ch samp le t h r o u g h v a r i o u s l o t s o f c h i c k e r y t h r o c y t e s , v i r u s e s and r e a g e n t s . The r e l e v a n c e o f HI t o immun i t y w i l l be d i s c u s s e d i n t h e summary and c o n c l u s i o n s s e c t i o n . R u b e l l a V i r u s N e u t r a l i z a t i o n N e u t r a l r e d s t a i n i n g o f t h e f o c i o f i n f e c t i o n o f r u b e l l a v i r u s on RK c e l l s (Nawa, 1979) was i n i t i a l l y emp loyed t o d e t e c t r u b e l l a i n d u c e d p l a q u e s , b u t p r o v e d i n a d e q u a t e when c e l l mono l a y e r s became o ve r g r own and " n a t u r a l " c e l l a g g r e g a t i o n a l s o was s t a i n e d . The r u b e l l a immunope rox i da se t e c h n i q u e d e s c r i b e d b r o u g h t a measure o f c o n f i d e n c e i n t o t h e p l a q u e c o u n t i n g p r o c e d u r e . I t may a l s o be a p p l i e d d i a g n o s t i c a l l y i n t h e d e t e c t i o n o f a c u t e r u b e l l a v i r u s i n f e c t i o n s v i a c e l l c u l t u r e g r ow th w i t h i n 48 t o 72 h o u r s , o r t o t h e d e t e c t i o n o f r u b e l l a s p e c i f i e d p r o t e i n s i n t h e c e l l membranes o f b i o p s y s pe c imens s u s p e c t e d i n p e r s i s t e n t r u b e l l a i n f e c t i o n . Raw l s ( 1 9 6 7 ) , d e t e r m i n e d t h a t t h e e l emen t s o f n o r m a l se rum w h i c h p romote " s p e c i f i c n e u t r a l i z a t i o n o f r u b e l l a v i r u s a r e h e a t l a b i l e " and t h a t t h e a c t i o n - 4 3 -may be due t o complement s i n c e t h e y f o u n d t h a t h e t e r o l o g o u s a n t i g e n - a n t i b o d y r e m o v a l o f complement a l s o r e d u c e d t h e r u b e l l a a n t i b o d y n e u t r a l i z a t i o n t i t r e . T h i s r e d u c t i o n i n a n t i b o d y t i t r e i n h e a t e d serum (56°C f o r 30 m i n u t e s ) was f o u n d t o be e f f e c t i v e l y r e v e r s e d b y t h e a d d i t i o n o f f i v e t o t e n p e r c e n t g u i n e a p i g se rum ( L e e r h o y , 1 9 6 8 ) , as w e l l , t h e a d d i t i o n o f t h e se rum r a i s e d t i t r e s f o u r t o e i g h t f o l d . I n my e x p e r i m e n t s , t h e a d d i t i o n o f 2.5% r a b b i t complement i n c r e a s e d t h e n e u t r a l i z a t i o n t i t r e s o f t h e p o l y v a l e n t immune serum b y a f a c t o r o f e i g h t . The a d d i t i o n o f r a b b i t complement s i g n i f i c a n t l y enhanced t h e n e u t r a l i z i n g a n t i b o d y t i t r e s o f f o u r MAbs (12B2D, 5 -20 ; 16D9D, Neg - 10 ; 21B8H, Neg - 10 ; and 21B9H, N e g - 2 0 ) , two e a ch f o r RA 27 /3 and M33 s t r a i n v i r u s e s . T h i s may d e m o n s t r a t e t h e a c c e s s i b i l i t y o f one o r more c r i t i c a l b i n d i n g s i t e s n o t a v a i l a b l e on t h e o t h e r s t r a i n o f v i r u s . I f o u n d i n my t r i a l s t h a t h e a t e d r a b b i t serum ha s no e f f e c t on t h e r u b e l l a n e u t r a l i z i n g a n t i b o d y t i t r e s o f t h e h e a t e d m o n o c l o n a l o r p o l y c l o n a l s e r a , b u t u n h e a t e d serum e l e v a t e d t h e t i t r e s 2 t o 3 f o l d . The t h r e e - f o l d d i f f e r e n c e i n t i t r e s b e tween m o n o c l o n a l and p o l y v a l e n t serum may be due t o a l a r g e r number o f s e n s i t i z i n g a n t i b o d i e s t h a t d i r e c t complement m e d i a t e d c e l l l y s i s , o r t o t h e e x i s t e n c e o f a m u l t i t u d e o f r u b e l l a n e u t r a l i z i n g a n t i b o d i e s ( b o t h complement i n d e p e n d e n t and d e p e n d e n t ) , c a u s i n g n e u t r a l i z a t i o n due t o an a c c u m u l a t i o n o f c o n f o r m a t i o n a l changes i n s i t e s c r i t i c a l f o r i n f e c t i v i t y , as s e q u e n t i a l a d d i t i o n o f n o n - n e u t r a l i z i n g MAbs have b e en shown t o n e u t r a l i z e r u b e l l a v i r u s (Ge r na , 1 9 8 7 ) . -44-X I . SUMMARY AND CONCLUSIONS Twenty f i v e m o n o c l o n a l a n t i b o d i e s t h a t were p r o d u c e d t o r u b e l l a v i r u s p r o t e i n s o f t h e RA 27 /3 v a c c i n e s t r a i n v i r u s were c h a r a c t e r i z e d b y means o f i m m u n o p r e c i p i t a t i o n o f r a d i o l a b e l l e d r u b e l l a v i r u s s t r u c t u r a l p r o t e i n s , IgG s u b - c l a s s i f i c a t i o n , h a e m a g g l u t i n a t i o n i n h i b i t i o n (HI ) and v i r u s n e u t r a l i z a t i o n (N t ) o f r u b e l l a v i r u s . The r u b e l l a v i r u s g l y c o p r o t e i n s p e c i f i c i t i e s o f t h i r t e e n o f t h e MAbs were f o u n d , and a l l we re d i r e c t e d t o t h e E l e n v e l o p e g l y c o p r o t e i n o f e i t h e r t h e RA 27 /3 a n d / o r t h e M33 w i l d s t r a i n ( T a b l e V I I ) . Mos t o f t h e MAbs o f u n i d e n t i f i a b l e s p e c i f i c i t y d e m o n s t r a t e d c o - p r e c i p i t a t i o n o f t h e E2 p r o t e i n w i t h E l . T h i s i s l i k e l y due t o d i s u l p h i d e l i n k a g e s w h i c h , when d i s r u p t e d , c au s e l o s s o f c o n f o r m a t i o n o f t h e a p p r o p r i a t e e p i t o p e , and n o n - p r e c i p i t a t i o n . O n l y t h r e e m o n o c l o n a l a n t i b o d i e s f a i l e d t o show any r e a c t i v i t y , e v en t hough a l l were f o u n d t o a t t a c h t o r u b e l l a i n f e c t e d c e l l s b y t h e immunope rox i da se t e c h n i q u e . E l e v e n (44%) were t y p e d as IgG.. , s i x (24%) as I g G . , and e i g h t (32%) as -L Z.EL I g G 2 b . N i n e m o n o c l o n a l s t e s t e d p o s i t i v e f o r t h e a b i l i t y t o i n t e r f e r e w i t h t h e h a e m a g g l u t i n a t i n g e f f e c t o f M33 r u b e l l a v i r u s . Ten MAbs n e u t r a l i z e d e i t h e r M33 o r RA 27 /3 s t r a i n s i n p l a q u e r e d u c t i o n t e s t s and t h e n e u t r a l i z i n g a b i l i t y (N t ) o f f o u r was enhanced two t o t h r e e f o l d w i t h t h e a d d i t i o n o f r a b b i t comp lement . I n a c c o r d a n c e w i t h o b s e r v a t i o n s o f o t h e r r e s e a r c h e r s , I f o u n d t h a t t h e r e was no c o r r e l a t i o n be tween HI and N t a b i l i t i e s o f t h e m o n o c l o n a l a n t i b o d i e s ; f o u r o f t e n a n t i b o d i e s t h a t h a d N t a c t i v i t y h a d HI a c t i v i t y and - 4 5 -T a b l e V I I . Summary o f r e s u l t s o f m o n o c l o n a l 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 RA 27 /3 v a c c i n e and M33 w i l d s t r a i n r u b e l l a v i r u s . P r o t e i n HI • IgG sub - N e u t r a l i z a t i o n S p e c i f i c i t y M33 c l a s s M33 RA 27 /3 M33 RA no comp* comp no comp comp 2F4B E l E l < 8 1 10 5 5 < 2F12F E l < 8 2a < < < < 3D5D ** ** 8192 2b < < < < 3D9F E l E l > 16384 2b < < < < 4G6A E l < 8 1 < < < < 4G12E 32 2a 5 < < < 12B2D E l 4096 2a 5 20 NA 5 12B3G E l E l < 8 2a < < 5 < 13A1F E l 32 2b < < < < 13A4H E l < 8 2b < < < 5 13G6H < 8 1 < < < < 14B1F E l E l < 8 1 < < < < 14B3D < 8 1 . < < < < 15C3E < 8 2b < < < < 15C11B < 8 1 < < < < 16A3C E l E l < 8 2b 20 NA 10 NA 16A10E E l E l 32 1 5 < < < 16B2C ** ** > 16384 2b < < < < 16B8D ** ** > 16384 2b < < < < 16D9D 64 1 < 10 < < 16D11C < 8 1 < < < < 21B8H E l < 8 1 < < < 10 21B9H E l < 8 1 < 5 < 20 21D9C < 8 2a < < < < 21D10B < 8 2a < < < < * comp lement . ** p r e c i p i t a t e s b o t h E l and E2 g l y c o p r o t e i n s . s i x o f n i n e t h a t h ad HI c a p a c i t y a l s o h ad N t a c t i v i t y . HI t i t r e s , and more r e c e n t l y E L I SA t i t r e s have b e en t h e benchmark o f c l i n i c a l r u b e l l a e x p o s u r e , b u t t h e y have l i t t l e c o r r e l a t i o n (as d e m o n s t r a t e d w i t h m o n o c l o n a l r e a c t i v i t i e s ) w i t h t h e p a t i e n t ' s immune s t a t u s o t h e r t h a n t h e s t a t i s t i c a l p r o b a b i l i t y t h a t a p p r o x i m a t e l y 95% o f e x po s ed ( i n f e c t e d o r immun i zed) p e o p l e b e com i ng r e s i s t a n t t o i n f e c t i o n o r r e - i n f e c t i o n . The n e u t r a l i z a t i o n b y p l a q u e r e d u c t i o n p r o c e d u r e i s a more a c c u r a t e r e f l e c t i o n o f immune s t a t u s , b u t howeve r i s a t e d i o u s and t i m e con sum ing p r o c e d u r e n o t s u i t e d t o mass s c r e e n i n g s . - 4 6 -Monoclonal antibodies, due to their well prescribed specificity are capable of part i a l l y unraveling the labyrinth of the immune response as well as dissecting the antigenic and biological properties of viruses. This study may enable the construction of monoclonal a f f i n i t y chromatography columns which can be used as a means for screening and purifying polypeptides, both native and those constructed through cDNA technology for analysis and possible sub-unit vaccine applications. Attenuated vaccines u n t i l recently, have been created with limited or no information on the sc i e n t i f i c basis or the molecular nature of virulence or attenuation. 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