Antibodies specific to a deoxyribomononucleotide (dpT) and a deoxyribodinucleotide (dpTpA)

Abstract

Thi,s t h e s i s dea ls w it h the s p e c i f i c i t y a n a l y s i s , a f f i n i t y c h r o m a t o g r a p h i c p u r i f i c a t i o n and a p p l i c a t i o n s of h a p t e n s p e c i f i c a n t i b o d i e s e l i c i t e d in r a b b i t s a g a i n s t p r o t e i n c o n j u g a t e s of the m o n o n u c l e o t i d e , dpT and t h e .d i n u c l e o t i d e , dpTpA. T h e s e s t u d i e s i n v o l v e d the p r e p a r a t i o n of n u c l e o t i d e d e r i v a t i v e s , o l i g o n u c l e o t i d e s , p r o t e i n - n u c l e o t i d e c o n j u g a t e s an d i m m u n o a d s o r b e n t s . O u c h t e r l o n y d o u b l e d i f f u s i o n , q u a n t i t a t i v e p r e c i p i t a t i o n , b i n d i n g of the r a d i o a c t i v e h a p t e n s a nd t h e i r i n h i b i t i o n w e r e the m e t h o d s e m p l o y e d for the c h a r a c t e r i z a t i o n of a n t i s e r a . The i n t e r a c t i o n of h a p t e n s w i t h a n t i b o d i e s w e r e a n a l y s e d by n i t r o c e l l u l o s e f i l t e r assay, e q u i l i b r i u m d i a l y s i s an d f l u o r e s c e n c e q u e n c h i n g m et h o d s . S om e of the i m p o r t a n t new f i n d i n g s mad e d u r i n g t h e s e s t u d i e s are the f o l l o w i n g : B o t h b o v i n e s e r u m a l b u m i n and t h y r o g l o b u l i n are e q u a l l y e f f e c t i v e as h a pt en c a r r i e r s for the e l i c i t a t i o n of dpT s p e c i f i c a n t i b o d i e s . dpT a n t i b o d i e s are s p e c i f i c for the w h o l e m o l e c u l e of dpT and are of l o w c r o s s r e a c t i v i t y w i t h ot he r m o n o n u c l e o t i d e s . T h e s e a n t i b o d e s can be use d for the r a d i o i m m u n o a s s a y of dpT. d pT a n t i b o d i e s b in d to dpT at 3 ' -e nd of o l i g o n u c l e o t i d e s w i t h a b o u t 10 tim e s m o r e a , f i n i t y than to the ^pT r e s i d u e at the 5 ' - en d or in the i n t e r n a l p o s i t i o n s . dpTpA a n t i b o d i e s are s e q u e n c e s p e c i f i c w i t h o u t any cr os s r e a c t i v i t y of the c o n s t i t u e n t m o n o n u c l e o tides. In a d d i t i o n to dpTpA. c o v a l e n t l y a t t a c h e d to AH S e ph ar os e, it held e l e c t r o s t a t i c a l l y on D E A E c e l l u l o s e can be used for the p u r i f i c a t i o n of d p T p A a n t i b o d i e s