UBC Theses and Dissertations

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UBC Theses and Dissertations

A non-radioactive method of detecting nucleic acids Kalmar, Gabriel Bela 1986

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A NON-RADIOACTIVE METHOD OF DETECTING NUCLEIC ACIDS by G a b r i e l B e l a K a l m a r B . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (The D e p a r t m e n t o f P l a n t S c i e n c e ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g •^.o the. vetquired s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA A p r i l , 1986 © G a b r i e l B e l a Kalmar 1986 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f P\GI/A4~ -Sng.vAC.-ff The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D a t e ii A b s t r a c t M o n o c l o n a l a n t i b o d i e s (MCAs) s p e c i f i c f o r i o d i n a t e d c y t i d i n e were produced and c h a r a c t e r i z e d f o r t h e i r p o s s i b l e a p p l i c a t i o n i n the development o f a s e r o l o g i c a l , n o n - r a d i o a c t i v e a s s a y f o r n u c l e i c a c i d s . The method r e q u i r e s the m o d i f i c a t i o n o f s i n g l e -s t r a n d e d r i b o n u c l e i c a c i d (ss-RNA) by a d d i t i o n o f an i o d i n e t o the C -5 p o s i t i o n o f c y t i d i n e v i a the Commerford r e a c t i o n . . The l e v e l o f i o d i n a t i o n was f o l l o w e d by u l t r a v i o l e t (UV) s p e c t r o s c o p y and X-ray d i f f r a c t i o n (XRF) and was found t o be h i g h e r f o r polyC than f o r c e l l u l a r RNA or ss-RNA of s o u t h e r n bean mosaic and cowpea c h l o r o t i c m o t t l e v i r u s . No e v i d e n c e was o b t a i n e d f o r i o d i n a t i o n o f d o u b l e - s t r a n d e d (ds) RNA o f t u r n i p y e l l o w mosaic v i r u s . Only one hybridoma, ( R l ) , of 2,500, t e s t e d s e c r e t e d MCAs s p e c i f i c f o r i o d i n a t e d c y t i d i n e . The s p e c i f i c i t y and a f f i n i t y o f t h i s MCA f o r i o d i n a t e d and n o n - i o d i n a t e d s y n t h e t i c p o l y -n u c l e o t i d e s and i o d i n a t e d c e l l u l a r ss-RNA were a s s e s s e d by s o l i d -phase and i n h i b i t i o n a s s a y s , u s i n g the enzyme-1 i n k e d immunosorbent assay ( E L I S A ) . Seven o t h e r hybridomas produced MCAs w i t h v a r y i n g s p e c i f i c i t i e s f o r i o d i n a t e d n u c l e o t i d e - c a r r i e r c o m b i n a t i o n s . 'The b i n d i n g of MCA R l t o i o d o c y t i d i n e - c a r r i e r was i n h i b i t e d by i o d o c y t i d i n e or i o d o c y t i d i n e - 5 ' - p h o s p h a t e and by ad e n o s i n e or adenosine-5'-phosphate (20 and 200 times l e s s so, r e s p e c t i v e l y ) but not by g u a n o s i n e , t h y m i d i n e , and u r i d i n e , or t h e i r monophosphates. As l i t t l e as 5 ng o f i o d o c y t i d i n e - c a r r i e r bound t o a n i t r o c e l l u l o s e membrane c o u l d be d e t e c t e d by MCA R l i n Ill an immune-blot a s s a y w h e r e a s no r e a c t i o n was o b s e r v e d w i t h 1-1000 ng o f c y t i d i n e - c a r r i e r . The l o w e r l e v e l s o f d e t e c t i o n f o r i o d i n a t e d c e l l u l a r n u c l e i c a c i d and i o d i n a t e d p o l y C w e r e 50 and 25 ng, r e s p e c t i v e l y , i n t h e same a s s a y . The m e t h o d may h a v e a p p l i c a t i o n s i n t h e d e v e l o p m e n t o f a s e r o l o g i c a l , n o n - r a d i o a c t i v e a s s a y f o r n u c l e i c a c i d s . iv Xi§ble p f Co n t e n t s Page T i t l e Page i A b s t r a c t i i Tab l e of C o n t e n t s i v L i s t o f F i g u r e s v L i s t o f Tab l e s v i i Acknowledgement v i i i I n t r o d u c t i o n 1 L i t e r a t u r e Review 4 Chemical M o d i f i c a t i o n of N u c l e i c A c i d s 4 Use o f A n t i b o d i e s For D e t e c t i n g N u c l e i c A c i d s 10 P r o d u c t i o n o f A n t i b o d i e s A g a i n s t N u c l e i c A c i d s 11 H a l o g e n a t i o n of N u c l e i c A c i d s 13 M a t e r i a l s and Methods 17 C o n j u g a t i o n of Hapten With P r o t e i n C a r r i e r 17 I o d i n a t i o n o f P o l y c y t i d i n e and ssRNA 19 I o d i n a t i o n o f dsRNA 22 P r o d u c t i o n o f Mo n o c l o n a l A n t i b o d y 23 C h a r a c t e r i z a t i o n o f MCAs 28 Membrane B l o t t i n g Assay of N u c l e o t i d e s and ssRNA ... 29 B i n d i n g of I o d i n a t e d RNA t o ELISA P l a t e s 31 R e s u l t s 32 C o n j u g a t i o n of Hapten With P r o t e i n C a r r i e r 32 I o d i n a t i o n o f P o l y c y t i d i n e and ssRNA 39 I o d i n a t i o n o f dsRNA 41 P r o d u c t i o n o f Mo n o c l o n a l A n t i b o d y 49 C h a r a c t e r i z a t i o n o f MCAs 50 Membrane B i n d i n g Assay of N u c l e o t i d e s and ssRNA .... 69 B i n d i n g of I o d i n a t e d RNA t o ELISA P l a t e s 72 D i s c u s s i o n 73 C o n c l u s i o n s 79 Summary o f Research R e s u l t s 80 B i b l i o g r a p h y 81 Appendix L i s t of A b b r e v i a t i o n s 90 V o f F i g u r e s F i g u r e C o n t e n t Page 1 T h i n l a y e r c h r o m a t o g r a p h y s h o w i n g t h e e f f e c t o f t h e c o n j u g a t i o n r e a c t i o n on i o d o c y t i d i n e a t v a r i o u s t i m e 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 . . 34 2 UV s p e c t r a o f i o d o c y t i d i n e b e f o r e and a f t e r i n c u b a t i o n i n t h e p r e s e n c e o f s o d i u m b o r o h y d r i d e 35 3 SDS-PAGE c o m p a r i n g t h e e f f e c t o f i n c u b a t i n g t h e c a r r i e r - c o n j u g a t e i n t h e p r e s e n c e o f s o d i u m b o r o h y d r i d e f o r v a r y i n g l e n g t h s o f t i m e 36 4 SDS-PAGE c o m p a r i n g t h e c a r r i e r - c o n j u g a t e b e f o r e and a f t e r t h e c o n j u g a t i o n r e a c t i o n 37 5 C o m p a r i s o n o f t h e UV s p e c t r a o f v a r i o u s c a r r i e r -c o n j u g a t e s 38 6 T h i n l a y e r c h r o m a t o g r a p h y s h o w i n g a c o m p a r i s o n o f f r a c t i o n s c o l l e c t e d f r o m an i o d i n a t i o n r e a c t i o n , u s i n g c y t i d i n e as t h e s u b s t r a t e . 42 7 XRF d a t a c o m p a r i n g i o d i n a t i o n r e a c t i o n s c o n t a i n i n g v a r i o u s amounts o f t h e k e y e l e m e n t s o f t h e r e a c t i o n 45 8 C o m p a r i s o n o f t h e UV s p e c t r a o f c y t i d i n e , i o d o -c y t i d i n e , p o l y - C and i o d i n a t e d p o l y - C . .. 46 9 A 5% a e r y l a m i d e - 8 M u r e a g e l c o m p a r i n g i o d i n a t e d and n o n - i o d i n a t e d ssRNA 48 10 > B i n d i n g o f p o l y c l o n a l s e r a and MCA R l w i t h v a r i o u s c a r r i e r s and c a r r i e r - c o n j u g a t e s 51 11 E l u t i o n p r o f i l e o f MCA R l p u r i f i e d f r o m a s c i t e s f l u i d by c h r o m a t o g r a p h y on D E A E - S e p h a c e l 52 12 SDS-PAGE c o m p a r i n g f r a c t i o n s f r o m a DEAE-S e p h a c e l column u s e d t o p u r i f y MCA R l 53 13 S o l i d - p h a s e E L I S A c o m p a r i n g t h e s p e c i f i c i t y o f s e l e c t e d MCAs and p o l y c l o n a l s e r a . . . . 55 14 I n h i b i t i o n o f b i n d i n g o f s e l e c t e d MCAs and p o l y c l o n a l s e r a t o i o d o c y t i d i n e by v a r i o u s n u c l e o t i d e s and n u c l e o s i d e s 56 15 I n h i b i t i o n o f MCA R l b i n d i n g t o i o d o c y t i d i n e by v a r i o u s n u c l e o t i d e s and n u c l e o s i d e s 57 vi 16 I n h i b i t i o n o f MCA R l b i n d i n g t o i o d o c y t i d i n e by v a r i o u s c o n c e n t r a t i o n s o f n u c l e o t i d e s and n u c l e o s i d e s 63 17 I n h i b i t i o n o f MCA R l b i n d i n g t o i o d o c y t i d i n e by a f i x e d c o n c e n t r a t i o n o f v a r i o u s n u c l e o t i d e s and n u c l e o s i d e s 65 18 S c a t c h a r d p l o t c o m p a r i n g t h e b i n d i n g c o n s t a n t s o f v a r i o u s n u c l e o t i d e s and n u c l e o s i d e s t o MCA R l 66 19 I n h i b i t i o n o f MCA R l b i n d i n g t o i o d o c y t i d i n e by i o d i n a t e d and n o n - i o d i n a t e d p o l y - C b e f o r e and a f t e r v a r i o u s r i b o n u c l e a s e d i g e s t i o n s 67 20 I n h i b i t i o n o f MCA R l b i n d i n g t o i o d o c y t i d i n e by i o d i n a t e d a nd n o n - i o d i n a t e d RNA b e f o r e and a f t e r v a r i o u s r i b o n u c l e a s e d i g e s t i o n s 68 21 I m m u n o b l o t s h o w i n g b i n d i n g o f MCA R l t o v a r i o u s a mounts o f i o d o c y t i d i n e - H S A c o n j u g a t e 70 22 I m m u n o b l o t s h o w i n g b i n d i n g o f MCA R l t o v a r i o u s a mounts o f i o d i n a t e d RNA 71 vii L i s t of Tables Table Content Page 1 XRF data comparing the resul t s of the i o d i -nation react ion using various RNAs as the substrate 43 2 S e n s i t i v i t y of the XRF system for detec t ing key elements involved in the i o d i n a t i o n react ion 44 3 XRF data from i o d i n a t i o n reactions conta ining d i f f e r e n t key elements of the reac t ion 47 4 Data from a so l id -phase ELISA to determine the binding s p e c i f i c i t y of MCA R5 with various h a p t e n - c a r r i e r conjugates 54 viii A c k n o w l e d g e m e n t s I w i s h t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o D r . R i c h a r d I . H a m i l t o n f o r e n c o u r a g e m e n t and a d v i c e w i t h r e s e a r c h a n d r e l a t e d m a t t e r s , and a l s o f o r h i s c r i t i c i s m and comments i n t h e p r e p a r a t i o n o f t h i s m a n u s c r i p t . H i s d i r e c t i o n a n d a d v i c e t h r o u g h o u t t h i s w ork w e r e i n v a l u a b l e . I w o u l d l i k e t o e x p r e s s g r a t i t u d e t o Mr. D a v i d W a k a r c h u k f o r v a l u a b l e d i s c u s s i o n s , t o M r s . Bea V a l e n t i n e f o r h e r u n w a n i n g s u p p o r t a nd a s s i s t a n c e w i t h t h e XRF s t u d i e s , a n d t o Dr. R o b e r t M a r t i n f o r h e l p f u l d i s c u s s i o n and comments t h r o u g h o u t t h i s w o r k . T e c h n i c a l s u p p o r t f r o m Mr. Andrew W i e c z o r e k i n t h e p r o d u c t i o n a n d m a i n t e n a n c e o f t h e h y b r i d o m a s was g r e a t l y a p p r e c i a t e d . The many h e l p f u l d i s c u s s i o n s w i t h Dr. R i c h a r d S t a c e - S m i t h , D r . J a c k T r e m a i n e and Mr. B i l l R o n a l d a r e g r a t e f u l l y a c k n o w l e d g e d . A p p r e c i a t i o n i s e x p r e s s e d t o Mr. Wes M a c D i a r m i d f o r h e l p f u l a s s i s t a n c e w i t h p r e p a r a t i o n o f g r a p h i c s f o r t h i s m a n u s c r i p t . G r a d i t u d e i s e x p r e s s e d t o members o f t h e s u p e r v i s i n g c o m m i t t e e : D r s . M. Shaw, R. A . J . W a r r e n , J.B. H u d s o n , a n d V.C. R u n e c k l e s , f o r t h e i r i n v a l u a b l e d i r e c t i o n and comments t h r o u g h o u t t h i s work. F i n a l l y , I w i s h t o e x p r e s s my a p p r e c i a t i o n t o D r . M a r v i n W e i n t r a u b , D i r e c t o r , f o r m a k i n g i t p o s s i b l e f o r me t o c o n d u c t t h i s work a t t h e V a n c o u v e r R e s e a r c h S t a t i o n o f A g r i c u l t u r e C a n a d a . 1 I n t r o d u c t i o n I n r e c e n t y e a r s many o f t h e t e c h n i q u e s d e v e l o p e d f o r u s e i n m o l e c u l a r b i o l o g y have become a v a i l a b l e f o r t h e r o u t i n e d i a g n o s i s and i d e n t i f i c a t i o n o f p l a n t v i r u s e s and v i r o i d s , and t h e d i s e a s e s t h e y c a u s e . The most common o f t h e s e t e c h n i q u e s i s t h e enzyme-l i n k e d i m m u n o s o r b e n t a s s a y ( E L I S A ) e m p l o y i n g b o t h p o l y c l o n a l ( C l a r k , 1981) and m o n o c l o n a l a n t i b o d i e s ( v a n R e g e n m o r t e l , 1 9 8 4 ) . H o wever, t o p r o d u c e a n t i b o d i e s i t i s f i r s t n e c e s s a r y t o be a b l e t o p u r i f y a p r o t e i n a c e o u s component a s s o c i a t e d w i t h t h e v i r u s ( v a n R e g e n m o r t e l , 1 9 8 2 ) . I n s e v e r a l c a s e s t h i s h a s p r o v e d t o be d i f f i c u l t : many v i r u s e s t h a t have d e v a s t a t i n g e f f e c t s on c r o p s c a n n o t be r e a d i l y p u r i f i e d , b e c a u s e t h e v i r u s i s e i t h e r u n s t a b l e i n v i t r o o r r e a c h e s a v e r y low t i t r e i n t h e p l a n t ( G i l m e r e t a l . , 1 9 7 6 ) ; and b e c a u s e v i r o i d s h a v e no known p r o t e i n a c e o u s c o a t a s s o c i a t e d w i t h them (Owens and D i e n e r , 1 9 8 1 ) . I n t h e s e c a s e s a t t e m p t s h a v e b e e n made t o p u r i f y n u c l e i c a c i d r e l a t e d t o t h e v i r u s and t o u s e i t ( K u r p p a and M a r t i n , 1985 and Dodds e t a l . , 1 9 8 4 ) , o r a r e c o m b i n a n t f o r m ( i e . c o m p l e m e n t a r y d e o x y r i b o n u c l e i c a c i d o r cDNA; P a l u k a i t i s e t a l . , 1 9 8 3 ) , i n a n u c l e i c a c i d h y b r i d i z a t i o n a s s a y t o d e t e c t t h e p a t h o g e n i n p l a n t m a t e r i a l . D e s p i t e t h e s u c c e s s f u l u s e o f h y b r i d i z a t i o n a s s a y s t o i d e n t i f y and d e t e c t d i s e a s e s i n m e d i c a l v i r o l o g y ( C o n n e r e t a l . , 1 9 8 3 ) , t h e t e c h n i q u e has n o t been u s e d on a l a r g e - s c a l e i n p l a n t p a t h o l o g y b e c a u s e , u n t i l r e c e n t l y , t h e o n l y m e thod o f d e t e c t i n g h y b r i d i z a t i o n s has been t o l a b e l t h e n u c l e i c a c i d p r o b e w i t h a r a d i o a c t i v e i s o t o p e and, c o n s e q u e n t l y , t o v i s u a l i z e h y b r i d i z a t i o n s b y a u t o r a d i o g r a p h y . T h i s has p r o v e n t o be a p r o b l e m b e c a u s e r o u t i n e d i a g n o s i s i s o f t e n done i n a f i e l d l a b o r a t o r y w h e r e t h e s t a f f o f t e n has n e i t h e r t h e f a c i l i t i e s n o r t h e t r a i n i n g t o p r o p e r l y and s a f e l y h a n d l e r a d i o a c t i v e m a t e r i a l . I n t h e p a s t t h e r e h a v e been a t t e m p t s t o l a b e l n u c l e i c a c i d s w i t h n o n - r a d i o a c t i v e r e a g e n t s . F l u o r o p h o r e s have been c o u p l e d t o RNA ( C o n n o l l y and R i d e r , 1 9 8 3 ) , b u t t h e s e h a v e t h e d i s a d v a n t a g e s o f r e q u i r i n g d e l i c a t e h a n d l i n g t e c h n i q u e s , n o t b e i n g as s e n s i t i v e as a u t o r a d i o g r a p h y , and r e q u i r i n g e x p e n s i v e e q u i p m e n t f o r p r o p e r r e c o r d i n g and q u a n t i t a t i o n o f r e s u l t s . Many s y s t e m s o f d e t e c t i o n h a v e b e e n d e v e l o p e d w h i c h t a k e a d v a n t a g e o f t h e e s s e n t i a l l y i r r e v e r s i b l e r e a c t i o n b e t w e e n b i o t i n and a v i d i n ( G r e e n , 1 9 6 3 ) . C o n s e q u e n t l y , b i o t i n has b e e n i n c o r p o r a t e d i n t o n u c l e i c a c i d p r o b e s b o t h e n z y m a t i c a 1 1 y ( L a n g e r e t a l . , 1 9 8 1 ) , a nd p h o t o -c h e m i c a l l y ( F o r s t e r e t a l . , 1 9 8 5 ) ; h y b r i d i z a t i o n s a r e t h e n v i s u a l i z e d by t h e u s e o f c o l o r i m e t r i c r e a g e n t s c o u p l e d t o a v i d i n . T h e s e t e c h n i q u e s h a v e a s e n s i t i v i t y e q u a l t o t h a t o f r a d i o a c t i v e m e t h o d s and t h e d e r i v a t i z e d n u c l e i c a c i d c a n be s t o r e d f o r months a t 4 C, w i t h o u t l o s s o f a c t i v i t y . However, none o f t h e s e p r o c e d u r e s , e x c e p t t h a t o f F o r s t e r e t a l . ( 1 9 8 5 ) , a l l o w s f o r t h e e a s y and c o s t - e f f i c i e n t p r e p a r a t i o n o f l a r g e amounts ( i e . g r e a t e r t h a n 100 ug) o f l a b e l l e d n u c l e i c a c i d p r o b e . M o r e o v e r , a l m o s t a l l o f t h e n o n - r a d i o a c t i v e l a b e l l i n g p r o c e d u r e s d e s c r i b e d t o d a t e i n v o l v e r e l a t i v e l y g r o s i s m o d i f i c a t i o n s o f t h e n u c l e i c a c i d , w h i c h may r e n d e r them u n s u i t a b l e f o r u s e i n o t h e r p o t e n t i a l a p p l i c a t i o n s ( e g . f l o w c y t o m e t r y ) . T h e r e f o r e , e x p e r i m e n t s w e re p e r f o r m e d t o d e v e l o p a n o n -r a d i o a c t i v e method o f d e t e c t i n g n u c l e i c a c i d s by s y n t h e s i z i n g a 3 n u c l e o t i d e a n a l o g , w h i c h c o u l d be u s e d p r a c t i c a l l y i n v i t r o a n d w h i c h w o u l d c a u s e t h e l e a s t d i s t u r b a n c e o f c e l l u l a r p r o c e s s e s i f i n t r o d u c e d i n v i v o . Thus t h e o b j e c t i v e s o f t h i s t h e s i s w e r e : o ne, t o i o d i n a t e RNA a t t h e C-5 p o s i t i o n o f c y t i d i n e by t h e C u m m e r f o r d r e a c t i o n ( C o m m e r f o r d , 1 9 7 1 ) ; two, t o p r o d u c e h y b r i d o m a s s e c r e t i n g MCAs a g a i n s t t h e m o d i f i e d n u c l e o t i d e ; a n d t h r e e , t o a s s e s s r e a c t i v i t y o f t h e MCAs w i t h i o d i n a t e d RNA. T h e s e MCAs c o u l d t h e n be u s e d v i a i m m u n o - c o l o r i m e t r i c t e c h n i q u e s t o d e t e c t an i o d i n a t e d n u c l e i c a c i d p r o b e i n a h y b r i d i z a t i o n a s s a y . T h i s a p p r o a c h was c h o s e n f o r s e v e r a l r e a s o n s : one, t h e r e a c t i o n i s s p e c i f i c t o s i n g l e - s t r a n d e d r i b o n u c l e i c a c i d ( s s R N A ) and t h e m a j o r i t y o f p l a n t v i r u s e s and a l l v i r o i d s c o n t a i n a ssRNA genome ( M a t t h e w s , 1 9 8 1 ) ; . two, i t has been d e m o n s t r a t e d t h a t h a l o g e n a t e d n u c l e o t i d e s a r e r e a d i l y i n c o r p o r a t e d i n t o c e l l u l a r DNA i n y i y o (Dunn and S m i t h , 1 9 5 7 ) ; and t h r e e , h i g h l y s p e c i f i c a n t i b o d i e s h a v e b e e n p r o d u c e d t o m o d i f i e d n u c l e o t i d e s (Munns and L i s z e w s k i , 1 9 8 0 ) , and s p e c i f i c a l l y t o h a l o g e n a t e d n u c l e o t i d e s ( G r a t z n e r , 1 9 8 2 ) . l i t e r a t u r e R e v i e w T r a d i t i o n a l l y , n u c l e i c a c i d s h a v e been d e t e c t e d t h r o u g h t h e u s e o f r a d i o - l a b e l l e d m a r k e r s . The most commonly u s e d i s o t o p e i s P32 ( M a n i a t i s e t a l . , 1 9 8 2 ) , b u t t r i t i u m ( B a r k e r e t . a l . , 1983) a n d 1125 ( J a c k s o n and M i l n e r , 1979) have a l s o b e e n u s e d t o a l e s s e r e x t e n t . More r e c e n t l y , h o w e v e r , t h e r e h a v e b e e n s e v e r a l a t t e m p t s t o p r o d u c e n o n - r a d i o a c t i v e p r o b e s f o r t h e d e t e c t i o n o f n u c l e i c a c i d s , and f o r use i n s t u d y i n g t h e c o n f o r m a t i o n a l s t r u c t u r e o f n u c l e i c a c i d s . C h e m i c a l M o d i f i c a t i o n o f N u c l e i c A c i d s The m a j o r i t y o f t h e n o n - r a d i o a c t i v e m ethods o f d e t e c t i n g n u c l e i c a c i d s i n v o l v e e i t h e r d i r e c t m o d i f i c a t i o n o f t h e n u c l e i c a c i d o r c h e m i c a l l y a t t a c h i n g a f o r e i g n e n t i t y ( e g . f e r r i t i n , f l u o r e s c e n t p r o b e s o r ' h i s t o n e s ) t o t h e n u c l e i c a c i d . E a r l y e x p e r i m e n t s by S t e i n e r e t a l . ( 1 9 7 3 ) , i n d i c a t e d t h a t p o l y r i b o a d e n y 1 i c a c i d c o u l d be c o n v e r t e d t o a f l u o r e s c e n t d e r i v a t i v e when t r e a t e d w i t h c h l o r o a c e t y l a l d e h y d e . The p h y s i c o -c h e m i c a l b e h a v i o r o f n u c l e i c a c i d m o d i f i e d by c h l o r o a c e t y l a l d e h y d e was s t u d i e d ( L e e and Wetraur, 1973) and i t was f o u n d t h a t , as w i t h o t h e r n u c l e i c a c i d s c o n t a i n i n g b a s e s m o d i f i e d w i t h l a r g e e x t e r n a l g r o u p s , t h e m e l t i n g t e m p e r a t u r e (Tm) a n d t h e r a t e o f r e n a t u r a t i o n w e re d e c r e a s e d . D e o x y r i b o n u c l e i c a c i d (DNA) m o d i f i e d i n t h i s way was u s e d t o s t u d y t h e i n t e r a c t i o n b e t w e e n DNA and a b i n d i n g p r o t e i n ( S i l v e r and F e r s h t , 1982; 1983) by q u a n t i t a t i n g c h a n g e s i n f l u o r e s c e n c e as t h e two c o m p o n e n t s b o u n d t o g e t h e r u n d e r v a r i o u s c o n d i t i o n s . Yang and S o i l ( 1 9 7 4 ) , 5 i n s e r t e d f l u o r e s c e n t p r o b e s i n t o v a r i o u s b a s e s i n t r a n s f e r RNA ( t R N A ) a nd u s e d d a t a r e c o r d e d f r o m s i n g l e t - s i n g l e t e n e r g y t r a n s f e r t o e l u c i d a t e t h e t e r t i a r y s t r u c t u r e o f t h e n u c l e i c a c i d . S c h r e i b e r e t a l . ( 1 9 7 9 ) , s t u d i e d t h e a c c e s s i b i l i t y o f t h e 3'-end o f RNA t o l a b e l l i n g w i t h a f l u o r e s c e n t p r o b e b e f o r e a n d a f t e r b i n d i n g t o a r i b o s o m e c o m p l e x , and c o n s e q u e n t l y h y p o t h e s i s e d a b o u t t h e s p a t i a l o r i e n t a t i o n o f t h e 3'-end o f t h e RNA w i t h i n t h e r i b o s o m e . S e v e r a l n o n - r a d i o a c t i v e m e t h o d s h a v e been d e v i s e d t o d e t e c t n u c l e i c a c i d h y b r i d i z a t i o n b o t h i n s i t u and i n v i t r o . E l e c t r o n -d e n s e f e r r i t i n was c o u p l e d t o t h e 3'-end o f RNA (Wu and D a v i d s o n , 1 9 7 3 ) , t h e m o d i f i e d RNA was t h e n h y b r i d i z e d t o ss-DNA and t h e d u p l e x was d e t e c t e d by e l e c t r o n m i c r o s c o p y . The t e c h n i q u e e n a b l e d t h e d e t e c t i o n o f tRNA genes i n b a c t e r i a l DNA. I t was f u r t h e r o b s e r v e d t h a t v a r i o u s c h e m i c a l g r o u p s c o u l d be a t t a c h e d t o 3' t e r m i n a l r i b o n u c l e o t i d e s o x i d i z e d w i t h p e r i o d a t e t h r o u g h t h e f o r m a t i o n o f a S c h i f f ' s b a s e w i t h a p r i m a r y a m i n e on t h e l a b e l l i n g c h e m i c a l (Munns and L i s z e w s k i , 1 9 8 0 ) . T h i s m e t h o d has b e e n u s e d t o a t t a c h f l u o r e s c e n t g r o u p s t o t h e 3'-ends o f RNA (Bauman e t a l . , 1 9 8 1 ) , o r DNA ( E s h a g h p o u r e t a l . , 1 9 7 9 ) , t h e l a t t e r f i r s t r e q u i r i n g t h e i n c o r p o r a t i o n o f a few r i b o n u c l e o t i d e s u s i n g t h e enzyme, d e o x y n u c l e o t i d y 1 t e r m i n a l t r a n s f e r a s e . Bauman e t a l . ( 1 9 8 1 ) , a t t a c h e d t h e f l u o r e s c e n t g r o u p , t e t r a m e t h y l r h o d a m i n e i s o t h i o c y a n a t e , and f o u n d t h a t t h e m e l t i n g t e m p e r a t u r e o f t h e m o d i f i e d n u c l e i c a c i d was u n c h a n g e d ; h o w e v e r t h e p r o b e d i d b r e a k down a t t h e h i g h e r t e m p e r a t u r e s r e q u i r e d f o r s t r i n g e n t h y b r i d i z a t i o n s . The method was n o t as s e n s i t i v e as a u t o r a d i o g r a p h y , b u t i t was f o u n d t o be r e l a t i v e l y e a s y t o q u a n t i t a t e f l u o r e s c e n c e , i f q u e n c h i n g c o u l d be c o n t r o l l e d , t h u s m a k i n g t h e p r o b e u s e f u l f o r v a r i o u s s t u d i e s u s i n g f l o w c y t o m e t r y . As n u c l e i c a c i d m o d i f y i n g enzymes became more commonly u s e d t h e i r e x c e l l e n t s p e c i f i c i t y , c o m p a r e d t o t h a t o f a c h e m i c a l r e a c t i o n , was e x p l o i t e d t o a t t a c h a l t e r e d n u c l e o t i d e s t o n u c l e i c a c i d , p r o d u c i n g m o d i f i c a t i o n s w i t h a much h i g h e r s p e c i f i c i t y . A d v a n t a g e was t a k e n o f t h e r e l a t i v e l y low s p e c i f i c i t y o f T4 RNA l i g a s e f o r i t s s u b s t r a t e t o c o u p l e two f l u o r e s c e n t and one b i o t i n y l a t e d d e r i v a t i v e s o f a r i b o n u c l e o t i d e t o t h e 3'-end o f RNA ( R i c h a r d s o n and Gumport, 1 9 8 3 ) . N u c l e i c a c i d w i t h any o f t h e s e m o d i f i c a t i o n s was d e t e c t a b l e a t p i c o g r a m l e v e l s . The same enzyme was u s e d t o a t t a c h n u c l e o t i d e s m o d i f i e d w i t h a p h o s p h o r o t h i o a t e g r o u p t o t h e 3'-end o f b o t h RNA and DNA ( C o s s t i c k e t a l . , 1 9 8 4 ) . F l u o r e s c e n t g r o u p s c o u l d be a t t a c h e d t o t h i s h i g h l y n u c l e o p h i l i c g r o u p b e f o r e o r a f t e r e n z y m a t i c i n c o r p o r a t i o n . C o n n o l l y and R i d e r ( 1 9 8 3 ) , h a v e d e s c r i b e d t h e s y n t h e s i s o f o l i g o n u c l e o t i d e s c o n t a i n i n g n u c l e o t i d e s w i t h a f r e e s u l f h y d r y l t o w h i c h a f l u o r o p h o r e c o u l d be a t t a c h e d . However f l u o r e s c e n c e i s n o t n e c e s s a r i l y t h e i d e a l d e t e c t i o n s y s t e m b e c a u s e c a r e must be t a k e n t o c o n t r o l q u e n c h i n g , and i f t h e s i g n a l i s t o be q u a n t i t a t e d , e x p e n s i v e and c o m p l i c a t e d e q u i p m e n t i s r e q u i r e d . T c h e n e t a l . ( 1 9 8 4 ) , a l s o d e v e l o p e d a s y s t e m o f l a b e l l i n g n u c l e i c a c i d s v i a a c h e m i c a l m o d i f i c a t i o n . However, i n s t e a d o f a t t a c h i n g a f l u o r e s c e n t l a b e l , t h e n u c l e i c a c i d was d e r i v a t i z e d w i t h N - a c e t o x y - N - 2 - a c e t y l a m i n o f l u o r e n e (AAF) and i t s 7 - i o d o d e r i v a t i v e ( I A A F ) . The l a b e l l e d n u c l e i c a c i d was t h e n d e t e c t e d i n an i m m u n o l o g i c a l a s s a y u s i n g a n t i b o d i e s s p e c i f i c f o r t h e d e r i v a t i z e d n u c l e o t i d e . The t e c h n i q u e was u s a b l e w i t h b o t h RNA a n d DNA and was as s e n s i t i v e as r a d i o a c t i v e l a b e l l e d p r o b e s , b u t w i t h a c l e a r e r s i g n a l d e t e c t i o n . However, t h e s y n t h e s i s o f AAF a n d IAAF i s c o m p l e x and b o t h compounds a r e c a r c i n o g e n i c ( K r i e k , 1 9 7 4 ) , t h e r e b y f u r t h e r c o m p l i c a t i n g t h e i r h a n d l i n g . G l u t a r a l d e h y d e c a n a l s o be u s e d t o c o n j u g a t e p r o t e i n s t o p o l y n u c l e o t i d e s t h r o u g h some unknown m e c h a n i s m (Hopwood e t a l . , 1 9 7 0 ) . T h i s t e c h n i q u e was u s e d t o c o n j u g a t e a low m o l e c u l a r w e i g h t p r o t e i n , h i s t o n e H I , t o n u c l e i c a c i d ( R e n z , 1 9 8 3 ) . S u r p r i s i n g l y , m e l t i n g c u r v e s o f t h e c o n j u g a t e d n u c l e i c a c i d d i f f e r e d o n l y s l i g h t l y c o mpared t o u n a l t e r e d n u c l e i c a c i d as l o n g as t h e mass r a t i o o f p r o t e i n t o n u c l e i c a c i d was k e p t b e l o w one. The h i s t o n e was t h e n l a b e l l e d w i t h e i t h e r 1125 o r b i o t i n a nd t h e c o n j u g a t e d p o l y n u c l e o t i d e u s e d i n h y b r i d i z a t i o n s . The d e t e c t i o n l e v e l o f b o t h o f t h e p r o t e i n - 1 a b e l 1 i n g t e c h n i q u e s was f o u n d t o be i n t h e r a n g e o f s t a n d a r d r a d i o - l a b e l l i n g t e c h n i q u e s , b u t t h e b a c k g r o u n d w i t h b i o t i n - l a b e l l e d HI was f o u n d t o be u n a c c e p t a b l y h i g h . F u r t h e r m o r e , i t was d i f f i c u l t t o a c c u r a t e l y and r e p r o d u c i b l y c o n t r o l t h e l e v e l o f c o n j u g a t i o n . The p r o c e d u r e was r e f i n e d by t h e s y n t h e s i s o f a s m a l l p o l y m e r w i t h s e v e r a l p r i m a r y a m i n e s t o w h i c h a p r o t e i n ( i e . h o r s e r a d i s h p e r o x i d a s e o r a l k a l i n e p h o s p h a t a s e ) c o u l d be a t t a c h e d , and w h i c h c o u l d be u s e d i n a c o l o r i m e t r i c a s s a y (Renz and K u r z , 1 9 8 4 ) . B a c k g r o u n d p r o b l e m s w e r e e l i m i n a t e d and t h e d e t e c t i o n l e v e l was i n t h e p i c o g r a m r a n g e , b u t q u e s t i o n a b l e s u r v i v a l o f t h e enzyme u n d e r c e r t a i n h y b r i d i z a t i o n c o n d i t i o n s d i m i n i s h e d t h e u s e f u l n e s s o f t h e s y s t e m 8 ( F o r s t e r e t a l . , 1 9 8 5 ) . The most s u c c e s s f u l s y s t e m s d e v e l o p e d f o r t h e n o n -r a d i o a c t i v e d e t e c t i o n o f n u c l e i c a c i d h a v e t a k e n a d v a n t a g e o f t h e r e m a r k a b l y s t r o n g i n t e r a c t i o n b e t w e e n b i o t i n and a v i d i n , w h i c h i s c h a r a c t e r i z e d by a d i s s o c i a t i o n c o n s t a n t o f 10-15 ( G r e e n , 1 9 6 3 ) . T h i s p a r t i c u l a r a p p r o a c h has b e e n u s e d i n i n s i t u h y b r i d i z a t i o n s ( M a n n i n g e t . a l . , 1 9 7 5 ) . B i o t i n was c o u p l e d t o c y t o c h r o m e C, w h i c h i n t u r n was c o u p l e d t o r i b o s o m a l RNA (rRNA) and u s e d i n a h y b r i d i z a t i o n a s s a y i n chromosome s q u a s h e s . C o m p l e m e n t a r y s e q u e n c e s w e re d e t e c t e d by a v i d i n l i n k e d t o l a t e x s p h e r e s w h i c h c o u l d be o b s e r v e d i n t h e s c a n n i n g e l e c t r o n m i c r o s c o p e . S e n s i t i v i t y was l e s s t h a n what w o u l d be d e t e c t e d by a u t o r a d i o g r a p h y u n d e r s i m i l i a r c o n d i t i o n s , b u t t h e e a s e , s a f e t y , a n d s p e e d o f p e r f o r m i n g t h e t e c h n i q u e w e r e f o u n d t o be s u p e r i o r . The t e c h n i q u e was f u r t h e r m o d i f i e d by c o u p l i n g t h e N-h y d r o x y s u c c i n i m i d e e s t e r o f b i o t i n t o t h e 3'-end o f RNA, and h y b r i d i z a t i o n s were v i s u a l i z e d v i a t h e e l e c t r o n m i c r o s c o p e f o l l o w i n g i n c u b a t i o n o f t h e c o n j u g a t e w i t h a v i d i n l i n k e d t o f e r r i t i n ( S o d j a and D a v i d s o n , 1 9 7 8 ) . However, c e r t a i n c h e m i c a l s i d e r e a c t i o n s w e r e o b s e r v e d d u r i n g m o d i f i c a t i o n o f t h e 3'-end o f t h e RNA, due t o s t e r i c h i n d r a n c e i n t h e d u p l e x t o b i n d i n g o f a v i d i n t o b i o t i n . The s t e r i c h i n d r a n c e was e l i m i n a t e d by u s i n g a l o n g e r l i n k e r m o l e c u l e , s u c h as c y t o c h r o m e C, and f u r t h e r m o r e , s e v e r a l b i o t i n s c o u l d be a t t a c h e d t o e a c h c y t o c h r o m e C l i n k e r m o l e c u l e , t h e r e b y i n c r e a s i n g s e n s i t i v i t y ( S o d j a and D a v i d s o n , 1 9 7 8 ) . L a n g e r e t a l . ( 1 9 8 1 ) , r e p o r t e d t h e s y n t h e s i s o f b i o t i n y l a t e d t h y m i d i n e - t r i p h o s p h a t e (TTP) and u r i d i n e - t r i p h o s p h a t e (UTP) t h a t 9 c o u l d be e n z y m a t i c a l l y i n c o r p o r a t e d i n t o DNA and RNA, r e s p e c t i v e l y . N u c l e i c a c i d c o n t a i n i n g the m o d i f i e d n u c l e o t i d e s was found to have m e l t i n g and h y b r i d i z a t i o n k i n e t i c s s i m i l i a r to those of unmodified n u c l e i c a c i d . The b i o t i n y l a t e d probe, with a v i d i n coupled to f l u o r e s c e n t i n d i c a t o r s , was used to study n u c l e i c a c i d s i n c e l l c u l t u r e s and p a r a f f i n - embedded t i s s u e ( B r i g a t i et a l . , 1983). Although not as s e n s i t i v e as a utoradiography, the technique was a t t r a c t i v e because of i t s ease of e x e c u t i o n and the long s h e l f - l i f e of the r e q u i r e d reagents. The s e n s i t i v i t y of the system c o u l d be i n c r e a s e d by the use of longer spacer arms in l i n k i n g the b i o t i n to the n u c l e o t i d e to remove any e f f e c t s of s t e r i c hindrance, and the use of polymers of b a c t e r i a l a l k a l i n e phosphatase l i n k e d to a v i d i n f o r the c o l o r i m e t r i c d e t e c t i o n of n u c l e i c a c i d h y b r i d i z a t i o n s (Leary et a l . , 1983). The s e n s i t i v i t y of the technique was found to be i n the picogram range, d e t e c t i n g s i n g l e copy genes i n mammalian DNA with a lower s i g n a l to noise r a t i o than corre s p o n d i n g r a d i o a c t i v e probes. D e l i u s et a l . (1985), l a b e l e d only the 5'-end of the p o l y n u c l e o t i d e with the b i o t i n y l a t e d analogue, thus a l l o w i n g the d e t e c t i o n of h y b r i d i z a t i o n i n the p a r t i c u l a r s t r a n d of i n t e r e s t ( M a n i a t i s et a l . , 1982). A u n i v e r s a l b i o t i n y l a t e d d e r i v a t i v e capable of b i n d i n g to any n u c l e i c a c i d has been s y t h e s i z e d ( F o r s t e r et a l . , 1985). B i o t i n was coupled to a l i n k e r arm c o n t a i n i n g a p h o t o - r e a c t i v e group, a l l o w i n g attachment of the l a b e l to the p o l y n u c l e o t i d e v i a a l i g h t - i n d u c e d r e a c t i o n . Frequency of l a b e l l i n g of more than one i n one-hundred n u c l e o t i d e s does not a f f e c t h y b r i d i z a t i o n 10 k i n e t i c s , and s e n s i t i v i t y i s i n t h e p i c o g r a m r a n g e . However, i t may be t e c h n i c a l l y d i f f i c u l t t o r e p r o d u c i b l y c o n t r o l t h e f r e q u e n c y o f l a b e l l i n g due t o t h e s e n s i t i v i t y o f p h o t o - d r i v e n c h e m i c a l r e a c t i o n s ( J o l l e y e t a l . , 1 9 6 7 ) . A l t h o u g h s e v e r a l s u c c e s s f u l n o n - r a d i o a c t i v e s y s t e m s have b e e n d e v e l o p e d f o r t h e l a b e l l i n g o f n u c l e i c a c i d s , o n l y t h e b i o t i n - a v i d i n s y s t e m a p p e a r s t o be u s e a b l e p r a c t i c a l l y . However, w i t h t h e e x c e p t i o n o f t h e p r o c e d u r e o u t l i n e d by F o r s t e r e t a l . ( 1 9 8 5 ) , none o f t h e p r o t o c o l s u s i n g b i o t i n y l a t e d d e r i v a t i v e s c a n be u s e d p r a c t i c a l l y t o p r e p a r e l a r g e q u a n t i t i e s o f l a b e l l e d n u c l e i c a c i d . M o r e o v e r a l l o f t h e n u c l e i c - a c i d l a b e l l i n g p r o c e d u r e s , i n c l u d i n g t h o s e u s i n g b i o t i n , i n v o l v e r a t h e r g r o s s m o d i f i c a t i o n s o f t h e n u c l e i c a c i d , w h i c h c o u l d a f f e c t t h e i r u s e f u l n e s s i n an i n v i v o a p p l i c a t i o n . Use o f A n t i b o d i e s F o r D e t e c t i n g N u c l e i c A c i d s A n t i b a d i e s h a v e p r e v i o u s l y b e e n p r o d u c e d t o t h e common and t h e m o d i f i e d b a s e s f o u n d i n n u c l e i c a c i d (Munns and L i s z e w s k i , 1 9 8 0 ) . T h e s e a n t i b o d i e s can be v e r y s p e c i f i c : R a i n e r and S t o l l a r ( 1 9 7 8 ) p r o d u c e d a n t i b o d i e s c a p a b l e o f d i f f e r e n t i a t i n g b e t w e e n i n t a c t and h y d r o l y z e d 7 - m e t h y l g u a n o s i n e . S p e c i f i c a n t i b o d i e s t o N - 6 - m e t h y l a d e n o s i n e and 7 - m e t h y l g u a n o s i n e h a v e b e e n a t t a c h e d t o a m a t r i x and u s e d f o r t h e a f f i n i t y p u r i f i c a t i o n o f n u c l e i c a c i d s c o n t a i n i n g t h e m o d i f i e d b a s e s (Munns e t a l . , 1 9 7 7 ) . I s o p e n t e n y l d e r i v a t i v e s o f a d e n o s i n e were i d e n t i f i e d i n RNase T l - d i g e s t e d RNA i s o l a t e d f r o m c o l i m u t a n t s by a n t i b o d i e s s p e c i f i c f o r t h e m o d i f i e d b a s e ( V o i d and N o l e n , 1 9 7 9 ) . G r a t z n e r ( 1 9 8 2 ) , a l s o u s e d a h a l o g e n t o l a b e l n u c l e i c a c i d 11 i n d e v e l o p i n g a n o n - r a d i o a c t i v e , f l u o r e s c e n t p r o b e f o r s t u d y i n g n u c l e i c a c i d s y n t h e s i s i n v i v o . I n c o r p o r a t i o n o f 5-b r o m o d e o x y u r i d i n e i n c e l l c u l t u r e s was q u a n t i t a t e d by f l o w c y t o m e t r y u s i n g a MCA s p e c i f i c f o r t h e m o d i f i e d b a s e , c o u p l e d w i t h a f l u o r e s c e n t i n d i c a t o r . S a w i c k i e t a l . ( 1 9 7 1 ) , u s e d s p e c i f i c a n t i b o d i e s t o 5-bromo and 5 - i o d o - u r i d i n e t o d e t e c t t h e h a l o g e n a t e d b a s e s i n d e n a t u r e d DNA. O t h e r n u c l e i c a c i d s c o n t a i n i n g b a s e s w i t h s l i g h t m o d i f i c a t i o n s h a v e b e e n d e t e c t e d w i t h a h i g h d e g r e e o f s e n s i t i v i t y u s i n g a n t i b o d i e s s p e c i f i c f o r t h e a p p r o p r i a t e m o d i f i e d b a s e . D e t e c t i o n o f DNA c o n t a i n i n g 5-m e t h y l c y t i d i n e by i m m u n o l o g i c a l a s s a y i s r e p o r t e d t o be a t t h e p i c o g r a m l e v e l ( B a l l e t a l . , 1983 and A c h w a l and C h a n d r a , 1 9 8 2 ) . A s i m i l i a r a p p r o a c h t o t h a t e m p l o y e d i n t h i s w o r k , was u s e d by Tchen e t a l . ( 1 9 8 4 ) . N u c l e i c a c i d was c h e m i c a l l y m o d i f i e d by a h i g h l y s p e c i f i c r e a c t i o n and h y b r i d o m a s were p r o d u c e d w h i c h s e c r e t e d m o n o c l o n a l a n t i b o d i e s (MCA) s p e c i f i c t o t h e m o d i f i e d b a s e , and w h i c h c o u l d be u s e d t o d e t e c t t h e m o d i f i e d n u c l e i c a c i d i n an i m m u n o l o g i c a l a s s a y . P r o d u c t i o n o f A n t i b o d i e s A g a i n s t N u c l e i c A c i d s S e v e r a l p r o c e d u r e s have b e e n d e s c r i b e d f o r s u c c e s s f u l l y p r o d u c i n g b o t h p o l y c l o n a l ( K i t a g a w a e t a l . , 1977) and m o n o c l o n a l a n t i b o d i e s ( J a c o b and T r o n , 1982) a g a i n s t n u c l e i c a c i d s . A n t i b o d i e s a g a i n s t d o u b l e - s t r a n d e d ( d s ) RNA h a v e b e e n u s e d t o d e t e c t F i j i d i s e a s e v i r u s i n i n f e c t e d l e a v e s o f s u g a r c a n e ( F r a n c k i and J a c k s o n , 1 9 7 2 ) , and S i n d b i s v i r u s i n f e c t i o n i n c h i c k , h a m s t e r , and m o s q u i t o c e l l s ( S t o l l a r e t a l . , 1 9 7 2 ) . 12 S i m i l i a r l y , a n t i b o d i e s a g a i n s t RNA-DNA h y b r i d s were u s e d i n an i m m u n o f l u o r e s c e n c e a s s a y t o d e t e c t h y b r i d i z a t i o n s i n chromosome s q u a s h e s ( R u d k i n and S t o l l a r , 1 9 7 7 ) . H owever, i t has been f o u n d t h a t when p o l y n u c l e o t i d e s a r e u s e d as t h e a n t i g e n , a n t i b o d i e s a r e s p e c i f i c t o e i t h e r t h e s u g a r -p h o s p h a t e b a c k b o n e ( B o r e l e t a l . , 1 9 7 0 ) , o r t o t h e s e c o n d a r y s t r u c t u r e o f t h e n u c l e i c a c i d ( S t o l l a r , 1 9 7 0 ) . J o h n s t o n e t a l . ( 1 9 7 5 ) , s t u d i e d t h e s p e c i f i c i t y o f a n t i b o d i e s a g a i n s t dsRNA and f o u n d t h a t t h e a n t i b o d i e s c o u l d b i n d t o t h e d u p l e x e v e n i f some o f t h e b a s e s i n t h e immunogen were a l t e r e d , s o l o n g as t h e c o n f o r m a t i o n o f t h e dsRNA was n o t c h a n g e d . F o r e x a m p l e , MCAs were p r o d u c e d a g a i n s t t h e a n t i g e n p o l y ( d G - 5 - B r C ) : p o l y ( d G - d C ) , w h i c h f o r m s t h e Z - h e l i c a l s t r u c t u r e o f DNA. S e v e r a l h y b r i d o m a s s e c r e t i n g MCAs s p e c i f i c f o r Z-DNA were f o u n d , b u t none w e r e r e a c t i v e a g a i n s t t h e h a l o g e n a t e d n u c l e o t i d e ( M o l e r e t a l . , 1 9 8 2 ) . P r o d u c t i o n o f a n t i b o d i e s t o n u c l e i c a c i d s i s g r e a t l y i m p r o v e d i f t h e n u c l e i c a c i d i s c o n j u g a t e d t o a p r o t e i n c a r r i e r . Many metho d s have b e e n d e s c r i b e d f o r c h e m i c a l l y c o n j u g a t i n g t o p r o t e i n c a r r i e r s s m a l l m o l e c u l e s ( h a p t e n s ) s u c h as n u c l e i c a c i d s ( B o r e l e t a l . , 1984; S t o l l a r , 1970) o r n u c l e o s i d e s and n u c l e o t i d e s (Munns and L i s z e w s k i , 1 9 8 0 ) . U n f o r t u n a t e l y , t h e m a j o r i t y o f t h e s e i n v o l v e c o m p l e x p r o c e d u r e s , and many l e a d t o u n a c c e p t a b l e m o d i f i c a t i o n s o f t h e h a p t e n . H a l l o r a m and P a r k e r ( 1 9 6 6 ) , d e s c r i b e d a c o n j u g a t i o n p r o c e d u r e u s i n g c a r b o d i i m i d e , w h i c h was s i m p l e i n e x e c u t i o n and d i d n o t m o d i f y e i t h e r t h e b a s e o r t h e s u g a r m o i e t y ; h o w e v e r , i t r e q u i r e d t h a t t h e n u c l e o s i d e be e s t e r i f i e d a t t h e 2'- o r 3'- p h o s p h a t e . The l a t t e r w o u l d h a v e 13 b e e n a p r o b l e m as t h i s d e r i v a t i v e o f i o d o c y t i d i n e i s n o t t c o m m e r c i a l l y a v a i l a b l e , and i s t e c h n i c a l l y d i f f i c u l t t o s y n t h e s i z e . When a n t i b o d i e s p r o d u c e d a g a i n s t a d e n o s i n e c o n j u g a t e d t o a c a r r i e r by t h e c a r b o d i i m i d e p r o c e d u r e were s t u d i e d , t h e r e w e r e t h r e e d i s t i n c t g r o u p s o f a n t i b o d i e s : t h o s e s p e c i f i c t o t h e b a s e , t h e s u g a r - p h o s p h a t e , and t h e n u c l e o s i d e (Humayun and J a c o b , 1973) . F u r t h e r m o r e , t h e a n t i b o d i e s h a d a low a f f i n i t y f o r a d e n o s i n e i n n u c l e i c a c i d . Thus i t was c o n c l u d e d t h a t t h e r e was no a d v a n t a g e t o p r e s e r v i n g i n t a c t t h e r i b o s e r i n g and c o n s e q u e n t l y , t h e p e r i o d a t e o x i d a t i o n r e a c t i o n d e s c r i b e d by E r l a n g e r and B e i s e r ( 1 9 6 4 ) was u s e d , w h i c h a l l o w e d c o n j u g a t i o n o f c o m m e r c i a l l y a v a i l a b l e i o d o c y t i d i n e . A n t i b o d i e s , p r o d u c e d a g a i n s t n u c l e o s i d e s c o n j u g a t e d t o a c a r r i e r by t h i s t e c h n i q u e , w e r e e x a m i n e d (Humayun and J a c o b , 1974) , and t h r e e g r o u p s w ere f o u n d : t h o s e s p e c i f i c t o t h e b a s e , t h e o x i d i z e d r i b o s e , o r t h e n u c l e o s i d e w i t h t h e o x i d i z e d r i b o s e ( s e e D i s c u s s i o n ) . The r e a c t i o n was c o n s i d e r e d t o be t e c h n i c a l l y u n c o m p l i c a t e d , r e q u i r i n g f e w e r m a n i p u l a t i o n s t h a n most o t h e r p r o t o c o l s (Munns and L i s z e w s k i , 1 9 8 0 ) . I n t h e method o f E r l a n g e r and B e i s e r ( 1 9 6 4 ) , c o u p l i n g i s b e t w e e n f r e e p r i m a r y a m i n e s on t h e c a r r i e r and t h e p e r i o d a t e - o x i d i z e d a l c o h o l s o f t h e n u c l e o t i d e . T h i s f o r m s a S c h i f f ' s b a s e , w h i c h i s t h e n s t a b i l i z e d by r e d u c t i o n w i t h b o r o h y d r i d e (Munns and L i s z e w s k i , 1 9 8 0 ) . H§ipgenation o f N u c l e i c A c i d I o d i n e i s a h a l o g e n commonly u s e d t o l a b e l n u c l e i c a c i d s w h i c h a r e t o be u s e d i n n u c l e i c a c i d h y b r i d i z a t i o n s ( T e r e b a a nd 14 M c C a r t h y , 1973) and i n RNA s e q u e n c e d e t e r m i n a t i o n s ( D i c k s o n e t a l . , 1 9 7 9 ) . F u r t h e r m o r e , most o f t h e l i t e r a t u r e on i o d i n a t i o n r e a c t i o n s a r e b a s e d on s i n g l e - s t r a n d e d p o l y n u c l e o t i d e s as t h e p r e f e r e n t i a l s u b s t r a t e f o r i o d i n a t i o n ( P r e n s k y , 1 9 7 6 ) , w h i c h i s a m a j o r a d v a n t a g e o f t h e t e c h n i q u e b e c a u s e more t h a n 90% o f a l l p l a n t v i r u s e s h a v e a s i n g l e - s t r a n d e d ( s s ) n u c l e i c a c i d genome ( M a t t h e w s , 1 9 8 1 ) . C o m m e r f o r d ( 1 9 7 1 ) , f o u n d t h a t s i n g l e - s t r a n d e d n u c l e i c a c i d c o u l d be i o d i n a t e d t o a r e l a t i v e l y h i g h l e v e l ; h o w e v e r , t h e p h y s i o c h e m i c a l p r o p e r t i e s ( i e . r e n a t u r a t i o n and m e l t i n g p r o f i l e ) o f ssDNA were n o t s i g n i f i c a n t l y a l t e r e d when 10% o f t h e c y t i d i n e r e s i d u e s were i o d i n a t e d . D u p l e x e s f o r m e d b e t w e e n h a l o g e n a t e d p o l y n u c l e o t i d e s and p o l y - i n o s i n e were more s t a b l e t h a n t h e h y b r i d f o r m e d by p o l y - i n o s i n e and p o l y - c y t i d i n e ( M i c h e l s o n and Monny, 1 9 6 6 ) . I o d o c y t i d i n e i s i n c o r p o r a t e d i n t o t h e genome o f ' c e l l ' s i n v i v o (Dunn and S m i t h , 1 9 5 7 ) , and i s an e f f e c t i v e s u b s t r a t e f o r DNA p o l y m e r a s e i n v i t r o ( S c h e r b e r g and R e k e t o f f , 1 9 7 4 ) . A v a r i e t y of- p r o c e d u r e s has b een p u b l i s h e d f o r t h e i o d i n a t i o n o f n u c l e i c a c i d s ( P r e n s k y , 1 9 7 6 ) ; h o w e v e r , o n l y C o m m e r f o r d ( 1 9 7 1 ) has d e s c r i b e d a p r o c e d u r e w h i c h c o m b i n e s s p e c i f i c i t y and r e a s o n a b l e y i e l d w i t h an a c c e p t a b l e s i m p l i c i t y o f e x e c u t i o n . L i p k i n e t a l . ( 1 9 8 3 ) , f o u n d t h a t c y t i d i n e c o u l d be i o d i n a t e d w i t h i o d i n e m o n o c h l o r i d e , t o a y i e l d o f a b o u t 85 p e r c e n t . T h e s e e x p e r i m e n t s w e re r e p e a t e d u s i n g n u c l e i c a c i d as t h e s u b s t r a t e f o r i o d i n a t i o n , and i t was f o u n d t h a t u r i d i n e and g u a n o s i n e w ere i o d i n a t e d w i t h g r e a t e r s p e c i f i c i t y t h a n c y t i d i n e ( A s c o l i and K a h a n , 1 9 6 6 ) . C y t i d i n e was a l s o i o d i n a t e d i n a r e a c t i o n w i t h N - i o d o s u c c i n i m i d e , b u t w i t h a low e f f i c i e n c y 15 (Brammer, 1 9 6 3 ) . M i c h e l s o n and Monny ( 1 9 6 6 ) , f o u n d t h a t c y t i d i n e m o n o - p h o s p h a t e c o u l d be i o d i n a t e d w i t h i o d i n e t r i c h l o r i d e ; h o w e v e r , t h e r e a c t i o n does n o t work when t h e n u c l e o t i d e i s i n a p o l y n u c l e o t i d e . C o m m e r f o r d ' s r e a c t i o n has been shown t o be v e r y s p e c i f i c f o r c y t i d i n e , h a v i n g a b o u t a f i f t y - f o l d l o w e r s p e c i f i c i t y f o r u r a c i l , and no a p p r e c i a b l e r e a c t i o n w i t h any o t h e r n u c l e o s i d e . The r e a c t i o n r e p l a c e s t h e h y d r o g e n a t t h e C-5 p o s i t i o n o f t h e p y r i m i d i n e r i n g w i t h i o d i n e , s i m i l i a r t o t h a t d e s c r i b e d f o r p y r i m i d i n e b r o m i n a t i o n (Brammer, 1 9 6 3 ) . Thus t h e m o d i f i c a t i o n s h o u l d n o t i n t e r f e r e i n t h e h y d r o g e n b o n d i n g o b s e r v e d i n n u c l e i c a c i d h y b r i d s ( W a t s o n and C r i c k , 1 9 5 3 ) . The s i t e o f s u b s t i t u t i o n was v e r i f i e d when p o l y m e r s o f p y r i m i d i n e m o d i f i e d a t C-5 w e r e u s e d as t h e n u c l e i c a c i d s u b s t r a t e and no i o d i n a t i o n was d e t e c t e d ( C o m m e r f o r d , 1 9 7 1 ) . A l t h o u g h , t h e r e a c t i o n has n o t b e e n s c a l e d -up, t h e r e i s no o b v i o u s r e a s o n why i t c o u l d n o t be. F u r t h e r m o r e , t h e r e a c t i o n has b e e n u s e d s u c c e s s f u l l y w i t h RNA as t h e s u b s t r a t e ( P r e n s k y e t a l . , 1973) The p o s t u l a t e d m e c h a n i s m o f t h e i o d i n a t i o n r e a c t i o n r e q u i r e s T I 3 4 as a c a t a l y s t t o i n i t i a t e t h e f o r m a t i o n o f i o d i n e h y d r o x i d e w h i c h , when r e a c t e d w i t h c y t i d i n e , r e s u l t s i n t h e c o v a l e n t a t t a c h m e n t o f i o d i n e t o f o r m t h e u n s t a b l e i n t e r m e d i a t e 5 - i o d o - 6 -h y d r o x y d i h y d r o c y t i d i n e , w h i c h i s t h e n d e h y d r a t e d t o f o r m t h e s t a b l e p r o d u c t , 5 - i o d o c y t i d i n e . U r a c i l i s a l s o c o n v e r t e d t o t h e u n s t a b l e i n t e r m e d i a t e 5 - i o d o - 6 - h y d r o x y d i h y d r o u r i d i n e , b u t t h i s i s n o t r e a d i l y d e h y d r a t e d t o f o r m a s t a b l e p r o d u c t . G e t z e t a l . ( 1 9 7 2 ) , f o u n d t h a t s e c o n d a r y h e a t i n g a t a h i g h e r pH (pH 8) was 16 n e c e s s a r y t o remove any i o d i n e t h a t was n o t c o v a l e n t l y b o u n d . They t r a c e d t h e r e a c t i o n w i t h 1125 , and f o u n d t h a t a h i g h p r o p o r t i o n o f t h e l a b e l s e p a r a t e d f r o m t h e p r o d u c t a f t e r t h e s e c o n d h e a t i n g . T h i s i s p o s t u l a t e d t o be a r e s u l t o f d e i o d i n a t i o n o f t h e r e m a i n i n g u n s t a b l e 5 - i o d o - 6 - h y d r o x y d i h y d r p y r i m i d i n e s . The s e c o n d h e a t i n g a l s o i n c r e a s e s t h e p e r c e n t a g e o f 5 - i o d o c y t i d i n e f o r m e d ( C o m m e r f o r d , 1 9 7 1 ) ; t h e r e f o r e , t h e e q u i l i b r i u m b e t w e e n t h e f o r m a t i o n o f 5 - i o d o p y r i m i d i n e and t h e n a t i v e p y r i m i d i n e must f a v o r t h e f o r m e r f o r t h e 5 - i o d o - 6 - h y d r o x y d i h y d r o c y t i d i n e , and t h e l a t t e r f o r t h e u n s t a b l e u r i d i n e ( o r o t h e r n u c l e o s i d e ) d e r i v a t i v e s ( O r o s z and Wetmur, 1 9 7 4 ) . F o r t h e f i r s t h e a t i n g t h e pH s h o u l d be k e p t a t o r n e a r t h e pKa o f t h e a c e t a t e i o n . pH 4.6; h o w e v e r , t h e a c t u a l pH c a n be i n c r e a s e d t o 5.0 t o a v o i d e x c e s s i v e d e p u r i n a t i o n a n d d e a m i n a t i o n o f t h e n u c l e o t i d e s ( P r e n s k y e t a l . , 1 9 7 3 ) . C o m m e r f o r d ( 1 9 7 1 ) , a l s o a d d e d s u l f i t e a f t e r t h e f i r s t h e a t i n g t o remove e x c e s s t h a l l i u m and i o d i n e i o n s , b u t O r o s z and Wetmur ( 1 9 7 4 ) , showed t h a t t h i s r e s u l t e d i n t h e d e a m i n a t i o n o f c y t i d i n e , a n d h y d r o l y s i s o f t h e N - g l y c o s i d i c l i n k a g e . S c h e r b e r g and R e k e t o f f ( 1 9 7 4 ) a l s o f o u n d t h a t t h e e x c e s s i o n s h a d no d e t r i m e n t a l e f f e c t . 17 M a t e r i a l s and Methods C o n j u g a t i o n o f H a p t e n W i t h P r o t e i n C a r r i e r I n i t i a l l y t h e p r o c e d u r e o u t l i n e d by ( E r l a n g e r and B e i s e r , 1964) was u s e d . L a t e r some o f t h e m o d i f i c a t i o n s o f D ' A l i s a and E r l a n g e r ( 1 9 7 4 ) were a d o p t e d r e s u l t i n g i n a m o d i f i e d p r o c e d u r e w h i c h y i e l d e d t h e g r e a t e s t number o f h a p t e n m o l e c u l e s p e r m o l e o f c a r r i e r . The c a r r i e r s u s e d were human serum a l b u m i n ( H S A ) , b o v i n e s e r u m a l b u m i n ( B S A ) , o r o v a l b u m i n ( S i g m a C h e m i c a l C o . ) . Two and one h a l f mg o f n u c l e o s i d e and 2.7 mg o f s o d i u m p e r i o d a t e w e r e d i s s o l v e d i n 330 u l o f d i s t i l l e d w a t e r ( d H 2 0 X and t h e s o l u t i o n was l e f t a t room t e m p e r a t u r e f o r t h i r t y m i n u t e s . To t h i s was a d d e d 20 u l o f 1M e t h y l e n e g l y c o l and t h e r e a c t i o n was f u r t h e r i n c u b a t e d f o r f i v e m i n u t e s . Then 6.7 mg o f c a r r i e r , d i s s o l v e d i n 660 u l o f d H 2 0 was added and t h e pH was a d j u s t e d t o 9.5 w i t h 5% p o t a s s i u m c a r b o n a t e . The s o l u t i o n was a g i t a t e d a t room t e m p e r a t u r e f o r one h o u r and t h e pH was m a i n t a i n e d b e t w e e n 9-10. The r e a c t i o n m i x t u r e was t h e n c h i l l e d on i c e and d r o p s o f a s o l u t i o n ( 3 . 4 mg s o d i u m b o r o h y d r i d e d i s s o l v e d i n 660 u l o f dH' 20) w e r e a d d e d t o i t o v e r a p e r i o d o f f i v e m i n u t e s . The s o l u t i o n was t h e n l e f t a t 4 C f o r one h o u r , a f t e r w h i c h i t was d i a l y z e d a g a i n s t PBS (137 mM N a C l , 1.5 mM K H 2 P 0 4 , 8 mM N a H P 0 4 . 7 H 2 0 , 3 mM KC1, pH 8.0) f o r t h i r t y - s i x h o u r s w i t h t h r e e c h a n g e s o f b u f f e r . The c o n j u g a t e was t h e n r e c o v e r e d f r o m t h e d i a l y s i s t u b i n g a n d f r e e z e - d r i e d . R e a c t i o n s w e re m o n i t o r e d by u l t r a - v i o l e t (UV) s p e c t r o s c o p y , a n d t h i n - l a y e r c h r o m a t o g r a p h y ( T L C ) . The TLC was done a c c o r d i n g t o T o m a s h e f s k i e t a l . ( 1 9 7 4 ) . B o t h s i l i c a and c e l l u l o s e p l a t e s , w i t h f l u o r e s c e n t i n d i c a t o r i n c o r p o r a t e d i n t o t h e m a t r i x , w e r e 18 spotted with sample one cm from the base and chromatographed in a 60:10:20:10 mixture of a c e t o n i t r i l e : n - b u t a n o l : 0 . 1 M ammonium acetate:28% ammonium hydroxide in a closed container u n t i l the solvent front had moved to within one cm from the top of the p l a t e . Location of conjugates was determined by examination of the chromatogram under short-wavelength (254 nm) UV l i g h t . Success of conjugations was further confirmed by sodium dodecyl sul fa te -polyacrylamide gel electrophoresis (SDS-PAGE) using the procedure of Lammeli (1970). B r i e f l y , 8% SDS-PAG were cast and electrophoresed at 120 V, at 4 C for three hours. A l l samples p r i o r to electrophoresis were mixed 1:1 with reduct ion mix (0.5M T r i s - H C l pH 6.8, 10% SDS, 10% B-mercaptoethanol, 20% g l y c e r o l , 0.001% Bromophenol Blue) and heated at 80 C for twenty minutes. Gels (15 cm x 10 cm x 0.75 cm) were stained e i t h e r by Coomassie B r i l l i a n t Blue or s i l v e r s t a i n i n g . For Coomassie s t a i n i n g , gels were soaked overnight in a s o l u t i o n of 45% methanol, 7% ace t i c a c i d , 0.1% Coomassie B r i l l i a n t Blue G-250, and destained in the same solut ion excluding s t a i n . S i l v e r s t a i n i n g was e s s e n t i a l l y by the procedure of M e r r i l l et a l . (1981). A l l steps were done at room temperature with constant a g i t a t i o n . Gels were soaked in 50% methanol, 7% acet ic ac id for t h i r t y minutes to f i x the proteins in the gels . The gels were then washed three times with 200 mis of 10% ethanol, 5% a c e t i c a c i d over t h i r t y minutes. They were soaked in 200 mis of 0.0034 M potassium dichromate for f i v e minutes, followed by a thorough r i n s i n g with two l i t r e s of water over a period of f i v e minutes. The gels were then incubated in 200 ml of 0.012 M s i l v e r n i t r a t e 19 f o r t h i r t y m i n u t e s , and developed i n 0.28 M sodium c a r b o n a t e c o n t a i n i n g 0.5 ml formaldehyde s o l u t i o n / 1 i t r e . One l i t r e o f t h e l a t t e r was added, w i t h c o n s t a n t change o f s o l u t i o n , u n t i l bands i n t he g e l s t a r t e d t o appear. The r e a c t i o n was quenched w i t h 20% a c e t i c a c i d and the g e l s were s t o r e d i n 45% methanol, IX a c e t i c a c i d p r i o r t o d r y i n g on heavy f i l t e r paper. P r o t e i n q u a n t i t a t i o n was done u s i n g the "Bio-Rad P r o t e i n Assay K i t " u s i n g the procedure o u t l i n e d by B r a d f o r d (1976), w h i c h measures the c o l o r change t h a t o c c u r s w i t h Coomassie B r i l l i a n t B l u e G-250 when i t r e a c t s w i t h p r o t e i n . The s p e c i f i c c o n d i t i o n s used were as d e s c r i b e d i n the "Bio-Rad" u s e r ' s g u i d e . I o d i n a t i o n o f P o l y c y t i d i n e and ssRNA The p o l y - c y t i d i n e ( p o l y C ; Sigma Chemical Co.) and random RNA (Ph a r m a c i a P-L B i o c h e m i c a l s ) used i n the r e a c t i o n s were p u r c h a s e d from a commercial s o u r c e and f u r t h e r e x t r a c t e d w i t h d i s t i l l e d phenol and c h l o r o f o r m : a m y l a l c o h o l ( 2 4 : 1 ) . The p o l y C and RNA were p r e c i p i t a t e d from the aqueous phase w i t h o n e - t e n t h volume o f 3M sodium a c e t a t e p H 5.0, and t h r e e volumes o f e t h a n o l at -70 C o v e r n i g h t . V i r a l RNAs were e x t r a c t e d from p u r i f i e d p r e p a r a t i o n s o f s o u t h e r n bean mosaic v i r u s (SBMV) and cowpea c h l o r o t i c m o t t l e v i r u s ( p r o v i d e d by R. R. M a r t i n , A g r i c u l t u r e Canada R e s e a r c h S t a t i o n , Vancouver) by a m o d i f i c a t i o n o f the method o f Zimmern (1 9 7 5 ) . B r i e f l y , one mg o f v i r u s was i n c u b a t e d i n 200 u l o f d i s s o c i a t i o n b u f f e r (100 mM NaCl, 10 mM T r i s - H C l , 3 mM EDTA, 2% SDS pH 7.8) at 55 C f o r f o r t y - f i v e m i n u t e s . Then p r o t e i n a s e K, wh i c h had been a u t o - d i g e s t e d at 37 C f o r f o r t y - f i v e m i n u t e s , was 20 i a d d e d a t 0.5 mg/ml, and i n c u b a t e d a t 37 C f o r a f u r t h e r f o r t y -f i v e m i n u t e s . The m i x t u r e was t h e n e x t r a c t e d w i t h an e q u a l v o l u m e o f p h e n o l , t h e aqueous l a y e r r e m o v e d and t h e o r g a n i c l a y e r r e -e x t r a c t e d t w i c e w i t h a o n e - t e n t h v o l u m e o f 10 mM T r i s - H C l pH 7.8. The a q u e o u s l a y e r s were t h e n p o o l e d and e x t r a c t e d t h r e e t i m e s w i t h c h l o r o f o r m : a m y l a l c o h o l and t h e RNA was p r e c i p i t a t e d . A l l RNA s a m p l e s were e l e c t r o p h o r e s e d i n u r e a - a c r y l a m i d e g e l s e s s e n t i a l l y as d e s c r i b e d by M a n i a t i s e t a l . ( 1 9 8 2 ) . S a m p l e s w e r e e l e c t r o p h o r e s e d i n t h e p r e s e n c e o f 8M u r e a i n TBE (180 mM T r i s , 100 mM b o r i c a c i d - s o d i u m b o r a t e , 10 mM EDTA pH 8 . 0 ) . P r i o r t o l o a d i n g , s a m p l e s were d i s s o l v e d i n d e n a t u r i n g s o l u t i o n (TBE, 8 0 % ' f o r m a m i d e , 0.1% x y l e n e c y a n o l e , 0.1% B r o m o p h e n o l B l u e ) a n d , h e a t e d a t 55 C f o r t w e n t y m i n u t e s . A f t e r e l e c t r o p h o r e s i s , s a m p l e s w e r e s t a i n e d i n r u n n i n g b u f f e r (TBE) w i t h e t h i d i u m b r o m i d e (2 u g / m l ) , and v i s u a l i z e d u n d e r UV l i g h t . The i o d i n a t i o n s were c a r r i e d o u t a c c o r d i n g t o C o m m e r f o r d ( 1 9 7 1 ) and P r e n s k y ( 1 9 7 6 ) , w i t h some m o d i f i c a t i o n s . Two s e p a r a t e r e a c t i o n s w e r e e v e n t u a l l y o p t i m i z e d f o r s m a l l ( 50 u g ) , and l a r g e ( 3 mg) s a m p l e s . W a t e r u s e d i n t h e r e a c t i o n s was d i s t i l l e d , a n d f i l t e r e d t h r o u g h c h a r c o a l and i o n - e x c h a n g e c o l u m n s . A l l g l a s s w a r e was t h o r o u g h l y c l e a n e d and s t e r i l i z e d a l o n g w i t h p l a s t i c w a r e , a n d b u f f e r s . T h a l l i u m n i t r a t e ( T 1 ( N 0 3 ) 3 ) , t h e s o u r c e o f t h a l l i u m i o n w h i c h i s *an e s s e n t i a l component o f t h e i o d i n a t i o n r e a c t i o n , was k e p t as a 0.1 M s t o c k s o l u t i o n i n 0.5 M n i t r i c a c i d a t 4 C and d i l u t e d p r i o r t o use t o 0.015M w i t h 1M s o d i u m a c e t a t e - a c e t i c a c i d b u f f e r pH 5.0. R e a c t a n t s were a s s e m b l e d a t 4 C, w i t h t h e t h a l l i u m a l w a y s a d d e d l a s t . The s m a l l s c a l e r e a c t i o n c o n s i s t e d o f : 1.5 uM 21 Nal, 0.15 uM c y t i d i n e or 0.44 mg/ml n u c l e i c a c i d , and 5 uM T 1 ( N 0 3 ) 3 i n a volume of 225 u l . The pH was monitored with pH paper to ensure that i t was between 4.5-5.0. The s o l u t i o n was incu b a t e d at 60 C f o r one hour, quenched on i c e , and the pH a d j u s t e d to 8.0 with 1 M T r i s - H C l pH 8.0, and rei n c u b a t e d at 60 C f o r f i f t e e n minutes. The r e a c t i o n was stopped on i c e , and the r e a c t i o n products were concentrated by ethanol p r e c i p i t a t i o n , f o l l o w e d by d e s a l t i n g over Sephadex G-100. The l a r g e s c a l e r e a c t i o n , f o r n u c l e i c a c i d s only, was done i n a two ml volume, and c o n s i s t e d of: 6.5 uM Nal, 1.0 mg/ml n u c l e i c a c i d , and 13 uM T 1 ( N 0 3 ) 3 . The r e s t of the parameters were as above; except, the n u c l e i c a c i d was always concentrated by ethanol p r e c i p i t a t i o n p r i o r to p u r i f i c a t i o n . N u c l e o s i d e s , were p u r i f i e d on a s i l i c a column (3 gm dry weight, 0.75 cm diameter column, 100-200 mesh) and e l u t e d with the b u f f e r p r e v i o u s l y d e s c r i b e d f o r TLC. One m i l l i l i t e r f r a c t i o n s were c o l l e c t e d and analyzed by UV spectroscopy and TLC. N u c l e i c a c i d s were d e - s a l t e d on Sephadex G-100, the column volume always b e i n g at l e a s t ten times the sample s i z e i n a one ce n t i m e t e r diameter column. The column was e l u t e d with 10 mM T r i s - H C l , 1 mM EDTA pH 7.8, at 1 ml/minute and the eluant c o l l e c t e d i n one m i l l i l i t r e f r a c t i o n s . The r e a c t i o n was then analyzed by UV sp e c t r o s c o p y and X-ray f l u o r e s c e n c e (XRF). The l a t t e r technique r e q u i r e s bombardment of the sample with p o l y c h r o m a t i c X-rays, causing e x c i t a t i o n of e l e c t r o n s i n outer o r b i t a l s . The energy of decay or f l u o r e s c e n c e of these e x c i t e d e l e c t r o n s i s then measured, and used to determine the i d e n t i t y of the element (Woldseth, 1973). XRF was done with a "T r a c o r 22 N o r t h e r n N S 8 8 0 " X - r a y a n a l y s i s s y s t e m ; t h e X - r a y s o u r c e was a " H i t a c h i S - 5 0 0 " s c a n n i n g e l e c t r o n m i c r o s c o p e . I o d i n a t i o n o f d s - R N A D o u b l e - s t r a n d e d - R N A was p u r i f i e d b y a m o d i f i c a t i o n o f t h e m e t h o d o f M o r r i s a n d Dodds ( 1 9 7 9 ) . T w o - w e e k o l d C h i n e s e c a b b a g e s e e d l i n g s w e r e i n o c u l a t e d w i t h t u r n i p y e l l o w m o s a i c v i r u s (TYMV) a n d l e a f t i s s u e was h a r v e s t e d t h r e e t o f o u r w e e k s l a t e r . T i s s u e ( 2 5 - 4 0 g) was g r o u n d t o a f i n e p o w d e r i n l i q u i d n i t r o g e n a n d a l l o w e d t o p a r t i a l l y t h a w . T h e n 1 . 5 v o l u m e s o f 2x STE ( 0 . 1 M N a C l , 0 . 0 5 M T r i s - H C l , 0 . 0 0 1 M N a 2 E D T A pH 7 . 0 ) ( w / v ) , 1 / 1 0 v o l u m e o f 10% S D S , a n d 1 . 5 v o l u m e s o f STE s a t u r a t e d p h e n o l ( w / v ) w e r e a d d e d a n d t h e m i x t u r e was a g i t a t e d f o r 30 m i n u t e s a t r o o m t e m p e r a t u r e . A f t e r c e n t r i f u g a t i o n a t 10K rpm ( S o r v a l l , GSA r o t o r ) f o r one h o u r , t h e a q u e o u s l a y e r was r e m o v e d , a d j u s t e d t o 17% e t h a n o l , a n d a l l o w e d t o f l o w t h r o u g h a c o l u m n o f C F - 1 1 ( W h a t m a n ; 2 . 5 gm d r y w e i g h t i n a 1 cm d i a m e t e r c o l u m n , p r e - e q u i l i b r a t e d w i t h S T E / 1 7 % e t h a n o l ) . The c o l u m n was w a s h e d w i t h 100 m l o f S T E / 1 7 % e t h a n o l , p u r g e d t o r e m o v e m o s t o f t h e p r e v i o u s s o l v e n t , a n d d s - R N A was e l u t e d w i t h 3 , 3 , a n d 5 m l w a s h e s o f S T E . The f i r s t w a s h was d i s c a r d e d , t h e l a t t e r t w o c o m b i n e d , a n d t r e a t e d w i t h 10 u n i t s / m l o f RNase T I ( S i g m a C h e m i c a l C o . ) f o r f o r t y - f i v e "'So m i n u t e s a t 37 C . Then 0 . 0 3 v o l u m e s o f 1M M g C l 2 , a n d 0 . 5 u n i t s / m l o f D N a s e 1 ( P h a r m a c i a P - L B i o c h e m i c a I s ) w e r e a d d e d a n d i n c u b a t e d a s a b o v e . An a m o u n t o f EDTA ( p H 8) e q u i m o l a r t o M g C l 2 was t h e n a d d e d a n d t h e s o l u t i o n was a d j u s t e d t o 17% e t h a n o l . To t h i s was a d d e d 0 . 5 g o f C e l l e x - N l ( B i o R a d ) n o n - i o n i c c e l l u l o s e p o w d e r , t h e 23 m i x t u r e was a g i t a t e d f o r t h i r t y m i n u t e s , a p p l i e d t o a c o l u m n and w a s h e d w i t h 500 ml o f STE/17% e t h a n o l . The c o l u m n was p u r g e d t o remove most o f t h e p r e v i o u s s o l v e n t and t h e ds-RNA was e l u t e d i n a s m a l l v o l u m e o f STE, and p r e c i p i t a t e d w i t h e t h a n o l . The ds-RNA was t h e n q u a n t i t a t e d by UV s p e c t r o s c o p y , and 25 ug was i o d i n a t e d by t h e s m a l l s c a l e i o d i n a t i o n p r o c e d u r e . The s a m p l e was t h e n d e - s a l t e d i n an " A m i c o n " m i c r o c o n c e n t r a t o r w i t h TE (10 mM T r i s - H C l , 1 mM EDTA pH 7.8) a c c o r d i n g t o t h e m a n u f a c t u r e r ' s i n s t r u c t i o n s . The c o n c e n t r a t e d s a m p l e was t h e n t r e a t e d w i t h 20 u n i t s o f RNase TI a t 37 C f o r one h o u r and t h e v o l u m e was a d j u s t e d t o 200 u l w i t h STE. E t h a n o l was a d d e d t o 17% and t h e s a m p l e was p a s s e d t h r o u g h a 100 mg p l u g o f C e l l e x - N l , and w a s h e d w i t h 5 ml o f STE/17% e t h a n o l . The ds-RNA was e l u t e d w i t h a s m a l l v o l u m e o f STE and e t h a n o 1 - p r e c i p i t a t e d . A p p r o x i m a t e l y 20 ug o f ds-RNA, as d e t e r m i n e d by UV s p e c t r o s c o p y , was r e c o v e r e d and t h i s was a n a l y z e d by XRF. P 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 y T h r e e - t o f o u r - m o n t h o l d BALB/c m i c e were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 50 ug o f c o n j u g a t e h o m o g e n i z e d w i t h an e q u a l v o l u m e o f F r e u n d ' s i n c o m p l e t e a d j u v a n t . T h i s was r e p e a t e d t h r e e t i m e s a t two week i n t e r v a l s . Two weeks a f t e r t h e l a s t i n j e c t i o n , p o l y c l o n a l s e r a w e re o b t a i n e d by t h e t a i l - b l e e d i n g m e t h o d , and t i t r e d by e n z y m e - l i n k e d i m m u n o s o r b e n t a s s a y ( E L I S A ) . I f t h e t i t r e was g r e a t e r t h a n 1/10,000, t h e mouse was g i v e n a b o o s t e r i n t r a p e r i t o n e a l i n j e c t i o n w i t h 50 ug o f c o n j u g a t e t h r e e d a y s p r i o r t o c e l l f u s i o n . The E L I S A p r o c e d u r e u s e d i s b a s e d on t h o s e o u t l i n e d by C l a r k 24 and Adams (1977) and DeBoer and Wieczorek (1984). "Immulon-2" 96 w e l l ELISA p l a t e s (Dynatech L a b o r a t o r i e s L t d . ) were c o a t e d w i t h c o n j u g a t e at 1 ug/ml i n PBS and i n c u b a t e d at 37 C f o r two hours or at 4 C o v e r n i g h t . P l a t e s were then washed w i t h tap wa t e r , as the y were between each o f the f o l l o w i n g s t e p s : p l a t e s were then c o a t e d w i t h PBS-Tween (PBS, 0.053; Tween-20) c o n t a i n i n g 1% ovalbumin at 37 C f o r one hour; a n t i s e r a were a p p l i e d at the d e s i r e d c o n c e n t r a t i o n i n PBS-Tween, and i n c u b a t e d at 37 C f o r two hours; s e c o n d a r y a n t i b o d y , p e r o x i d a s e - c o n j u g a t e d goat anti-mouse ( d i l u t e d t o r e s u l t i n an O.D. r e a d i n g o f 1.500-2.000 under o p t i m a l c o n d i t i o n s ) , was added i n PBS-Tween and i n c u b a t e d at 37 C f o r two hours; and s u b s t r a t e (4mg/ml o f o-p h e n y l e n e d i a m i n e and 4 u l / 1 0 ml o f a 30% s o l u t i o n of hydrogen p e r o x i d e ) was added i n 0.1 M c i t r a t e - p h o s p h a t e b u f f e r pH 5.0. C o l o r u s u a l l y d e v e l o p e d i n f i f t e e n minutes at which time the r e a c t i o n was s t o p p e d by a d d i t i o n o f 2.5 M s u l p h u r i c a c i d . S p e c t r o p h o t o m e t r i c r e a d i n g o f the p l a t e s was done i n a " T i t e r t e k " ELISA p l a t e r e a d e r at 492 nm. A l l s o l u t i o n s were added i n volumes o f 100 u l / w e l l , e x cept the b l o c k i n g s o l u t i o n and the s u l p h u r i c a c i d , which were added at 200 and 20 u l / w e l l , r e s p e c t i v e l y . M o n o c l o n a l a n t i b o d i e s (MCAs), were produced a c c o r d i n g t o the methods o u t l i n e d by Goding (1980) and Oi and He r z e n b e r g (1980) w i t h m o d i f i c a t i o n s . A l l m a n i p u l a t i o n s were done a s e p t i c a l l y i n a b i o - h a z a r d l a m i n a r - f l o w hood. C e l l s were c u l t u r e d i n supplemented D u l b e c c o ' s M o d i f i e d E a g l e s Medium (DMEM, supplemented w i t h 40 mM sodium b i c a r b o n a t e , 2 mM L - g l u t a m i n e , 1 mM p y r u v a t e , 50 mg/1 25 g e n t a m y c i n s u l f a t e ) c o n t a i n i n g 1 0 - 2 0 % h e a t - t r e a t e d (60 C, 30 m i n ) f e t a l c a l f s e r u m ( F C S ) , a t 37 C i n a 10% C02 e n v i r o n m e n t . S o l u t i o n s u s e d i n t h e f u s i o n p r o t o c o l were k e p t a t 37 C. C e l l f u s i o n was c a r r i e d o u t w i t h c u l t u r e d NS-1 myeloma c e l l s h a r v e s t e d a t a l o g p h a s e o f g r o w t h . The i m m u n i z e d mouse was s a c r i f i c e d and a s p l e n e c t o m y was done. The s p l e e n was m a c e r a t e d i n 10 ml o f DMEM and t h e c e l l s were c o l l e c t e d u s i n g an 18-guage n e e d l e . The NS-1 and s p l e e n c e l l s were t h e n c e n t r i f u g e d a t 800 g f o r t e n m i n u t e s , t h e s u p e r n a t a n t removed, and t h e c e l l s r e s u s p e n d e d i n DMEM. T h i s was r e p e a t e d t w i c e and, a f t e r t h e s e c o n d w a s h i n g , t h e c e l l s w e r e c o m b i n e d i n a c o n i c a l c e n t r i f u g e t u b e , and p e l l e t e d . The s u p e r n a t a n t f l u i d was t h e n removed, and t h e s l u r r y o f c e l l s was b r i e f l y a g i t a t e d i n a t u b e m i x e r ( V o r t e x ) . Ten ml o f f r e s h l y a u t o c l a v e d p o l y e t h y l e n e g l y c o l (PEG 1 5 0 0 ) , d i l u t e d t o 3 5 % w i t h DME, was a d d e d s l o w l y t o t h e s l u r r y o v e r one m i n u t e . The m i x t u r e was c e n t r i f u g e d a t 800 g f o r t h r e e m i n u t e s , and a l l o w e d t o s t a n d u n d i s t u r b e d f o r f o u r m i n u t e s . The s u p e r n a t a n t f l u i d was t h e n r e m o v e d , and 10 ml o f DMEM added t o t h e p e l l e t e d c e l l s i n one s u d d e n b u r s t i n o r d e r t o d i s r u p t t h e c e l l s , b u t t h e c e l l s w e r e n o t r e s u s p e n d e d by any f u r t h e r p i p e t t i n g a c t i o n . The m i x t u r e was t h e n c e n t r i f u g e d as d e s c r i b e d a b o v e . The r e s u l t i n g p e l l e t was r e s u s p e n d e d , u s i n g a t u b e m i x e r , i n 50 ml HAT medium ( 8 0 % DMEM, 2 0 % FCS, 1.6 mM t h y m i d i n e , 10 mM h y p o x a n t h i n e , 0.04 mM a m i n o p t e r i n , and 5 mM 4 - ( 2 - h y d r o x y e t h y 1 ) - 1 -p i p e r a z i n e e t h a n e s u l f o n i c a c i d ( H E P E S ) , pH 7 . 0 ) , f o r t i f i e d w i t h t h y m o c y t e s o b t a i n e d f r o m two f o u r - t o s i x - w e e k o l d m i c e , a n d was h e d as d e s c r i b e d f o r t h e s p l e e n c e l l s . A l i q u o t s ( 1 00 u l ) o f t h e s u s p e n s i o n were t h e n d i s p e r s e d i n t o f i v e , p l a t e s a n d i n c u b a t e d a t 37 C. The a p p r o x i m a t e c e l l s t o myeloma c e l l s a t f u s i o n was 4:1. T w e n t y - f o u r h o u r s l a t e r , 100 u l o f HAT a d d e d . Ten t o f o u r t e e n d a y s p o s t - f u s i o n , t h e medium was remove d f r o m t h o s e w e l l s c o n t a i n i n g h y b r i d o m a s , and r e p l a c e d w i t h 200 u l / w e l l o f HT (DMEM, 2 0 % FCS, 1.6 mM t h y m i d i n e , and 10 mM h y p o x a n t h i n e ) . The p l a t e s w e r e t h e n r e - i n c u b a t e d f o r a p p r o x i m a t e l y two d a y s , a f t e r w h i c h t h e c u l t u r e s u p e r n a t a n t f l u i d s w e r e a s s a y e d f o r a n t i - i o d o c y t i d i n e a c t i v i t y b y E L I S A . H y b r i d o m a s , t h e c u l t u r e f l u i d s o f w h i c h were p o s i t i v e f o r a n t i -i o d o c y t i d i n e a c t i v i t y , were r e c l o n e d by s e r i a l d i l u t i o n i n DMEM c o n t a i n i n g 20% FCS and s u p p l e m e n t e d w i t h t h y m o c y t e s f r o m one mouse d i s t r i b u t e d among two 9 6 - w e l l c u l t u r e p l a t e s . C l o n e s w h i c h s t i l l t e s t e d p o s i t i v e were p r e s e r v e d i n DMEM, 20% FCS, 10% d i m e t h y l s u l f o x i d e , a t -70 C f o r t w e n t y - f o u r h o u r s , t h e n t r a n s f e r e d t o l i q u i d n i t r o g e n f o r l o n g - t e r r a s t o r a g e . A s c i t i c f l u i d , w h i c h c o n t a i n s l a r g e q u a n t i t i e s o f a n t i b o d y , was p r o d u c e d i n m i c e , a t l e a s t f o u r - m o n t h s o l d , w h i c h h a d be e n p r i m e d w i t h e i t h e r 0.5 ml o f p r i s t a n e o r 0.01% t r y p a n b l u e s i x t o t w e l v e d a y s o r t h r e e d a y s p r i o r , r e s p e c t i v e l y b e f o r e i n j e c t i o n o f h y b r i d o m a s . A p p r o x i m a t e l y 10 h y b r i d o m a c e l l l i n e s w e r e i n j e c t e d i n t r a p e r i t o n e a l 1 y , and f l u i d was h a r v e s t e d one t o two weeks l a t e r . MCAs were p u r i f i e d f r o m e i t h e r t h e a s c i t i c f l u i d o r f r o m c u l t u r e s u p e r n a t a n t f l u i d s , i n w h i c h t h e h y b r i d o m a s h a d b e e n grown t o n e a r c e l l d e a t h . A n t i b o d i e s were p r e c i p i t a t e d o u t o f c u l t u r e o r a s c i t e s f l u i d s by a d d i t i o n o f an e q u a l v o l u m e o f s a t u r a t e d ammonium s u l f a t e , ( J a c o b and T r o n , 1 9 8 2 ) , a t 4 C f o r a 2b 9 6 - w e l l c u l t u r e i r a t i o o f s p l e e n m e d i u m / w e l l was 27 minimum o f one h o u r . The p r e c i p i t a t e was p e l l e t e d , r e s u s p e n d e d i n a s m a l l v o l u m e o f PBS, and d i a l y z e d a g a i n s t PBS o v e r n i g h t . MCA w e r e t h e n a s s a y e d by E L I S A t o e n s u r e t h a t t h e y w e r e s t i l l a c t i v e . The m e t h o d o f f u r t h e r p u r i f i c a t i o n was t h e n d e t e r m i n e d b y t h e i s o t y p e o f t h e MCA. I s o t y p i n g was done by m i c r o - p r e c i p i t a t i o n o r g e l d i f f u s i o n ( B e n a c e r r a f and Unanue, 1979) w i t h a c o m m e r c i a l k i t ( B i o n e t i c s L a b s . ) , t h a t c o n t a i n e d r a b b i t a n t i - m o u s e i m m u n o g l o b u l i n s IgM, I g A , I g G l , I g G 2 a , IgG2b, and I g G 3 . F o r t h e m i c r o - p r e c i p i t i n a s s a y , i m m u n o g l o b u l i n (20 ug i n 20 u l ) was m i x e d w i t h 20 u l o f t h e d e s i r e d d i l u t i o n o f e a c h o f t h e t y p i n g a n t i s e r a i n a p l a s t i c p e t r i d i s h . The r e a c t i o n m i x t u r e s w e re o v e r l a y e d w i t h m i n e r a l o i l , and i n c u b a t e d a t 37 C f o r one h o u r . R e s u l t s w e r e t h e n r e c o r d e d , and, i f n e c e s s a r y , t h e r e a c t i o n m i x t u r e was f u r t h e r i n c u b a t e d a t 4 C. The d o u b l e d i f f u s i o n a s s a y was done i n 0.7% a g a r , PBS, 0.02% s o d i u m a z i d e , on m i c r o s c o p e s l i d e s w h i c h w e r e i n c u b a t e d a t room t e m p e r a t u r e i n a m o i s t a t m o s p h e r e . R e s u l t s w e r e r e c o r d e d a f t e r f o r t y - e i g h t h o u r s . I s o t y p e s I g G l and IgM were p u r i f i e d on D E A E - S e p h a c e l , ( F a h e y a nd H o r b e t t 1 9 5 9 ) , i n a 1.5 cm x 10 cm c o l u m n . A n t i s e r a w e r e d i a l y z e d a g a i n s t 10 mM T r i s - H C l pH 7.8, and t h e c o l u m n was e q u i l i b r a t e d w i t h t h e same b u f f e r . A n t i b o d i e s w e re e l u t e d f r o m t h e c o l u m n w i t h a g r a d i e n t o f 0 t o 0.3 M N a C l , 10 mM T r i s - H C l pH 7.8. E l u a n t f r a c t i o n s were t h e n a s s a y e d by SDS-PAGE, and E L I S A . A l l o t h e r i s o t y p e s w e re p u r i f i e d on a c o l u m n o f p r o t e i n A-S e p h a r o s e CL-4B, ( G o d i n g 1978) as f o l l o w s : 500 mg o f t h e m a t r i x was s w o l l e n i n PBS, and p o u r e d i n t o a 1 cm d i a m e t e r c o l u m n ; an 28 a n t i s e r u m was t h e n a p p l i e d t o t h e c o l u m n i n PBS, and i n c u b a t e d a t room t e m p e r a t u r e f o r one h o u r ; t h e c o l u m n was washed w i t h 50 ml o f PBS; t h e a n t i b o d y was e l u t e d w i t h a s m a l l v o l u m e o f 0.1 M g l y c i n e - H C l , 0.5 M N a C l pH 2.8, i n t o a t u b e c o n t a i n i n g 100 u l o f 1M T r i s - C l pH 8; and t h e a n t i b o d y was d i a l y z e d a g a i n s t PBS o v e r n i g h t . I f n e c e s s a r y , e l u a n t s f r o m e i t h e r o f t h e two c o l u m n s c o u l d be f u r t h e r c o n c e n t r a t e d by a n o t h e r ammonium s u l f a t e p r e c i p i t a t i o n . C h a r a c t e r i z a t i o n o f MCAs MCAs were c h a r a c t e r i z e d w i t h r e s p e c t t o t h e i r a f f i n i t y and s p e c i f i c i t y . A f f i n i t y was c h a r a c t e r i z e d i n a s o l i d - p h a s e a s s a y , u s i n g t h e p r e v i o u s l y o u t l i n e d E L I S A p r o c e d u r e ; e x c e p t , t h a t t h e c o n j u g a t e was a p p l i e d i n a s e r i a l d i l u t i o n . S p e c i f i c i t y was d e t e r m i n e d i n an i n h i b i t i o n a s s a y (Munns and L i s z e w s k i , 1 9 8 0 ) . E q u a l v o l u m e s o f a n t i b o d y and i n h i b i t o r were i n c u b a t e d a t 37 C f o r 18 h o u r s , t h e n t h e amount o f f r e e a n t i b o d y a v a i l a b l e f o r r e a c t i o n w i t h a n t i g e n was a s s a y e d by t h e s t a n d a r d E L I S A p r o c e d u r e . The i n h i b i t i o n a s s a y , w i t h m o d i f i c a t i o n s , was a l s o u s e d t o d e t e r m i n e MCA s p e c i f i c i t y w i t h r e s p e c t t o p o l y n u c l e o t i d e s . A l l n u c l e i c a c i d s a m p l e s w ere d e n a t u r e d i n b o i l i n g w a t e r f o r one t o two m i n u t e s , t h e n c o o l e d on i c e p r i o r t o any o t h e r m a n i p u l a t i o n . N u c l e i c a c i d s w e r e u s e d e i t h e r w i t h o r w i t h o u t an enzyme t r e a t m e n t . I f an enzyme t r e a t m e n t was done, t h e n i n h i b i t i n g n u c l e i c a c i d and enzyme s o l u t i o n were i n c u b a t e d f o r two h o u r s a t 37 C, p r i o r t o a d d i t i o n o f a n t i s e r a . Enzyme d i g e s t i o n s w e r e done e s s e n t i a l l y as o u t l i n e d i n M a n i a t i s e t a l . ( 1 9 8 2 ) . RNase A and 29 RNase T I d i g e s t s were done i n 250 u l o f TE c o n t a i n i n g 20 ug o f enzyme powder (RNase A) o r 20 u n i t s (RNase T I ) . N u c l e a s e P I ( S i g m a C h e m i c a l Co.) d i g e s t s w e r e done i n 250 u l o f 100 mM NaAc pH8, 10 mM Z n C l 2 , c o n t a i n i n g 20 u n i t s o f enzyme. To e n s u r e t h a t t h e v a r i o u s s o l u t i o n s u s e d i n enzyme d i g e s t i o n d i d n o t i n t e r f e r e w i t h t h e i n h i b i t i o n a s s a y , n u c l e o t i d e s and n u c l e o s i d e s , p r e v i o u s l y a s s a y e d u n d e r s t a n d a r d c o n d i t i o n s , were a s s a y e d i n t h e enzyme d i g e s t i o n b u f f e r s . RNA w h i c h had been d e n a t u r e d b y g l y o x a l a t i o n ( N a k a y a e t a l . , 1 968) was a l s o u s e d . G l y o x a l was d e i o n i z e d i m m e d i a t e l y b e f o r e u s e b y m i x i n g 10 ml w i t h 3 g o f "MB-1" ( S i g m a C h e m i c a l C o m p a n y ) , m i x e d b e d i o n - e x c h a n g e r e s i n , and a l l o w e d t o s t a n d f o r two h o u r s , a t w h i c h t i m e t h e g l y o x a l was r e c o v e r e d . RNA was d e n a t u r e d i n 10 mM p h o s p h a t e b u f f e r pH 7, c o n t a i n i n g 20 mM g l y o x a l , a t 60 C f o r two h o u r s and t h e n e t h a n o l - p r e c i p i t a t e d . P r i o r t o u s e , t h e RNA was h e a t e d a t 60 C f o r f i f t e e n m i n u t e s , t h e n q u e n c h e d on i c e . Membrane B l o t t i n g A s s a y o f N u c l e o t i d e s and ssRNA I n o r d e r t o f u r t h e r c h a r a c t e r i z e t h e MCAs, a c o l o r i m e t r i c i m m u n o b l o t t i n g t e c h n i q u e was e m p l o y e d . A l l b l o t s w e r e done w i t h e i t h e r n i t r o c e l l u l o s e o r " G e n e - S c r e e n " membranes (New E n g l a n d N u c l e a r ) , w h i c h were p r e - s o a k e d i n t h e a p p r o p r i a t e b u f f e r . E l e c t r o b l o t s w e r e done as d e s c r i b e d by T o wbin e t a l . , ( 1 9 7 9 ) , w i t h m o d i f i c a t i o n s f o r n u c l e i c a c i d s as o u t l i n e d i n t h e "Gene-S c r e e n " u s e r ' s g u i d e . P r o t e i n b l o t s w e r e f r o m SDS-PAG b l o t t e d o v e r n i g h t a t 100 raA a t 4 C, i n 20 mM T r i s , 150 mM g l y c i n e , a n d 2 0 % m e t h a n o l . P r i o r t o b l o t t i n g , g e l s were s o a k e d i n b u f f e r f o r 30 t w e n t y m i n u t e s , and a f t e r t r a n s f e r t h e membrane was a i r - d r i e d , a n d s t o r e d i n d e f i n i t e l y i n a d u s t - f r e e e n v i r o n m e n t . RNA e l e c t r o b l o t s w e r e done o u t o f u r e a - a c r y l a m i d e g e l s i n 25 mM p h o s p h a t e b u f f e r pH 6.5 a t 300 mA a t 4 C, f o r s i x h o u r s . The t r a n s f e r membrane was t h e n a i r - d r i e d and b a k e d u n d e r vacuum a t 80 C f o r two h o u r s . S p o t - b l o t s were done i n PBS ( p r o t e i n s ) and TE ( n u c l e i c a c i d s ) on membranes t h a t were a i r - d r i e d a f t e r b e i n g s o a k e d i n b u f f e r . S a m p l e s were e i t h e r a p p l i e d d i r e c t l y , o r i n a d o t - b l o t m a n i f o l d . Where m u l t i p l e s p o t t i n g s w e r e done w i t h o u t a m a n i f o l d , s a m p l e s i z e f o r any s i n g l e s p o t t i n g was l i m i t e d t o 2 u l . P r i o r t o s p o t - b l o t t i n g , n u c l e i c a c i d s w e re a l w a y s d e n a t u r e d by h e a t i n g a t 98 C f o r one t o t h r e e m i n u t e s , t h e n q u e n c h e d on i c e . I m m u n o - d e t e c t i o n o f s a m p l e s on membranes was b a s e d on t h e p r o c e d u r e o u t l i n e d by Hawkes e t a l . , ( 1 9 8 2 ) . The membrane was i n c u b a t e d i n PBS-Tween/1% FCS f o r f o r t y - f i v e m i n u t e s , t h e n a n t i b o d y was a d d e d t o t h i s s o l u t i o n , and i n c u b a t i o n was c o n t i n u e d f o r a f u r t h e r two h o u r s . The membrane was t h e n washed f o r t e n m i n u t e s : o n c e w i t h PBS-Tween, t h r i c e PBS-Tween/1% FCS, and o n c e w i t h PBS-Tween. The membrane was t h e n p l a c e d i n a s o l u t i o n o f PBS-Tween c o n t a i n i n g p e r o x i d a s e o r p h o s p h a t a s e - c o n j u g a t e d s e c o n d a r y a n t i b o d y f o r two h o u r s . The w a s h i n g p r o c e d u r e was t h e n r e p e a t e d w i t h a f i n a l r i n s e i n d i s t i l l e d w a t e r . The s u b s t r a t e , 6 mg o f 4 - c h l o r o - l - n a p h t h o l i n 2 ml o f m e t h a n o l , 10*ml o f PBS, and 10 u l o f a 3 0 % s o l u t i o n o f h y d r o g e n p e r o x i d e o r 30 mg o f F a s t Red i n PBS, r e s p e c t i v e l y , d e p e n d i n g on t h e s e c o n d a r y c o n j u g a t e u s e d , w e r e a d d e d i n a d a r k e n e d e n v i r o n m e n t . I n c u b a t i o n was f r o m t w e n t y m i n u t e s t o o v e r n i g h t . E n d p o i n t d e t e r m i n a t i o n was e s t i m a t e d 31 v i s u a l l y . B i n d i n g of I o d i n a t e d RNA To ELISA P l a t e s Attempts were made to develop a r a p i d i n i t i a l s c r e e n i n g of MCAs by the d i r e c t c o a t i n g of ELISA p l a t e s with i o d i n a t e d RNA. RNA was e n d - l a b e l l e d with y~[P32„]-ATP u s i n g p o l y n u c l e o t i d e k i n a s e as d e s c r i b e d by M a n i a t i s et a l . , (1982). L a b e l l e d RNA, i n PBS or STE, was then incubated i n uncoated Immulon-2 ELISA p l a t e s at 37 C f o r fou r hours and 4 C overnight Lee et a l . , (1981). P l a t e s were then washed e x t e n s i v e l y with PBS-Tween, and autoradiographed to determine i f any RNA had been bound to the p l a t e . P l a t e s were a l s o coated with 1% methylated-BSA i n PBS ( B a l l a r d et a l . , 1984), and p o l y - L - l y s i n e (Sigma Chemical Co.) at 100 ug/ml i n 150 mM T r i s - H C l pH 7.6 (Aotsuka et a l . , 1979), and incubated at 4 C o v e r n i g h t . They were then e x t e n s i v e l y washed with PBS-Tween, and t r e a t e d as d e s c r i b e d f o r uncoated p l a t e s . 32 R e s u l t s j y ^ a t i on o f Hapten W i t h P r o t e i n C a r r i e r C o n j u g a t i o n o f h a p t e n t o c a r r i e r was done i n i t i a l l y as d e s c r i b e d ( E r l a n g e r and B e i s e r , 1964), b u t t h e method r e s u l t e d i n a p o o r y i e l d o f c o n j u g a t e d p r o d u c t . The c h e m i s t r y o f t h e method a p p e a r e d t o be sound, but t h e h a r s h n e s s o f i t s e x e c u t i o n was most l i k e l y t h e c a u s e o f t h e low y i e l d . V a r i o u s m o d i f i c a t i o n s o f t h e p r o c e d u r e were t h e r e f o r e e v a l u a t e d . When t h e m i x t u r e , a f t e r sodium b o r o h y d r i d e r e d u c t i o n , was d i a l y z e d a g a i n s t w a t e r a f l o c u l a n t , w h i t e p r e c i p i t a t e was f o r m e d . I t was f o u n d t h a t t h e p r e c i p i t a t e c o u l d be s o l u b i l i z e d by r a i s i n g t h e pH. SDS-PAGE o f t h e s o l u b i l i z e d p r e c i p i t a t e r e s u l t e d i n t h e d e t e c t i o n o f a band o f 66-71 x 1 0 3 d a l t o n s , c h a r a c t e r i s t i c o f t h e c o n j u g a t e . Thus c o n j u g a t e s were d i a l y z e d a g a i n s t PBS pH 8, t o a v o i d f o r m a t i o n o f t h e p r e c i p i t a t e . The o r i g i n a l p r o c e d u r e c a l l e d f o r t h e a d d i t i o n o f f o r m i c a c i d a f t e r t h e c o n j u g a t i o n t o d e g r a d e u n r e a c t e d n u c l e o s i d e s ; however, even when added by d i a l y s i s , t h i s r e s u l t e d i n e x c e s s f o a m i n g t h a t l e d t o d e g r a d a t i o n o f t h e p r o t e i n a c e o u s c o n j u g a t e . Two c o n j u g a t i o n s were done s i m u l t a n e o u s l y , one w i t h and one w i t h o u t f o r m i c a c i d , t o d e t e r m i n e t h e e f f e c t s o f i t s o m i s s i o n f r o m t h e r e a c t i o n . The p r o d u c t s o f t h e r e a c t i o n s were a n a l y z e d by UV s p e c t r o s c o p y and g e l e l e c t r o p h o r e s i s ; t h e e f f i c i e n c y was e q u a l f o r b o t h r e a c t i o n s , b u t t h e y i e l d was c o n s i d e r a b l y h i g h e r when t h e f o r m i c a c i d was o m i t t e d . The e f f i c i e n c y o f t h e r e a c t i o n was s t i l l b e l o w t h a t o f some p u b l i s h e d r e s u l t s , and i t was f e l t n e c e s s a r y t o m a x i m i z e 33 e f f i c i e n c y b e c a u s e i t w o u l d i n c r e a s e t h e c h a n c e s o f o b t a i n i n g t h e v e r y s p e c i f i c immune r e a c t i o n d e s i r e d . An e x p e r i m e n t was done t o d e t e r m i n e i f t h e h a p t e n , s p e c i f i c a l l y i o d o c y t i d i n e ( I - C ) , was b e i n g d e g r a d e d d u r i n g t h e r e a c t i o n . The r e a c t i o n was done w i t h a l l c o m p o n e n t s e x c e p t t h e c a r r i e r , and t h e h a p t e n was t r a c e d t h r o u g h t h e r e a c t i o n by UV s p e c t r o s c o p y and TLC. The h a p t e n a p p e a r e d t o b r e a k down i n t h e p r o l o n g e d p r e s e n c e o f s o d i u m b o r o h y d r i d e , as i s shown by b o t h t h e TLC p l a t e ( F i g . 1 ) , and t h e UV s p e c t r a ( F i g . 2 ) . An e x p e r i m e n t was t h e n c o n d u c t e d t o d e t e r m i n e t h e minimum e x p o s u r e t o s o d i u m b o r o h y d r i d e t h a t s t i l l g a v e an a c c e p t a b l e y i e l d o f p r o d u c t . The c o n j u g a t i o n was c a r r i e d o u t w i t h one, two, and f o u r h o u r i n c u b a t i o n s i n t h e p r e s e n c e o f b o r o h y d r i d e , t h e p r o d u c t s b e i n g a n a l y z e d by SDS-PAGE ( F i g . 3 ) . No d i f f e r e n c e s w e r e o b s e r v e d b e t w e e n t h e v a r i o u s t r e a t m e n t s w i t h b o r o h y d r i d e . When t h e c a r r i e r was p a s s e d t h r o u g h a c o n j u g a t i o n r e a c t i o n w i t h o u t any h a p t e n p r e s e n t , i t was n o t e d a f t e r SDS-PAGE t h a t t h e c a r r i e r was m i l d l y d e n a t u r e d ( i e . i t s m o l e c u l a r w e i g h t h a d d e c r e a s e d ) as a r e s u l t o f t h e r e a c t i o n ( F i g . 4 ) . M o r e o v e r , t h i s g e l shows t h a t t h e r e i s a d e f i n i t e d i f f e r e n c e i n t h e d i s t a n c e o f m i g r a t i o n , w h i c h i s a f u n c t i o n o f m o l e c u l a r w e i g h t , b e t w e e n t h e c o n t r o l and t h e h a p t e n - c a r r i e r c o n j u g a t e . C o m p a r i s o n o f t h e UV s p e c t r u m o f t h e c o n j u g a t i o n p r o d u c t w i t h t h a t o f t h e c a r r i e r ( F i g . 4 ) , c l e a r l y showed t h a t c o n j u g a t i o n had o c c u r e d . The r e l a t i v e l e v e l o f c o n j u g a t i o n was d e t e r m i n e d f r o m t h e UV s p e c t r u m o f t h e c o n j u g a t e , ( E r l a n g e r and B e i s e r , 1 9 6 4 ) . An a v e r a g e o f f i f t e e n t o t w e n t y h a p t e n m o l e c u l e s were c o n j u g a t e d p e r c a r r i e r 34 o o o o (j o o o o o o o o o o o ^ - s o l v e n t f r o n t s o l v e n t f l o w A A 1 2 A A A A A A A A 3 4 5 6 7 8 9 10 A ii A A 12 13 A 14 A 15 A A 16 17 A 18 A 19 4 - o r i g i n F i g . l T h i n l a y e r c h r o m a t o g r a p h y s h o w i n g t h e e f f e c t o f t h e c o n j u g a t i o n r e a c t i o n on i o d o - c y t i d i n e (I-C) a t v a r i o u s t i m e s d u r i n g t h e r e a c t i o n . . R e a c t i o n m i x t u r e c o n t a i n e d a l l c o n s t i t u e n t s , e x c e p t c a r r i e r p r o t e i n . 1) c y t i d i n e c o n t r o l ; 2) I-C c o n t r o l ; 3 ) a f t e r s o d i um p e r i o d a t e ; 4) a f t e r e t h y l e n e g l y c o l ; 5) i m m e d i a t e l y a f t e r a d d i t i o n o f c a r b o n a t e ; 6) 7 m i n u t e s l a t e r ; 7) 20 m i n u t e s l a t e r ; 8) 30 m i n u t e s l a t e r ; 9) 45 m i n u t e s l a t e r ; 10) 60 m i n u t e s l a t e r ; 11) i m m e d i a t e l y a f t e r a d d i t i o n o f s o d i u m b o r o h y d r i d e ; 12) 15 m i n u t e s l a t e r ; 13) 30 m i n u t e s l a t e r ; 14) 1 h o u r l a t e r ; 15) 2 h o u r s l a t e r ; 16) 9 h o u r s l a t e r ; 17) 12 h o u r s l a t e r ; 18) 16 h o u r s l a t e r ; 19) s o l u b o l i z e d p r o d u c t a f t e r f r e e z e -d r y i n g : , # , 0 and Q r e p r e s e n t s v a r y i n g i n t e n s i t y f r o m br ight to dull. 35 340 330 320 310 300 290 280 270 260 250 240 230 w a v e l e n g t h (nm) F i g . 2 UV spectrum of I-C, a f t e r r e a c t i o n with sodium p e r i o d a t e ( • ) , and a f t e r f o u r hours of i n c u b a t i o n i n the presence of sodium borohydride ( • ). 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 myosin B-galactosidase phosphorylase B bovine albumin egg albumin carbonic anhydrase F i g . 3 Comparison o f c o n j u g a t i o n r e a c t i o n s between i o d o c y t i d i n e and b o v i n e serum albumin a f t e r i n c u b a t i o n w i t h (NaBH 4) f o r v a r y i n g l e n g t h s of tim e . E l e c t r o p h o r e s i s was i n an 8% SDS-Page, s t a i n e d w i t h Coomassie B r i l l i a n t B l u e . Lane 1: m o l e c u l a r weight s t a n d a r d s i n o r d e r o f d e c r e a s i n g m o l e c u l a r w e i ght ( t o p t o bot t o m ) , myosin (205,000 d ) , B - g a l a c t o s i d a s e (116,000 d ) , p h o s p h o r y l a s e B (97,000 d ) , b o v i n e serum albumin (66,000 d ) , egg albumin (45,000 d ) , and c a r b o n i c anhydrase (29 000 d ) ; l a n e 2: b o v i n e serum albumin (BSA) s t a n d a r d (5 ug): l a n e s 3,4, and 5: BSA i n NaBH 4 f o r one hour (5,10,20 ug); l a n e s 6,7, and 8: BSA i n NaBH 4 f o r two hours (5,10,20 ug); l a n e s 9,10, and 11: BSA i n NaBH 4 f o r f o u r hours (5,10,20 ug); l a n e s 12,13, and 14: BSA-i o d o c y t i d i n e i n NaBH 4 f o r one hour (5,10,20 ug); l a n e s 15,16, and 17: B S A - i o d o c y t i d i n e i n NaBH 4 f o r two hours (5,10,20 ug); l a n e s 18,19, and 20: B S A - i o d o c y t i d i n e i n NaBH 4 f o r f o u r hours (5,10,20 ug) . 37 1 2 3 4 5 6 7 8 9 10 • myosin B-galactosidase phosphorylase B bovine albumin egg albumin carbonic anhyd rase F i g . 4 An 8% SDS-po1yaery1amide g e l comparing the m o l e c u l a r weight d i f f e r e n c e s between b o v i n e serum a l b u m i n (BSA) and BSA c o n j u g a t e d w i t h i o d o c y t i d i n e . S t a i n i n g was w i t h Coomassie B r i l l i a n t B l u e . Lanes 1,2, and 3: B S A - i o d o c y t i d i n e (20,10,5 ug) ; l a n e s 4,5, and 6: BSA from a c o n j u g a t i o n r e a c t i o n e x c l u d i n g hapten (20,10,5 ug); l a n e s 7,8, and 9: BSA s t a n d a r d (20,10,5 ug); and l a n e 10: m o l e c u l a r w e i ght s t a n d a r d (see F i g . 3 ) . 38 A A A A A A A A A A A 340 320 . . 300 280 260 240 wavelength (nm) i F i g . 5 C o m p a r i s o n o f t h e UV s p e c t r a o f : b o v i n e serum a l b u m i n ( B S A ) - a f t e r a c o n j u g a t i o n r e a c t i o n c o n t a i n i n g no h a p t e n ( • ), B S A - c y t i d i n e ( • ) ' , B S A - i o d o c y t i d i n e ( • ) . The c o n c e n t r a t i o n o f e a c h sample was 50 ug/ml i n PBS, as d e t e r m i n e d by t h e " B i o - R a d " p r o t e i n a s s a y . 39 m o l e c u l e . Serum a l b u m i n f r o m human and b o v i n e s o u r c e s and o v a l b u m i n w e r e t h e c a r r i e r s w i t h t h e b e s t e f f i c i e n c y o f c o u p l i n g . H e m o c y a n i n and p o l y - l y s i n e were a l s o t r i e d , b u t i n t h e s e e x p e r i m e n t s t h e y had an u n a c c e p t a b l y l o w e f f i c i e n c y o f c o u p l i n g . I o d i n a t i o n o f P o l y c y t i d i n e and ssRNA A c c o r d i n g t o C o m m e r f o r d ( 1 9 7 1 ) , t h e p r o p o r t i o n s o f t h e i n i t i a l c o m p o n e n t s o f t h e r e a c t i o n a r e c r i t i c a l . I n t h e s c a l e d - u p v e r s i o n , t y p i c a l o f t h e p r e s e n t e x p e r i m e n t , t h i s d i d n o t a p p e a r t o be t h e c a s e . The c o n d i t i o n s o f t h e r e a c t i o n w e r e v a r i e d , b u t o n l y t h e r a t i o o f i o d i n e t o c y t i d i n e a p p e a r e d t o i n f l u e n c e t h e y i e l d . H o wever, t h i s r a t i o was n o t g r e a t l y a l t e r e d f r o m C o m m e r f o r d ' s i n o r d e r t o a v o i d d r a s t i c c h a n g e s i n t h e p h y s i c o -c h e m i c a l s t r u c t u r e o f t h e n u c l e i c a c i d . The r e a c t i o n was o p t i m i z e d u s i n g c y t i d i n e as t h e s u b s t r a t e , and t h e p r o d u c t s were a s s a y e d by UV s p e c t r o s c o p y and TLC. A TLC p l a t e s h o w i n g t h e p r o d u c t s o f t h e r e a c t i o n as o u t l i n e d i n M a t e r i a l s and M e t h o d s , i s shown i n F i g . 6 . The r e a c t i o n d i d n o t go t o c o m p l e t i o n and t h e y i e l d e s t i m a t e d f r o m UV s p e c t r o s c o p y was no g r e a t e r t h a n 15%. G e t z e t a l . , ( 1 9 7 2 ) and R o b e r s o n and C h e e v e r s ( 1 9 8 2 ) f o u n d t h a t n u c l e i c a c i d , n o t r i g o r o u s l y p u r i f i e d p r i o r t o i o d i n a t i o n c o n t a i n e d u n i d e n t i f i e d i m p u r i t i e s , w h i c h were n o n - s p e c i f i c a l l y i o d i n a t e d t o a h i g h l e v e l . T hus, a l l n u c l e i c a c i d s a c q u i r e d f r o m c o m m e r c i a l s o u r c e s were p u r i f i e d by p h e n o l - ^ c h l o r o f o r m e x t r a c t i o n , f o l l o w e d by e t h a n o l p r e c i p i t a t i o n . V i r a l RNA was p r o t e i n a s e -t r e a t e d , e x t r a c t e d w i t h p h e n o l - c h l o r o f o r m , and e t h a n o l -p r e c i p i t a t e d . P o l y n u c l e o t i d e s were d e n a t u r e d p r i o r t o i o d i n a t i o n , 40 b e c a u s e C o m m e r f o r d ( 1 9 7 1 ) , showed t h a t i n c o r p o r a t i o n o f i o d i d e i n c r e a s e d t h r e e - f o l d , upon d e n a t u r a t i o n . T h e r e f o r e , RNA was d i s s o l v e d i n l o w s a l t , d e n a t u r e d i n b o i l i n g w a t e r f o r t h i r t y s e c o n d s t o t h r e e m i n u t e s , d e p e n d i n g on t h e s a m p l e , and t h e n q u e n c h e d on i c e . I o d i n a t i o n o f RNA was m o n i t o r e d by XRF and UV s p e c t r o s c o p y . Two p r o b l e m s a r o s e , w i t h XRF. F i r s t l y , b e c a u s e t h e a r e a and t h i c k n e s s s c a n n e d was n o t c o n s i s t e n t f r o m s a m p l e t o s a m p l e , d i f f e r e n t s a m p l e s c o u l d n o t be d i r e c t l y c o m p a r e d . T h e r e f o r e s a m p l e s w ere com p a r e d on t h e b a s i s o f t h e r a t i o o f p h o s p h o r o u s t o i o d i n e i n t h e s a m p l e ( T a b l e 1 ) . T h i s r a t i o r e p r e s e n t s t h e a r e a u n d e r t h e peak c o r r e s p o n d i n g t o p h o s p h o r o u s d i v i d e d by t h e t o t a l o f t h e a r e a s u n d e r a l l t h e p e a k s c o r r e s p o n d i n g t o i o d i n e , as d e t e r m i n e d by XRF. S e c o n d l y , b e c a u s e t h e X - r a y s o u r c e was n o t m o n o c h r o m a t i c i t was n o t p o s s i b l e t o s p e c i f i c a l l y e x c i t e e l e c t r o n s i n a p a r t i c u l a r s h e l l . Thus some e l e m e n t s w e r e b e t t e r d e t e c t e d t h a n o t h e r s ( T a b l e 2 ) . F o r e x a m p l e , i o d i n e was d e t e c t e d , b u t t h a l l i u m was n o t . T h i s p r o v e d t o be a p r o b l e m b e c a u s e i t was n o t p o s s i b l e . , t o i n f e r , f r o m t h e a b s e n c e o f t h a l l i u m , t h a t t h e f i n a l d e s a l t i n g s t e p was s u c c e s s f u l i n r e m o v i n g a l l o f t h e r e m a i n i n g u n w a n t e d c o m p o n e n t s o f t h e i o d i n a t i o n r e a c t i o n . Thus one c o u l d n o t s a y w i t h c e r t a i n t y t h a t i o d i n e d e t e c t e d by XRF was c o v a l e n t l y l i n k e d t o t h e RNA, and n o t a l i n g e r i n g component o f t h e i o d i n a t i o n r e a c t i o n . Thus an i o d i n a t i o n r e a c t i o n was done w i t h o u t t h a l l i u m , t h a t w o u l d s e r v e as a n e g a t i v e c o n t r o l t o e n s u r e t h a t t h e f i n a l p r o d u c t was p u r e . B e c a u s e t h a l l i u m i o n i s t h e e s s e n t i a l c a t a l y s t 41 f o r t h e r e a c t i o n , no i o d i n e s h o u l d be d e t e c t e d i n t h e f i n a l p r o d u c t o f t h e c o n t r o l r e a c t i o n i f t h e p u r i f i c a t i o n i s s u c c e s s f u l . XRF a n a l y s i s o f t h e c o n t r o l and a c o m p l e t e i o d i n a t i o n r e a c t i o n ( T a b l e i 3, F i g . 7) d e t e c t e d no i o d i n e i n t h e f o r m e r , i n d i c a t i n g t h a t t h e p u r i f i c a t i o n was e f f i c i e n t . R e c o v e r y o f RNA a f t e r t h e i o d i n a t i o n r e a c t i o n was v e r y e f f i c i e n t ; c o n s i s t e n t l y a b o v e 90% as d e t e r m i n e d f r o m UV s p e c t r a . I o d i n a t i o n o f p o l y - C was f o u n d t o be v e r y e f f i c i e n t , a n d c o u l d be c o n f i r m e d b y UV s p e c t r o s c o p y . F i g . 8 ( b ) , shows a c o m p a r i s o n b e t w e e n s p e c t r a o f t h e i o d i n a t e d and n o n - i o d i n a t e d p o l y - C . As w o u l d be e x p e c t e d by c o m p a r i s o n o f t h e s p e c t r a o f t h e i n d i v i d u a l n u c l e o s i d e s , F i g . 8 ( a ) , t h e a b s o r b a n c e maximum f o r i o d i n a t e d p o l y - C i s s h i f t e d t o a l o n g e r w a v e l e n g t h . O t h e r RNA's were i o d i n a t e d and a s s a y e d by XRF, ( T a b l e 1 ) . The c e l l u l a r RNA c o n s i s t e n t l y had a l o w e r l e v e l o f i n c o r p o r a t i o n c o m p a r e d t o e i t h e r t h e p o l y - C o r t h e v i r a l RNA. I o d i n a t e d n u c l e i c a c i d was r e g u l a r l y a s s a y e d by e l e c t r o p h o r e s i s t o d e t e r m i n e i f any d e g r a d a t i o n h a d o c c u r r e d ( C u m m e r f o r d , 1 9 7 1 ) . None o f t h e i o d i n a t e d n u c l e i c a c i d a p p e a r e d t o be d e g r a d e d by t h e r e a c t i o n i n a q u a n t i t y l a r g e e n o u g h t o be d e t e c t e d ( F i g . 9 ) . I o d i n a t i o n o f ds-RNA The r e a c t i v i t y o f t h e a n t i b o d i e s w i t h i o d o c y t i d i n e i n a d u p l e x s t r u c t u r e o f n u c l e i c a c i d , b e c a u s e o f i t s c o n f i g u r a t i o n , w o u l d s i m u l a t e a n u c l e i c a c i d h y b r i d i z a t i o n . T h e r e f o r e t h e i o d i n a t i o n o f ds-RNA was a t t e m p t e d . The p u r i f i c a t i o n p r o c e d u r e e n s u r e d t h a t o n l y ds-RNA was 42 solvent-^ front solvent flow origin-^ A A A A cytidine iodo- 1 2 cytidine A 3 A A A A 4 5 6 7 fraction no. A A A 10 A 15 F i g . 6 T h i n l a y e r c h r o m a t o g r a p h y p l a t e s h o w i n g a c o m p a r i s o n o f f r a c t i o n s c o l l e c t e d f r o m an i o d i n a t i o n r e a c t i o n , w i t h c y t i d i n e as t h e s u b s t r a t e . N u c l e o s i d e s w e re p u r i f i e d on a s i l i c a c o l u m n p r i o r t o i o d i n a t i o n 0 , O a n d (_) r e p r e s e n t s vary-ing in tens i ty f r o m br ight to dul l . 43 E l e m e n t L i n e * E n e r g y ( K e V ) A r e a P / I a) Na KA 1.047 65535 S i KA 1.731 3942 P KA 2.021 61928 C I KA 2.631 8996 7.2 I LA 3.945 5879 I LB 4.245 2265 I LB2 4.530 509 Fe KA 6.409 481 b) Na KA 1.038 114107 S i KA 1.720 5873 P KA 2.021 104208 C I KA 2.620 34680 12.0 I LA 3.933 6258 I LB 4.240 2434 Fe KA 6.396 1854 Cu KA 8.035 764 c ) Na KA 1.042 113778 S i KA 1.714 1798 P KA 2.016 161272 C I KA 2.627 41221 16.0 I LA 3.946 6535 I LB 4.245 2784 I LB2 4.546 709 d) Na KA 1.036 218725 A l KA 1.472 1003 S i KA 1.716 5075 P KA 2.012 167258 C I KA 2.625 21148 9.0 I LA 3.938 13188 I LB 4.238 5217 I LB2 4.516 1047 Fe KA 6.387 987 T a b l e 1 XRF d a t a f r o m v a r i o u s i o d i n a t i o n s : a) P o l y - C , f r o m a s m a l l s c a l e r e a c t i o n ; b) P o l y - C , f r o m a l a r g e s c a l e r e a c t i o n ; c ) c e l l u l a r RNA ( P h a r m a c i a P-L B i o c h e m i c a l s ) , f r o m a s m a l l s c a l e r e a c t i o n ; d) RNA f r o m cowpea c h l o r o t i c m o t t l e v i r u s , f r o m a s m a l l s c a l e r e a c t i o n . P h o s p h o r o u s / i o d i d e r a t i o ( P / I ) i s an a v e r a g e f o r t h a t p a r t i c u l a r n u c l e i c a c i d u n d e r t h o s e r e a c t i o n c o n d i t i o n s . * R e f e r s t o t h e e l e c t r o n s h e l l f r o m w h i c h e x c i t a t i o n was b e i n g d e t e c t e d . 44 Element L i n e * Energy(KeV) Area a) Na KA 1.044 75148 S i KA 1.447 973 A l KA 1.750 14384 b) U n i * * — .783 275 Na KA 1.052 1632 A l KA 1.450 1378 S i KA 1.752 11037 I LA 3.941 5364 I LB 4.253 1877 Hg LA 9.988 296 T a b l e 2 D e t e c t i o n by XRF, o f I but not T I , the two key elements i n v o l v e d i n the i o d i n a t i o n r e a c t i o n . a) T1(N03)3 (1 ug) i n 3M NaAcetate pH 5, a p p l i e d i n a 1 u l s p o t ; b) N a l (1 ug) i n dH20, a p p l i e d i n a 1 u l s p o t . * R e f e r s t o the e l e c t r o n s h e l l from which e x c i t a t i o n was b e i n g d e t e c t e d . ** U n i = U n i d e n t i f i e d 45 PHH12 NON .828KEV/CH 180.SECS BUG* 2. 188KEV, 1261. 186. c C n c 3 O o >» Q : I x X-Ray Energy (KeV) F i g . 7 XRF data of i o d i n a t i o n r e a c t i o n s c o m p a r i n g ; a ) , a r e a c t i o n i n c l u d i n g t h a l l i u m ; b ) , a r e a c t i o n not i n c l u d i n g t h a l l i u m . 46 340 320 300 280 260 2.40 Wavelength (nm) F i g . 8 UV s p e c t r a o f : a) c y t i d i n e ( A ) , i o d o - c y t i d i n e ( 0 )» a m i x t u r e c o n t a i n i n g an e q u a l amount o f b o t h ( O ) l b) p o l y - C ( O ) . and i o d i n a t e d p o l y - C ( • ) . 47 F r a c t i o n E1emen t L i n e Energy Area Energy Area 3 Na KA a) 1.050 67807 b) 1.052 97269 A l KA 1.490 537 S i KA 1.730 4751 1.731 4359 P KA 2. 028 132894 2. 029 178685 C l KA 2. 639 4448 2. 634 15917 I LA 3. 953 6863 I LB 4. 256 2563 Fe KA 6.444 843 4 Na KA 1.052 102009 1. 053 33335 A l KA 1. 487 1020 1.475 2334 S i KA 1 . 737 2902 ' 1.756 26090 P KA 2. 028 38788 2. 048 3264 C l KA 2.639 4898 2. 638 5535 I LA 3. 979 644 Fe KA 6.400 693 5 Na KA 1. 052 118996 1 . 054 22710 A l KA 1.479 4047 S i KA 1. 747 4759 1 . 757 46325 P KA 2. 033 11065 2. 052 3750 C l KA 2.639 4603 2 . 635 "45 0 0 Fe KA 6.420 6334 6 Na KA 1. 052 103616 1 . 053 18889 A l KA 1. 475 1287 1 . 489 3510 S i KA 1. 755 11874 1 . 760 25981 P KA 2.039 4308 2 . 054 948 C l KA 2 . 641 11119 2 . 639 3281 Fe KA 6. 383 539 6.421 827 T a b l e 3 ' XRF d a t a from two i o d i n a t i o n r e a c t i o n s : a) r e a c t i o n m i x t u r e c o n t a i n e d I , and RNA; b) r e a c t i o n m i x t u r e c o n t a i n e d T I , I, and RNA. F r a c t i o n s (1 ml) c o l l e c t e d from a 15x1 cm G-100 column, w i t h c o n s t a n t g r a v i t y f l o w at 1 ml/minute. 48 mol. weight (xlO 6 daltons) -*1.15 -<1.00 -«0.85 F i g . 9 A 5% a c r y l a m i d e - 8 M urea g e l comparing i o d i n a t e d and non-i o d i n a t e d ssRNA from cowpea c h l o r o t i c mott.le v i r u s . S t a i n i n g was w i t h e t h i d i u m bromide. Lanes 1 and 2 : heat d e n a t u r e d i o d i n a t e d RNA (8,4 ug); l a n e 3: heat d e n a t u r e d RNA (4 ug); l a n e s 4 and 5: i o d i n a t e d RNA (8,4 ug); and l a n e 6: u n t r e a t e d RNA (8 ug). 49 a s s a y e d f o r p r e s e n c e o f i o d i n e . D i g e s t i o n w i t h RNase TI a l l o w e d t h e r e m o v a l o f s i n g l e s t r a n d e d r e g i o n s w i t h o u t a l t e r i n g t h e d o u b l e - s t r a n d e d r e g i o n s ( L i m and R o b e r t s , 1 9 8 2 ) . M o r e o v e r t h e f i n a l p u r i f i c a t i o n by c e l l u l o s e c h r o m a t o g r a p h y has b e e n shown t o be s p e c i f i c f o r d s - n u c l e i c a c i d u n d e r t h e c o n d i t i o n s u s e d h e r e ( B a r b e r 1 9 6 6 ) . T wenty ug o f ds-RNA was a s s a y e d by XRF f o r t h e p r e s e n c e o f i o d i n e . I o d i n a t i o n was n o t d e t e c t e d , b u t i t i s l i k e l y t h a t i o d i n a t i o n o f ds-RNA d i d o c c u r s i n c e ds-DNA c a n be i o d i n a t e d . P 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 y P r i o r t o f u s i o n , s e r a were o b t a i n e d f r o m i m m u n i z e d m i c e and t i t e r e d w i t h r e s p e c t t o a n t i b o d i e s f o r c a r r i e r and c a r r i e r -n u c l e o s i d e c o n j u g a t e s ( F i g . 1 0 a ) . The p l a t e s w e re c o a t e d w i t h a n t i g e n (10 u g / m l ) and t h e a n t i s e r u m f r o m m i c e i n i t i a l l y i n o c u l a t e d w i t h H S A - i o d o c y t i d i n e c o n j u g a t e , d i l u t e d t o one i n t e n - t h o u s a n d . A s u b s t a n t i a l l y h i g h e r t i t e r was d e t e c t e d f o r i o d o c y t i d i n e t h a n f o r c y t i d i n e o r o t h e r n u c l e o t i d e s . T h i s i s e s p e c i a l l y o b v i o u s i f n u c l e o t i d e c o n j u g a t e s o f o v a l b u m i n a r e c o m p a r e d , b e c a u s e t h i s c a r r i e r i s n o t a n t i g e n i c a l 1 y r e l a t e d t o t h e o t h e r c a r r i e r s ( b o v i n e and human se r u m a l b u m i n s ) . Thus a s e r o l o g i c a l r e a c t i o n w i t h t h e h a p t e n i s n o t masked by one o c c u r r i n g w i t h t h e c a r r i e r . A b o o s t e r i n j e c t i o n v i a t h e i n t r a p e r i t o n e a l r o u t e r a t h e r t h a n one by an i n t r a v e i n o u s r o u t e i n t h e t a i l was s u f f i c i e n t t o p r o d u c e an a c c e p t a b l e t i t r e f o r c e l l f u s i o n . I n i t i a l s c r e e n i n g o f MCAs was by an E L I S A p r o c e d u r e u s i n g c y t i d i n e and i o d o c y t i d i n e c o n j u g a t e s o f t h e i m m u n i z i n g c a r r i e r 50 and o v a l b u m i n as a n t i g e n s . T hose c l o n e s r e a c t i n g ( p r e f e r e n t i a l l y w i t h b o t h i o d o c y t i d i n e c o n j u g a t e s were f u r t h e r c h a r a c t e r i z e d . A s i m i l i a r i n i t i a l s c r e e n i n g p r o c e d u r e was u s e d by Lee e t a l . , ( 1 9 8 1 ) , i n s e l e c t i n g MCAs a g a i n s t v a r i o u s a d e n o s i n e d e r i v a t i v e s . From t w e l v e f u s i o n s a p p r o x i m a t e l y t w e n t y - f i v e h u n d r e d c l o n e s w e re p r o d u c e d a n d a f t e r i n i t i a l s c r e e n i n g , e i g h t o f t h e s e were s e l e c t e d f o r f u r t h e r c h a r a c t e r i z a t i o n . F o r p r o d u c t i o n o f a s c i t e s f l u i d , m i c e were p r i m e d w i t h t r y p a n b l u e o r p r i s t a n e p r i o r t o i n j e c t i o n o f t h e " h y b r i d o m a , b u t t h e l a t t e r was f o u n d t o be much more e f f i c i e n t . H o wever, t r y p a n b l u e h a d t h e a d v a n t a g e o f a c o n s i d e r a b l y s h o r t e r l a g t i m e b e t w e e n p r i m i n g t h e a n i m a l and i n j e c t i o n o f h y b r i d o m a c e l l s . MCAs were p u r i f i e d f r o m t h e a s c i t e s f l u i d by p r o t e i n A - S e p h a r o s e o r DEAE-S e p h a c e l c h r o m a t o g r a p h y , t h e c h o i c e o f method d e p e n d i n g e n t i r e l y on t h e i s o t y p e o f t h e p a r t i c u l a r c l o n e . The e l u t i o n p r o f i l e o f MCA R l p u r i f i e d on D E A E - S e p h a c e l i s shown i n F i g . 11. As c a n be s e e n f r o m t h e g e l e l e c t r o p h o r e s i s ( F i g . 12) t h e m e t h o d y i e l d e d a q u a n t i t y o f homogeneous a n t i b o d y ( f r a c t i o n numbers 3 3 - 4 4 ) , t h a t c o r r e s p o n d e d t o t h e f r a c t i o n s (numbers 33-46) c o n t a i n i n g t h e peak i m m u n o l o g i c a l a c t i v i t y ( F i g . 1 1 ) . C h a r a c t e r i z a t i o n o f MCAs Of t h e e i g h t MCAs s e l e c t e d a f t e r t h e i n i t i a l s c r e e n i n g one l o s t t h e a b i l i t y t o p r o d u c e a n t i b o d i e s d u r i n g t h e r e c l o n i n g p r o c e d u r e . The r e m a i n i n g MCAs were f i r s t c h a r a c t e r i z e d i n an a f f i n i t y a s s a y ( T a b l e 4, F i g . 1 3 ) , where t h e c o n c e n t r a t i o n o f a n t i g e n was s e r i a l l y d i l u t e d w h i l e t h e c o n c e n t r a t i o n o f a n t i b o d y 51 a) C a r r i e r - C o n j u g a t e b) 1 0 0 * -* * "55 80*> c « Q — 60H m o +^  Q . O 4 0 > 20>-~WBO H B O H B O H B O H B O H B O H B O H B O 1 2 3 4 5 6 7 C a r r i e r - C o n j u g a t e Fig. 1 0 B i n d i n g "of a) p o l y c l o n a l s e r a , b) MCA R l t o ELISA p l a t e s c o a t e d w i t h c a r r i e r s : HSA (H), BSA ( B ) , and ovalbumin ( 0 ) , and c o n j u g a t e s of each c a r r i e r t o : c y t i d i n e ( 1 ) , i o d o c y t i d i n e ( 2 ) , a d e n o s i n e ( 3 ) , guanosine ( 4 ) , t h y m i d i n e ( 5 ) , u r i d i n e ( 6 ) , and i o d o u r i d i n e ( 7 ) . % O p t i c a l D e n s i t y = O p t i c a l D e n s i t y v a l u e / O p t i c a l D e n s i t y v a l u e f o r H S A - i o d o c y t i d i n e . 52 IP 20 30 40 50 60 70 80 90 fraction number Fig . f 1 . E l u t i o n o f MCA ( R l ) f r o m a s c i t e s f l u i d a f t e r a d s o r p t i o n on D E A E - S e p h a c e l c o l u m n . P l o t c o m p a r e s c o l u m n f r a c t i o n e l u a t e ( T ) , s c a n n e d a t 280 nm, t o a c t i v i t y o f c o l u m n f r a c t i o n s ( • ) , as d e t e r m i n e d by E L I S A , u s i n g c a r r i e r - i o d o c y t i d i n e as t h e a n t i g e n . 53 1 2 3 4 5 6 7 8 9 10 1112 13 myosin B-galactosidase phosphorylase B bovine albumin IgG heavy chain egg albumin carbonic anhydrase IgG light chain Fig.12 An 8% S D S - p o l y a c r y l a m i d e g e l o f f r a c t i o n s c o l l e c t e d f r o m t h e D EA E - S ep h a c e I c o l u m n d e s c r i bed i n F i g . 11 . S t a i n i ng was w i t h C o o m a s s i e B r i l l i a n t B l u e . L a n e s 1-5: p o o l e d f r a c t i o n s numbered 33-44 ( 2 . 5 , 5 , 1 0 , 1 5 , 2 0 u g ) ; l a n e s 6-10: p o o l e d f r a c t i o n s numbered 45-55 ( 2 . 5 , 5 , 1 0 , 1 5 , 2 0 u g ) : l a n e s 11-13: m o l e c u l a r w e i g h t s t a n d a r d ( 1 0 , 1 5 , 2 0 u g ) , as d e s c r i b e d i n l e g e n d t o s e e F i g.3. 54 Amount of coating antigen . ( h apten - c arrief) H S A -iodocyt idine H a p t e n - C a r r i e r ( O D 4 g 5 va lues from E L I S A ) H S A -cyt idine Ovalbumin iodocytidine Ovalbumin cytidine H S A - iodocytidine H S A - c y t idine 3.3 x 10 1 1.1 x 10" 1, 3.7 x 10"2 1.2 x 1 0 - 2 4.1 x 1 0 - 3 1.4 x 1 0 " 3 4.6 x 1 0 - 4 1.5 x 1 0 - 4 5.2 x 1 0 " 5 1.7 x 1 0 - 5 ug 6.0 x 10 - 6 2.874 2.892 2.880 2.648 2.337 1 .699 1 .001 .549 .357 .290 .269 .2 57 1.771 1.407 .727 .400 • 336 .333 .291 .286 .288 .295 .268 .270 2.192 2.088 2.034 1 .874 1 .543 .973 .606 .379 .302 .279 .268 .252 .170 .105 .082 .063 .061 .053 .061 .054 .049 .053 .0 57 .046 1.623 2 .055 3.961 6.620 6 .955 « s„va2.... 3.440 1.920 1.240 .983 1.003 . 9 59 T a b l e 4 S p e c i f i c i t y o f MCA R5 b i n d i n g w i t h v a r i o u s h a p t e n -c a r r i e r c o n j u g a t e s as d e t e r m i n e d b y s o l i d - p h a s e E L I S A . S i m i l i a r d a t a were c o l l e c t e d f o r a l l o f t h e d i f f e r e n t a n t i b o d i e s , a n d u s e d t o c o m p a r e t h e i r s p e c i f i c i t i e s ( F i g . 1 3 ) . The p o i n t ( i e . a m o u n t o f c o a t i n g antigen ) a t w h i c h d a t a was o b t a i n e d f o r t h e c o m p a r i s o n w e r e d e t e r m i n e d i n d i v i d u a l l y f o r e a c h a n t i s e r u m b y s e l e c t i n g t h e p o i n t w h e r e t h e d i f f e r e n c e b e t w e e n i o d o c y t i d i n e -c a r r i e r / c y t i d i n e - c a r r i e r ( c a r r i e r u s e d t o i m m u n i z e t h e m o u s e ) was t h e g r e a t e s t ( e g . * ) . comparative fraction _t ro co ^ cn cn -J oo <Q © Y Y Y Y Y Y Y Y Y Y o o a co r r "i e n rt-CD CD c CO fi-n -es 3 O Ci a o • a c SS CD CO CL CD ca o -i H-a" fD a. H-3 >-3 CO a" O a CD 5 zr o o sr cr CD o 4* H-• 3 3 C 3 H-N CD n-CD a i o CO "S H-CD \ O a ri-p-CD 3 a* 3 CO - r r H-O 03 3 o << -3 rt- 3 -H- CD H-3 -O - CD I—' I CS O r r O CB CD CA o a. o o >-b •< *S r r 03 H -O rt-el-s' CD 3 O C CO CD O H-Cfl O a o o •< CD X. <-i CD CD r r O sr o CD CO 3 «3 a H-> Oa. Cfl • t—' CO CO Cfl 3 O H-a i T3 T3 O 3" I—i (fl •< CO O CD I—' O P3 3 t-i CO >-i I—1 CO > o Cfl o CD 3 3 CD 3 T H-3 <W O r r *I 3* CD Cfl <: -o CO CD "1 o CD 3" S Cfl CD 3 Cfl -o •-s H - C ! cr ^ CD O O" r r << H -CX H-• rt- 3 3* CD CD I 3* H* o > cr w Cfl o 3" H- CO O X) Ou r r O O >•< r r C Cfl CD H-a. a 3 r f CD O I > cr CD Cfl o Cfl CD I—J CD o rt-CD (X •o < o 3 2L > cr > 73 M -* o o 73 ro > o 33 n 7) 4^  73 > 03 O O > CO o 5 3] n a =° o > DO O O J 59 B C R1 B C D A B C D A B C R2 R3 R4 monoclonal Abs B c R5 B C R6 B C R8 A B C D polyclonal Ab F i g . 1 4 I n h i b i t i o n o f b i n d i n g o f v a r i o u s MCAs and p o l y c l o n a l serum t o i o d o c y t i d i n e - H S A (10 ug/ml) by 75 n m o l e s o f : A) c y t i d i n e , B) i o d o c y t i d i n e , C) c y t i d i n e - 5 ' - p h o s p h a t e , D) i o d o c y t i d i n e - 5 ' - p h o s p h a t e . I n h i b i t i o n was a s s a y e d by s o l i d - p h a s e E L I S A . 57 100>-80> 70> 60> c o £ 50*-n m o l e s o f i n h i b i t o r F i g . 1 5 I n h i b i t i o n o f MCA R3 b i n d i n g t o i o d o c y t i d i n e - H S A (10 u g / m l ) by: A) c y t i d i n e ( A ) , B) i o d o c y t i d i n e ( • ) , C) c y t i d i n e -5 ' - p h o s p h a t e ( • ) , D) i o d o c y t i d i n e - 5 ' - p h o s p h a t e ( 0 ) . i n a s o l i d -p h a s e E L I S A . 58 was h e l d c o n s t a n t . From t h i s a s s a y t h e r e a p p e a r e d t o be f o u r c l o n e s , R l , R5, R6, and R8 w h i c h c o u l d be p o t e n t i a l l y u s e f u l i n d e t e c t i n g i o d o c y t i d i n e . The c l o n e s w e r e t h e n a n a l y z e d i n a b i n d i n g i n h i b i t i o n a s s a y ( F i g . 1 4 ) . The r e s u l t s were f o u n d t o c o n f l i c t w i t h t h o s e o b t a i n e d i n t h e a f f i n i t y a s s a y ( F i g . 1 3 ) , b e c a u s e o n l y R l a p p e a r e d t o be i n h i b i t e d by i o d o c y t i d i n e and i t s m o n o - p h o s p h a t e d e r i v a t i v e . H o wever, b e c a u s e R5, R6, and R8 had no a f f i n i t y f o r any o f t h e p r o t e i n a c e o u s c a r r i e r s , t h e y were p r o b a b l y r e a c t i n g w i t h an e p t i t o p e t h a t was r e p r e s e n t i n g some c o m b i n a t i o n o f t h e c a r r i e r a n d h a p t e n . R e s u l t s f r o m t h e i n h i b i t i o n a s s a y a l s o i n d i c a t e d t h a t t h e b i n d i n g o f R3 was a t l e a s t p a r t i a l l y i n h i b i t e d by i o d o c y t i d i n e . Thus t h e a s s a y was r e p e a t e d w i t h R3 u s i n g a b n o r m a l l y h i g h c o n c e n t r a t i o n s o f t h e i n h i b i t o r s ( F i g . 1 5 ) . The i n h i b i t i o n o f b i n d i n g was n o t i n c r e a s e d s i g n i f i c a n t l y e v e n w i t h v e r y h i g h l e v e l s o f i n h i b i t o r ; t h e r e f o r e , t h e b i n d i n g b e t w e e n R3 and i o d o c y t i d i n e must be one o f low s p e c i f i c i t y t h a t r e a d i l y i n t e r c h a n g e s w i t h o t h e r a n t i g e n s . MCA R l was t h e n s e l e c t e d f o r f u r t h e r e v a l u a t i o n o f i t s p o t e n t i a l t o be u s e d f o r d e t e c t i n g i o d o c y t i d i n e i n n u c l e i c a c i d . The a f f i n i t y a s s a y was r e p e a t e d u s i n g a l l t h e n u c l e o s i d e - c a r r i e r c o n j u g a t e s and t h e r e s u l t s were c o m p a r e d w i t h t h o s e o b t a i n e d w i t h p o l y c l o n a l s e r a ( F i g . 1 0 ) . R l a p p e a r e d t o h a v e a f f i n i t y o n l y f o r i o d o c y t i d i n e c o n j u g a t e s . An i n h i b i t i o n a s s a y was a l s o done u s i n g a r a n g e o f e a c h n u c l e o t i d e and n u c l e o s i d e ( F i g . 1 6 ) , and t h e r e s u l t s w e r e a l s o c ompared t o t h o s e o b t a i n e d w i t h p o l y c l o n a l s e r a ( F i g . 1 7 ) . I n h i b i t i o n was by f a r s t r o n g e s t when u s i n g 59 • i i o d o c y t i d i n e a n d i t s m o n o - p h o s p h a t e d e r i v a t i v e ( F i g . 1 6 a , F i g . 1 7 b ) , b u t a d e n o s i n e and i t s mon o - p h o s p h a t e d e r i v a t i v e a l s o a p p e a r e d t o i n h i b i t b i n d i n g ( F i g . 16b, F i g 1 7 b ) . Due t o t h i s a m b i g u i t y t h e d a t a were c o n v e r t e d t o a S c a t c h a r d p l o t , w h i c h a l l o w s f o r t h e c a l c u l a t i o n o f a b i n d i n g c o n s t a n t . T h i s i s b e s t e x p r e s s e d , as a r e l a t i v e v a l u e w h i c h i s s e p a r a t e l y c a l c u l a t e d f o r a l l members o f a s e t , and r e p r e s e n t s t h e r e l a t i v e s t r e n g t h o f a s s o c i a t i o n b e t w e e n two components ( F e r d i n a n d , 1 9 7 6 ) . The e q u a t i o n u s e d i n t h e c a l c u l a t i o n s was: B/Bo=X n/X +K , where B= s a m p l e o p t i c a l d e n s i t y ; Bo= o p t i c a l d e n s i t y when no i n h i b i t o r was u s e d ; X= n m o l e s o f i n h i b i t o r ; K= b i n d i n g c o n s t a n t ; n= number o f b i n d i n g s i t e s / m o l e c u l e . The r e s u l t s f r o m t h i s c a n be p l o t t e d i n two s e p a r a t e g r a p h s ( F i g . 1 8 ) . F i g . 18a, co m p a r e s t h e b i n d i n g c o n s t a n t s o f t h e n u c l e o t i d e s and n u c l e o s i d e s w i t h R l . The b i n d i n g o f i o d o c y t i d i n e and i o d o c y t i d i n e - 5 ' - p h o s p h a t e t o R l was a p p r o x i m a t e l y 20 and 200 t i m e s s t r o n g e r t h a n t h a t o f a d e n o s i n e o r a d e n o s i n e - 5 ' - p h o s p h a t e , r e s p e c t i v e l y . F i g . 18b, i s a r e c a l c u l a t i o n o f a p o r t i o n o f t h e d a t a p r e s e n t e d i n F i g . 16, u s i n g t h e a b o v e d e s c r i b e d e q u a t i o n . I n t h e c a l c u l a t i o n o f Bo a s e p a r a t e v a l u e was u s e d f o r ft.each i n h i b i t t i n g n u c l e o t i d e o r n u c l e o s i d e , w h e r e a s i n a l l t h e o t h e r c a l c u l a t i o n s o f % i n h i b i t i o n t h e v a l u e c o r r e s p o n d i n g t o Bo was an a v e r a g e o f t h e v a l u e s f r o m a l l t h e n e g a t i v e c o n t r o l s i n t h a t e x p e r i m e n t . The g r a p h c l e a r l y shows t h a t b i n d i n g s i t e s c a n n o t be s a t u r a t e d w i t h a d e n o s i n e o r a d e n o s i n e - 5 ' - p h o s p h a t e , r e g a r d l e s s o f t h e c o n c e n t r a t i o n o f i n h i b i t o r . T h i s i n d i c a t e s t h a t t h e b i n d i n g i s weak and t h a t t h e i n h i b i t o r w i l l r e a d i l y d i s s o c i a t e f r o m t h e a n t i b o d y . 6 0 MCA R l was t h e n a s s a y e d f o r i t s a b i l i t y t o b i n d t o i o d o c y t i d i n e i n c e l l u l a r RNA. An i n h i b i t i o n a s s a y was i n i t i a l l y u s e d w i t h i o d i n a t e d and n o n - i o d i n a t e d n u c l e i c a c i d as t h e i n h i b i t o r . Then t h e a s s a y was r e p e a t e d w i t h v a r i o u s enzyme d i g e s t i o n s o f t h e n u c l e i c a c i d i n o r d e r t o d e t e r m i n e w h e t h e r o r n o t s e c o n d a r y s t r u c t u r e o f t h e n u c l e i c a c i d had an i n h i b i t o r y e f f e c t on t h e b i n d i n g o f t h e MCA t o t h e a n t i g e n . Enzyme d i g e s t s were done i n b u f f e r s w h i c h p r o v i d e d c o n d i t i o n s o p t i m a l f o r d i g e s t i o n by t h e p a r t i c u l a r enzyme, and t h u s t h e i n h i b i t i o n a s s a y s were no l o n g e r b e i n g done i n PBS. To e n s u r e t h a t d i f f e r e n t b u f f e r s were riot i n t e r f e r i n g w i t h t h e i n h i b i t i o n a s s a y , i n h i b i t i o n a s s a y s w i t h a l l n u c l e o s i d e s and n u c l e o t i d e s w e re r e p e a t e d u s i n g t h e v a r i o u s d i f f e r e n t b u f f e r s . None o f t h e b u f f e r s was f o u n d t o i n t e r f e r e w i t h t h e a s s a y . A l l d i g e s t i o n s were done a t pH 6 o r g r e a t e r b e c a u s e p o l y C h a s b e e n shown t o have e x t e n s i v e s e c o n d a r y s t r u c t u r e b e l o w pH 6, w h i c h r e n d e r s i t r e s i s t a n t t o some o f t h e enzymes u s e d i n t h e p r e s e n t e x p e r i m e n t s ( L i m and R o b e r t s 1 9 8 2 ) . N u c l e a s e P I , w h i c h i s a n o n -s p e c i f i c e n d o n u c l e a s e , was u s e d t o p r o d u c e n u c l e o t i d e s w i t h a 5'~ p h o s p h a t e ( F u j i m o t o e t a l . , 1 9 7 4 ) . A l t h o u g h , t h e o p t i m a l a c t i v i t y o f n u c l e a s e P I i s a t pH 5, RNA was c o m p l e t e l y d i g e s t e d b y t h e enzyme u n d e r c o n d i t i o n s o f t h e i n h i b i t i o n a s s a y (pH 6) as d e t e r m i n e d by PAGE o f e n z y m e - t r e a t e d RNA. RNase A d i g e s t i o n s w e r e done i n l o w s a l t b e c a u s e i t was f o u n d t h a t u n d e r t h e s e c o n d i t i o n s t h e enzyme w o u l d d i g e s t d o u b l e - s t r a n d e d r e g i o n s o r r e g i o n s o f c o m p l e x s e c o n d a r y s t r u c t u r e i n t h e RNA w h i c h a r e r e s i s t a n t t o d i g e s t i o n i n h i g h s a l t ( H a m i l t o n e t a l . , 1 9 8 0 ) . T h i s enzyme d i g e s t s p r e f e r e n t i a l l y a t t h e 3'-end o f p y r i m i d i n e n u c l e o t i d e s . 61 A l s o u s e d was RNAse T I , w h i c h c l e a v e s p r e f e r e n t i a l l y a t t h e 3'-e n d o f g u a n o s i n e r e s i d u e s , b u t w i l l c l e a v e o n l y i n s i n g l e -s t r a n d e d r e g i o n s ( L i m and R o b e r t s , 1 9 8 2 ) . R e s u l t s o b t a i n e d when, i o d i n a t e d and n o n - i o d i n a t e d p o l y C w e r e u s e d as i n h i b i t o r s a r e shown i n F i g . 19. P o l y C d i d n o t i n h i b i t b i n d i n g e i t h e r i n i t s n a t i v e f o r m o r when d i g e s t e d t o n u c l e o t i d e s . T h e s e r e s u l t s c o r r e l a t e w i t h t h o s e o b t a i n e d when c y t i d i n e e i t h e r as n u c l e o t i d e o r c y t o s i n e n u c l e o s i d e was u s e d as i n h i b i t o r . I o d o - p o l y C p r o d u c e d a maximum o f a b o u t 50% i n h i b i t i o n , w h i c h c o u l d n o t be e x c e e d e d a t h i g h e r i n h i b i t o r c o n c e n t r a t i o n s . B u t when i o d o - p o l y C was d i g e s t e d w i t h v a r i o u s r i b o n u c l e a s e s t h e n u c l e o t i d e p r o d u c t s c o m p l e t e l y i n h i b i t e d b i n d i n g o f R l . S i m i l i a r r e s u l t s were o b s e r v e d when i o d i n a t e d RNA was u s e d as t h e i n h i b i t o r ( F i g . 2 0 ) . B o t h n u c l e i c a c i d s showed i n c r e a s e d i n h i b i t i o n upon RNase TI d i g e s t i o n , b u t maximum i n h i b i t i o n was o n l y a p p r o a c h e d when a n u c l e a s e , t h e m echanism o f a c t i o n o f w h i c h was n o t d e p e n d e n t on RNA s t r u c t u r e , was u s e d . T h e r e was a h i g h e r l e v e l o f i n h i b i t i o n by e q u a l q u a n t i t i e s o f n u c l e a s e P l - d i g e s t e d p r o d u c t s t h a n by s i m i l i a r RNase A d i g e s t s , e v e n t h o u g h b o t h enzymes d i g e s t RNA w i t h s e c o n d a r y s t r u c t u r e . T h i r t y n m o l e s o r 15 ug o f i o d o c y t i d i n e - 5 ' - p h o s p h a t e a l l o w e d f o r maximum i n h i b i t i o n o f R l , and 100 ug o f n u c l e a s e P l - d i g e s t e d i o d o - p o l y C was r e q u i r e d t o a c h i e v e t h e same l e v e l o f i n h i b i t i o n . T h e r e f o r e , a p p r o x i m a t e l y one i n e v e r y s e v e n ( 1 0 0 / 1 5 ) n u c l e o t i d e s i n i o d o - p o l y C was i o d i n a t e d . The maximum l e v e l o f i n h i b i t i o n o f R l w i t h n u c l e a s e - P l d i g e s t e d i o d o - R N A o c c u r e d w i t h 800 ug o f i n h i b i t o r . Thus one i n e v e r y f i f t y - t h r e e n u c l e o t i d e s was 6 2 i o d i n a t e d . A s s u m i n g t h a t e a c h o f t h e f o u r b a s e s : c y t i d i n e , u r i d i n e , g u a n o s i n e , and a d e n o s i n e , r e p r e s e n t e d o n e - q u a r t e r o f t h e t o t a l n u c l e o t i d e s i n t h e RNA, a p p r o x i m a t e l y one i n e v e r y t h i r t e e n c y t i d i n e r e s i d u e s ( [ 80-0/15 ]'/4) was i o d i n a t e d . As c a n be s e e n ( F i g . 19, F i g . 20) e n z y m e - d i g e s t e d , i o d i n a t e d RNA r e s u l t e d i n g r e a t e r i n h i b i t i o n o f R l t h a n an e q u a l q u a n t i t y o f u n d i g e s t e d i o d i n a t e d RNA, i n d i c a t i n g t h a t i n t h e i n t a c t r i b o -n u c l e o t i d e p o l y m e r some o f t h e p o t e n t i a l a n t i g e n i c s i t e s w e r e n o t a v a i l a b l e f o r r e a c t i o n w i t h t h e a n t i b o d y . Even t h o u g h t h e RNA was h e a t - d e n a t u r e d i t was p r e s u m e d t h a t d e n a t u r a t i o n may n o t h a v e b e e n c o m p l e t e , and t h a t s e c o n d a r y s t r u c t u r e o f t h e RNA was p r e v e n t i n g some o f t h e p o t e n t i a l a n t i g e n - a n t i b o d y r e a c t i o n s . I t w o u l d h a v e been b e n e f i c i a l t o d e t e r m i n e t o what e x t e n t s e c o n d a r y s t r u c t u r e was i n t e r f e r i n g w i t h t h e i m m u n o l o g i c a l r e a c t i o n . V a r i o u s m ethods o f c h e m i c a l d e n a t u r a t i o n o f RNA w e r e i n v e s t i g a t e d , b u t most were f o u n d t o r e a c t i n d i s c r i m i n a t e l y w i t h t h e b a s e s , c a u s i n g c o n s i d e r a b l e a l t e r a t i o n s o f t h e o r i g i n a l c h e m i c a l s t r u c t u r e ( L e h r a c h e t a l . , 1 9 7 1 ) . G l y o x a l , w h i c h h a s b e e n shown t o c h e m i c a l l y d e n a t u r e RNA ( C a r m i c h a e l , 1 9 8 0 ) , p r o d u c e d a s t a b l e r e a c t i o n p r o d u c t o n l y w i t h g u a n o s i n e u n d e r t h e c o n d i t i o n s o f t h e r e a c t i o n ( N a k a y a e t a l . , 1968; B r o u d e a n d Budowsky, 1 9 7 1 ) . The g l y o x a l a t i o n r e a c t i o n was f i r s t done w i t h t h e f o u r n u c l e o s i d e s n o r m a l l y f o u n d i n RNA, and w i t h i o d o c y t i d i n e , t h e e f f e c t s o f t h e r e a c t i o n b e i n g m o n i t o r e d b y UV s p e c t r o s c o p y . O n l y g u a n o s i n e showed a s p e c t r a l s h i f t as a r e s u l t o f t h e r e a c t i o n , t h u s i t was assumed t h a t t h e r e a c t i o n was i n d e e d s p e c i f i c . However, when g l y o x a l a t e d iodo-RNA was u s e d i n t h e i n h i b i t i o n a s s a y , t h e r e was no s i g n i f i c a n t i n c r e a s e i n t h e l e v e l iooH n m o l e s o f I n h i b i t o r 64 C) 100>" 80»-c o !5 60*-c 100 200 300 nmoles of Inhibitor F i g . 1 6 I n h i b i t i o n o f MCA R l b i n d i n g t o i o d o c y t i d i n e - H S A (10 ug / m l ) w i t h v a r y i n g c o n c e n t r a t i o n s o f t h e n u c l e o s i d e s : a) c y t i d i n e ( I ) , c y t i d i n e - 5 ' - p h o s p h a t e ( T ) , i o d o c y t i d i n e ( • ) , i o d o c y t i d i n e - 5 ' - p h o s p h a t e ( X ) ; b) a d e n o s i n e ( T ) , a d e n o s i n e - 5 ' -p h o s p h a t e ( X ) , g uanos i n e ( • ) , gu a n o s i n e - 5 ' - p h o s p h a t e ( • ); c ) t h y m i d i n e ( • ) , t h y m i d i n e - 5 ' - p h o s p h a t e ( tfr ) , u r i d i n e ( X ) , u r i d i n e - 5 ' - p h o s p h a t e ( T ) , i o d o u r i d i n e ( • ) . A s s a y was done b y s o l i d - p h a s e E L I S A . a) 100> >• 80> >• c o 60*-15 *-c 40»> *• 20*-*-F G H I n h i b i t o r b) 100*->• 80*-*-c o 60** '3 *-IE c 401* e* *-20*-»-F G H I n h i b i t o r F i g . 1 7 I n h i b i t i o n o f b i n d i n g t o i o d o c y t i d i n e - H S A (10 ug/ml) by: a) p o l y c l o n a l s e r a and b) MCA R l i n t h e p r e s e n c e o f 75 n m o l e s o f : A) c y t i d i n e , B) i o d o c y t i d i n e , C) c y t i d i n e - 5 ' - p h o s p h a t e , D) i o d o c y t i d i n e - 5 ' - p h o s p h a t e , E) a d e n o s i n e , F) a d e n o s i n e - 5 ' -p h o s p h a t e , G) g u a n o s i n e , H) g u a n o s i n e - 5 ' - p h o s p h a t e , I ) t h y m i d i n e , J ) t h y m i d i n e - 5 ' - p h o s p h a t e , K) u r i d i n e , L) u r i d i n e - 5 ' - p h o s p h a t e , M) i o d o u r i d i n e . I n h i b i t i o n was a s s a y e d by s o l i d - p h a s e E L I S A . a) 66 •o c A B C D E F G H I J h • L M I n h i b i t o r b) 100 200 300 n m o l e s o f I n h i b i t o r Fig . 1 8 a) C o m p a r i s o n o f t h e b i n d i n g c o n s t a n t o f MCA R l w i t h t h e f o l l o w i n g n u c l e o s i d e s : A) c y t i d i n e , B) i o d o c y t i d i n e , C) c y t i d i n e -5 ' p h o s p h a t e , D) i o d o c y t i d i n e - 5 ' - p h o s p h a t e , E) a d e n o s i n e , F) a d e n o s i n e - 5 ' - p h o s p h a t e , G) g u a n o s i n e , H) g u a n o s i n e - 5 ' - p h o s p h a t e , I ) t h y m i d i n e , J ) t h y m i d i n e - 5 ' - p h o s p h a t e , K) u r i d i n e , L) u r i d i n e -5 ' - p h o s p h a t e , M) i o d o u r i d i n e . b) O p t i c a l d e n s i t y o f MCA R l when i n h i b i t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f n u c l e o s i d e s : c y t i d i n e ( + ) , i o d o c y t i d i n e ( T ) , c y t i d i n e - 5 ' - p h o s p h a t e ( X ) , i o d o c y t i d i n e - 5 ' - p h o s p h a t e ( • ) , a d e n o s i n e (*) , a d e n o s i n e - 5 ' - p h o s p h a t e (•) . 67 a) 100> 200 400 600 800 u g o f I n h i b i t o r b) :  200 400 600 800 u g o f I n h i b i t o r F i g . 1 9 a) I n h i b i t i o n o f MCA R l b i n d i n g w i t h p o l y - C ( # ) and i o d o p o l y - C ( • ) , and w i t h RNase A d i g e s t e d p o l y - C ( • ) and i o d o p o l y - C (*) . : b) I n h i b i t i o n o f MCA R l b i n d i n g w i t h u n d i g e s t e d i o d o p o l y - C (•) , and i o d o p o l y - C d i g e s t e d w i t h RNase A (*) , RNase TI ( • ) , and N u c l e a s e P I (T). B o t h a s s a y s were s o l i d - p h a s e E L I S A , u s i n g HSA-i o d o c y t i d i n e ( a t 10 ug/ml) as t h e . c o a t i n g / a n t i g e n . 68 10C*-ug of Inhibitor F i g . 2 0 I n h i b i t i o n o f MCA R l b i n d i n g t o i o d o c y t i d i n e - H S A ( 10 u g / r a l ) w i t h u n d i g e s t e d ( • ) , and RNase A- ( T ) , RNase T I - ( ) . N u c l e a s e P I - ($|fr) d i g e s t e d i o d i n a t e d c e l l u l a r RNA. C o n t r o l v a l u e s (RNA d i g e s t e d i n d i v i d u a l l y w i t h e a c h enzyme) a r e r e p r e s e n t e d by a s i n g l e l i n e ( • ) , b e c a u s e v a l u e s a t any p o i n t v a r i e d by l e s s t h a n 5% o f a v e r a g e OD f o r any o f t h e enzyme d i g e s t i o n s . A s s a y s w e r e s o l i d - p h a s e E L I S A . 69 o f i n h i b i t i o n as c o m p a r e d t o iodo-RNA w h i c h was d e n a t u r e d o n l y by h e a t i n g . Membrane B l o t t i n g A s s a y o f N u c l e o t i d e s and ssRNA I t was f o u n d t h a t t h e s e n s i t i v i t y o f d e t e c t i n g t h e a n t i g e n on n i t r o c e l l u l o s e o r n y l o n membranes was d i r e c t l y d e p e n d e n t on t h e p a r t i c u l a r p r e c i p i t a t i n g s u b s t r a t e and s e c o n d a r y a n t i b o d y b e i n g u s e d . P h o s p h a t a s e - c o n j u g a t e d s e c o n d a r y a n t i b o d y was u s e d w i t h F a s t Red, and p e r o x i d a s e c o n j u g a t e s were u s e d w i t h 3-3'-d i a m i n o b e n z i d i n e , and w i t h 4 - c h l o r o - l - n a p h t h o l . The l a t t e r was f o u n d t o h a v e t h e l o w e s t s i g n a l - t o - n o i s e r a t i o , and was t h e f a s t e s t t o d e v e l o p c o l o r . However, i t has t h e d r a w b a c k o f l o s i n g i t s c o l o r q u i c k l y i n t h e p r e s e n c e o f l i g h t . I o d o c y t i d i n e - c a r r i e r c o n j u g a t e s were d e t e c t e d , w i t h o u t a n y c r o s s r e a c t i o n w i t h o t h e r n u c 1 e o s i d e - c a r r i e r s , o r t h e c a r r i e r s t h e m s e l v e s , a t a l e v e l o f l e s s t h a n f i v e nanograms by R l i n t h e membrane b i n d i n g a s s a y ( F i g . 2 1 ) . I n t h e same a s s a y , i o d i n a t e d n u c l e i c a c i d was d e t e c t a b l e a t l e s s t h a n o n e - h u n d r e d n a n o g r a m s by R l , w h i l e n o n - i o d i n a t e d n u c l e i c a c i d was n o t d e t e c t e d a t a l l ( F i g . 2 2 ) . D e t e c t i o n o f i o d o - p o l y C was s l i g h t l y b e t t e r t h a n i o d i n a t e d - R N A , w h i c h i s t o be e x p e c t e d s i n c e i o d o - p o l y C c o n t a i n s more i o d i n a t e d n u c l e o t i d e s p e r u n i t o f RNA. When i o d i n a t e d c e l l u l a r RNA was s t a i n e d w i t h e t h i d i u m b r o m i d e a f t e r e l e c t r o p h o r e s i s i n a u r e a p o l y a c r y l a m i d e g e l , a b r o a d z o n e was d e t e c t e d , r e p r e s e n t i n g p o l y n u c l e o t i d e s v a r y i n g f r o m a p p r o x i m a t e l y 50-700 n u c l e o t i d e s i n l e n g t h . I o d o - p o l y C was n o t s t a i n a b l e w i t h e t h i d i u m b r o m i d e , b u t t o l u i d i n e b l u e s t a i n i n g 70 HSA-cytidine HSA-iodocytidine Fig.21 Immunoblot showing b i n d i n g of MCA R l to v a r i o u s amounts (ug) of i o d o c y t i d i n e - H S A c o n j u g a t e . R e a c t i o n was d e v e l o p e d w i t h goat anti-mouse p e r o x i d a s e - c o n j u g a t e u s i n g 4 - c h l o r o - l - n a p h t h o l as the s u b s t r a t e . F i g . 2 2 Immunoblot showing b i n d i n g o f MCA R l to v a r i o u s amounts o f i o d i n a t e d c e l l u l a r RNA. R e a c t i o n was dev e l o p e d w i t h goat a n t i -mouse p e r o x i d a s e - c o n j u g a t e u s i n g 4 - c h l o r o - l - n a p h t o l as the s u b s t r a t e . 72 r e s u l t e d i n a s i m i l i a r zone r e p r e s e n t i n g a range of s i z e s to a pproximately 350 n u c l e o t i d e s . When i o d i n a t e d RNA was e l e c t r o p h o r e t i c a l l y t r a n s f e r e d from the u r e a - d e n a t u r i n g g e l to a membrane, MCA R l b i n d i n g to the i o d i n a t e d n u c l e i c a c i d was observed throughout the m olecular weight range of the RNA. T h i s i n d i c a t e d that the i o d i n a t i o n r e a c t i o n was not d i s c r i m i n a t o r y to any p a r t i c u l a r l e n g t h of RNA. B i d d i n g of I o d i n a t e d RNA to ELISA P l a t e s I t was found that RNA c o u l d not be bound to any of the ELISA p l a t e s t r i e d , whether or not they were coated with any of the macromolecules supposedly used f o r enhancing the b i n d i n g between n u c l e i c a c i d s and the p o l y s t y r e n e s u r f a c e of the p l a t e s . 73 D i s c u s s i o n The p r e s e n t s t u d y d e s c r i b e d t h e m o d i f i c a t i o n and r e f i n e m e n t o f an e s t a b l i s h e d p r o c e d u r e f o r i o d i n a t i o n o f n u c l e i c a c i d t o a l l o w t h e r e p r o d u c i b l e m o d i f i c a t i o n o f n u c l e i c a c i d i n q u a n t i t i e s ( m i c r o g r a m s t o m i l l i g r a m s ) s u f f i c i e n t f o r r e s e a r c h and d i a g n o s t i c p u r p o s e s . H y b r i d o m a s s e c r e t i n g m o n o c l o n a l a n t i b o d i e s r e a c t i n g s p e c i f i c a l l y w i t h t h e m o d i f i e d n u c l e i c a c i d were i s o l a t e d , and t h e i r s p e c i f i c i t y and r e a c t i v i t y were c h a r a c t e r i z e d u n d e r a v a r i e t y o f c o n d i t i o n s . The i o d i n a t i o n r e a c t i o n d e s c r i b e d by C o m m e r f o r d ( 1 9 7 1 ) , was a l t e r e d and r e f i n e d t o a l l o w r e p r o d u c i b l e m o d i f i c a t i o n o f RNA a t v a r y i n g c o n c e n t r a t i o n s . I n t h e p r e s e n t s t u d y , i t was o b s e r v e d t h a t c e l l u l a r RNA c o n s i s t e n t l y had l e s s i o d i d e i n c o r p o r a t e d t h a n v i r a l RNA ( T a b l e 1 ) . T h i s d e c r e a s e was p o s t u l a t e d t o be a r e s u l t o f t h e s e c o n d a r y s t r u c t u r e o f RNA s t e r i c a l l y i n t e r f e r i n g w i t h t h e i o d i n a t i o n r e a c t i o n . T h i s w o u l d a p p l y more t o c e l l u l a r RNA t h a n v i r a l RNA b e c a u s e t o t a l c e l l u l a r RNA i s composed l a r g e l y o f r i b o s o m a l - R N A (rRNA) and t r a n s f e r - R N A ( t RNA) ( W h i t e e t a l . , 1 9 6 8 ) , b o t h o f w h i c h a r e c h a r a c t e r i z e d by c o n s i d e r a b l e s e c o n d a r y s t r u c t u r e ( S a e n g e r , 1 9 8 3 ) . A l t h o u g h t h e n u c l e i c a c i d i s d e n a t u r e d p r i o r t o t h e r e a c t i o n , t h e s e c o n d a r y s t r u c t u r e i s p a r t i a l l y r e f o r m e d a t t h e t e m p e r a t u r e o f t h e i o d i n a t i o n r e a c t i o n ( S t a n l e y " and^sr V a s s e l i n k o , 1 9 7 8 ) . F u r t h e r m o r e , g r e a t e r t h a n 7 5 % o f t h e c e l l u l a r RNA was e s t i m a t e d by g e l e l e c t r o p h o r e s i s t o be l e s s t h a n o n e - h u n d r e d f i f t y b a s e s i n l e n g t h , and RNA o f t h i s s i z e r e f o r m s s e c o n d a r y s t r u c t u r e f a s t e r t h a n l a r g e r p i e c e s o f RNA ( i e . v i r a l -74 RNA) ( B r i t t e n a n d Kohne, 1 9 6 8 ) . S i n c e C o m m e r f o r d ( 1 9 7 1 ) showed t h a t s e c o n d a r y s t r u c t u r e i n t e r f e r e d e x t e n s i v e l y w i t h t h e i o d i n a t i o n r e a c t i o n u s i n g DNA as a s u b s t r a t e , i t i s r e a s o n a b l e t o assume t h a t t h i s e f f e c t w o u l d be m a g n i f i e d when RNA i s u s e d as a s u b s t r a t e b e c a u s e RNA has more c o m p l e x s t r u c t u r e t h a n DNA ( S a e n g e r , 1 9 8 3 ) . The i o d i n a t i o n o f RNA d e r i v e d f r o m d u p l e x m o l e c u l e s o f dsRNA was a l s o a t t e m p t e d , b e c a u s e t h i s w o u l d have p e r m i t t e d t h e d e t e r m i n a t i o n o f t h e r e a c t i v i t y o f t h e MCAs w i t h i o d o c y t i d i n e i n a d u p l e x s t r u c t u r e ( i e . s i m i l i a r t o t h e s t r u c t u r e f o r m e d i n a n u c l e i c a c i d h y b r i d i z a t i o n ) . I o d i n a t i o n o f t h i s m a t e r i a l was n o t d e t e c t a b l e by t h e methods u s e d h e r e . C o m m e r f o r d ( 1 9 7 1 ) r e p o r t e d t h a t dsDNA c a n be i o d i n a t e d o n l y a f t e r i t has b e e n c o m p l e t e l y d e n a t u r e d . However, i t was f o u n d i n t h i s s t u d y t h a t some dsRNAs were d i f f i c u l t t o d e n a t u r e ( u n p u b l i s h e d r e s u l t s ) , and t h u s i t i s p o s s i b l e t h a t t h e r e a c t i o n f a i l e d b e c a u s e t h e dsRNA was i n c o m p l e t e l y o r n o t a t a l l d e n a t u r e d . Of t h e h y b r i d o m a s s c r e e n e d , many were f o u n d t o r e a c t w i t h t h e i m m u n i z i n g c a r r i e r - c o n j u g a t e c o m b i n a t i o n ; h o w e v e r , o n l y a s m a l l p e r c e n t a g e ( l e s s t h a n 1) o f t h e c l o n e s a p p e a r e d t o be s e c r e t i n g a n t i b o d i e s w h i c h r e a c t e d s p e c i f i c a l l y w i t h i o d o c y t i d i n e ( F i g s . 13 and 14).- S e v e n o f t h e more s p e c i f i c c l o n e s were s e l e c t e d f o r f u r t h e r c h a r a c t e r i z a t i o n , and o f t h e s e , o n l y one ( R l ) f o r m e d a s p e c i f i c a n t i g e n - a n t i b o d y c o m p l e x w i t h i o d o c y t i d i n e . E v e n R l bound p r e f e r e n t i a l l y t o t h e 5 ' - p h o s p h a t e d e r i v a t i v e o f i o d o c y t i d i n e ( F i g . 1 6 ) . T h i s was p o s t u l a t e d t o be a r e s u l t o f t h e s i z e d i f f e r e n c e b e t w e e n t h e d e s i r e d a n t i g e n ( i e . i o d o c y t i d i n e ) and t h e a n t i g e n - a n t i b o d y b i n d i n g s i t e . The s i t e o f 75 i i n t e r a c t i o n on an i m m u n o g l o b u l i n m o l e c u l e i s a p p r o x i m a t e l y 20 x o o 25 A ( A m i t e t a l . , 1 9 8 5 ) , o r , t h r e e - d i m e n s i o n a l l y , 15 x 20 x 12 A ( S e g a l e t a l . , 1 9 7 4 ) , w h e r e a s t h e t h r e e - d i m e n s i o n a l s p a c e o o c c u p i e d by an a v e r a g e n u c l e o s i d e i s a p p r o x i m a t e l y 5 x 5 x 3 A ( M a h l e r and C o r d e s , 1 9 6 6 ) . I t has b een shown t h a t b o t h d i n u c l e o t i d e s (Khan e t a l . , 1 9 7 7 ) , and t r i n u c l e o t i d e s ( K h a n and J a c o b , 1977) c a n be u s e d t o p r o d u c e a n t i b o d i e s w h i c h a r e s p e c i f i c f o r t h e i m m u n i z i n g s e q u e n c e . J o h n s t o n and S t o l l a r ( 1 9 7 8 ) , f o u n d t h a t when a n t i b o d i e s a r e p r o d u c e d a g a i n s t dsRNA t h e t y p i c a l a n t i g e n i c s i t e s p a n s f o u r b a s e p a i r s . Thus i t was n o t s u r p r i s i n g t h a t t h e m a j o r i t y o f t h e c l o n e s p r o d u c e d a n t i b o d i e s w h i c h b o u n d t o a l a r g e r a n t i g e n i c s i t e t h a n i o d o c y t i d i n e . MCA R l b o u n d t o i o d o c y t i d i n e and i t s 5 ' - p h o s p h a t e d e r i v a t i v e v e r y s p e c i f i c a l l y , s h o w i n g no a p p r e c i a b l e c r o s s - r e a c t i o n w i t h e i t h e r t h y m i d i n e , i o d o u r i d i n e , o r t h e i r r e s p e c t i v e 5 ' - p h o s p h a t e d e r i v a t i v e s ; e v e n t h o u g h , t h e i r t h r e e - d i m e n s i o n a l s t r u c t u r e s a r e c l o s e l y r e l a t e d . T h i s i n d i c a t e d t h a t t h e s u b s t i t u t i o n a t C-5 was n o t t h e o n l y e p i t o p e i n v o l v e d i n t h e a n t i g e n i c r e a c t i o n . From t h e r e s u l t s o f t h e i n h i b i t i o n a s s a y ( F i g s . 19 and 20) w h e r e b i n d i n g o f MCA R l t o i o d o c y t i d i n e - H S A was i n h i b i t e d w i t h i o d i n a t e d p o l y C and c e l l u l a r RNA, i t was e v i d e n t t h a t b o t h t h e s e c o n d a r y s t r u c t u r e o f t h e RNA and t h e s t e r i c h i n d r a n c e o f t h e p o l y n u c l e o t ide.»&!interf e r e d i n t h e a n t i b o d y - a n t i g e n i n t e r a c t i o n . When t h e i o d i n a t e d RNA was d i g e s t e d w i t h e i t h e r P I n u c l e a s e o r RNase A ( t w o r i b o n u c l e a s e s w h i c h d i g e s t RNA w i t h s e c o n d a r y s t r u c t u r e ) p r i o r t o t h e i n h i b i t i o n a s s a y , i t s i n h i b i t o r y e f f e c t on b i n d i n g o f MCA R l i n c r e a s e d . I t was n o t e d t h a t i o d i n a t e d 76 n u c l e i c a c i d d i g e s t e d by PI n u c l e a s e had a g r e a t e r i n h i b i t o r y e f f e c t than t h o s e d i g e s t e d by RNase A. This.was a t t r i b u t e d t o one of two f a c t o r s , or t h e i r combined e f f e c t s . F i r s t l y , RNase A p r e f e r e n t i a l l y h y d r o l y z e s p y r i m i d i n e n u c l e o t i d e s whereas PI n u c l e a s e has no s p e c i f i c s u b s t r a t e (Lim and R o b e r t s , 1982), and thus RNase A d i g e s t i o n may not r e s u l t i n complete breakdown o f the RNA. S e c o n d l y , i t i s p o s s i b l e t h a t the 3'-phosphate d i g e s t i o n p r o d u c t of RNase A i s not as good an i n h i b i t o r of MCA R l b i n d i n g as i s the 5'-phosphate n u c l e o t i d e produced by PI n u c l e a s e d i g e s t i o n . The l a t t e r c o u l d not be e m p i r i c a l l y t e s t e d because the 3'-phosphate d e r i v a t i v e o f i o d o c y t i d i n e i s not c o m m e r c i a l l y a v a i l a b l e . D i g e s t i o n o f i o d i n a t e d c e l l u l a r RNA by RNase T I , an enzyme which d i g e s t s o n l y s i n g l e - s t r a n d e d RNA and not r e g i o n s o f h i g h e i — o r d e r s t r u c t u r e (Lim and R o b e r t s , 1982), r e s u l t e d i n i n c r e a s e d i n h i b i t i o n o f MCA R l b i n d i n g to i o d o c y t i d i n e - H S A as compared to t h a t o f u n d i g e s t e d i o d i n a t e d RNA. However, the i n h i b i t i o n o f RNase T l - d i g e s t e d n u c l e i c a c i d was a p p r o x i m a t e l y one t h i r d t h a t of the same n u c l e i c a c i d d i g e s t e d by n u c l e a s e P I . T h i s i n d i c a t e s t h a t secondary s t r u c t u r e of the n u c l e i c a c i d p r e v e n t e d b i n d i n g of the MCA to p o t e n t i a l e p i t o p e s i n the n u c l e i c a c i d . However at the e q u i v a l e n t t o t a l n u c l e i c a c i d c o n c e n t r a t i o n , t h e e f f e c t on i n h i b i t i o n was a p p r o x i m a t e l y one q u a r t e r o f t h a t when n u c l e i c a c i d was d i g e s t e d w i t h PI n u c l e a s e ( 400 ug of n u c l e i c a c i d , F i g . 2 0 ) , but an e f f e c t due to s t e r i c i n t e r f e r e n c e was e x p e c t e d ( L e a r y et a l . , 1983). Attempts were made t o r e p e a t the i n h i b i t i o n a s s a y s u s i n g i o d i n a t e d RNA de n a t u r e d by g l y o x a l a t i o n , a c o n t r o l l e d c h e m i c a l r e a c t i o n , but the d e n a t u r a t i o n d i d not appear t o be s u c c e s s f u l . 77 The r e a c t i o n was c o n t r o l l e d t o a l t e r o n l y g u a n o s i n e r e s i d u e s , and i t i s p o s s i b l e t h a t t h i s a l t e r a t i o n a l o n e was n o t e n o u g h t o c a u s e s i g n i f i c a n t b r e a k d o w n o f t h e s e c o n d a r y s t r u c t u r e o f t h e n u c l e i c a c i d . I t i s a l s o p o s s i b l e t h a t t h e d e n a t u r a n t r e a c t e d w i t h o t h e r n u c l e o t i d e s , i n c l u d i n g i o d o c y t i d i n e , b u t t h e m o d i f i c a t i o n was n o t o f a s u f f i c i e n t l e v e l t o be d e t e c t e d by UV s p e c t r o s c o p y . The l i m i t o f d e t e c t i o n o f i o d i n a t e d n u c l e i c a c i d i n a membrane b i n d i n g a s s a y was f o u n d t o be i n t h e n a nogram r a n g e ( F i g . 2 2 ) . The r e a c t i o n was s p e c i f i c and t h e r e was no d e t e c t a b l e c r o s s - r e a c t i o n w i t h n o n - i o d i n a t e d n u c l e i c a c i d . The s e n s i t i v i t y c o u l d p r o b a b l y have been i n c r e a s e d by u s i n g v a r i o u s i m m u n o l o g i c a l a m p l i f y i n g t e c h n i q u e s ; f o r e x a m p l e , an a v i d i n - b i o t i n p e r o x i d a s e c o n j u g a t e as a s e c o n d a r y d e t e c t i o n s y s t e m ( M a d r i and B a r w i c k , 1 9 8 3 ) . A l t h o u g h c h e m i c a l d e n a t u r a t i o n o f t h e n u c l e i c a c i d b e f o r e b i n d i n g i t t o t h e membrane m i g h t have i n c r e a s e d s e n s i t i v i t y o f a b i n d i n g a s s a y , h e a t d e n a t u r a t i o n was u s e d b e c a u s e o f r e p o r t s ( M a u l e e t a l . , 1983; M c M a s t e r and C a r m i c h a e l , 1977; and Thomas, 1980) i n d i c a t i n g d e c r e a s e d b i n d i n g o f c h e m i c a l l y d e n a t u r e d RNA t o n i t r o c e l l u l o s e membranes. A l t h o u g h i t was n o t d i r e c t l y a s s e s s e d i n t h i s work b e c a u s e o f t i m e c o n s t r a i n t s , t h e r e i s e v i d e n c e o f a n t i b o d i e s b i n d i n g t o s p e c i f i c n u c l e o t i d e s i n t h e d u p l e x s t r u c t u r e o f p o l y n u c l e o t i d e s ( S a w i c k i e t a l . , 1971 and B a l l e t a l . , 1 9 8 3 ) . As p r e v i o u s l y s t a t e d , t h i s p r o c e d u r e w o u l d be v e r y a p p l i c a b l e i n d e t e c t i n g v i r a l p l a n t p a t h o g e n s b e c a u s e t h e m a j o r i t y o f them h a v e a ssRNA genome. The v i r a l RNA c a n be m o d i f i e d on a l a r g e s c a l e ( i e . m i l l i g r a m s ) and i s s t a b l e i n t h e m o d i f i e d f o r m i n d e f i n i t e l y i f 78 s t o r e d d r y . The i o d i n a t e d RNA c o u l d t h u s be u s e d as a h y b r i d i z a t i o n p r o b e . I f a r e c o m b i n a n t DNA o f t h e v i r u s i s a v a i l a b l e , t h e n i t c o u l d be u s e d i n e i t h e r t h e b a c t e r i o p h a g e SP6 o r T7 p o l y m e r a s e s y s t e m t o g e n e r a t e t r a n s c r i p t s w i t h i o d o c y t i d i n e i n c o r p o r a t e d i n t h e p l a c e o f a t l e a s t some o f t h e c y t i d i n e r e s i d u e s ( A x e l r o d and K r a m e r , 1 9 8 5 ) . T h i s t e c h n i q u e i s h o t p r a c t i c a l l y a p p l i c a b l e f o r d e t e c t i n g p l a n t v i r u s e s w h i c h h a v e a low t i t r e i n t h e i r h o s t , and most v i r u s e s w i t h a h i g h t i t r e c a n be d e t e c t e d by s t a n d a r d s e r o l o g i c a l m e t h o d s . However, e v e n t h o u g h s e r o l o g y i s u s u a l l y t h e f a s t e s t and s i m p l e s t t e c h n i q u e i n a l a r g e s c a l e p r a c t i c a l a p p l i c a t i o n , t h i s i s n o t a l w a y s t h e c a s e . F o r e x a m p l e , i n d i s e a s e s c a u s e d by v i r o i d s (Owens and D i e n e r , 1981) and i n i n f e c t i o n s c a u s e d by i n c o m p l e t e o r d e f e c t i v e v i r u s e s t h e r e a r e no s t r u c t u r a l p r o t e i n s a s s o c i a t e d w i t h d i s e a s e d t i s s u e s ( H a r r i s o n e t a l . , 1 9 8 3 ) . I n o t h e r v i r a l d i s e a s e s t h e v i r u s c a n n o t be i s o l a t e d , b u t t h e g e n o m i c r e p l i c a t i v e f o r m ( i e . dsRNA) can ( K u r p p a and M a r t i n , 1 9 8 5 ) , and i t c o u l d t h e n be m a n i p u l a t e d i n v i t r o t o p r o d u c e a ssRNA p r o b e . I n o t h e r v i r a l i n f e c t i o n s t h e v i r u s a t t a i n s a h i g h t i t r e i n t h e p l a n t , b u t i s n o t a n t i g e n i c ( e g . t o m a t o s p o t t e d w i l t v i r u s ) ( M a t t h e w s , 1 9 8 1 ) . I n e i t h e r o f t h e s e e x a m p l e s , h y b r i d i z a t i o n w i t h d e r i v a t i z e d n u c l e i c a c i d , f o l l o w e d by s e r o l o g i c a l d e t e c t i o n u s i n g MCA R l w o u l d p r o v i d e an a l t e r n a t i v e t o t h e u s e o f r a d i o - l a b e l l e d n u c l e i c a c i d as a h y b r i d i z a t i o n p r o b e . 79 i C o n c l u s i o n s A n o n - r a d i o a c t i v e t e c h n i q u e was d e v e l o p e d f o r d e t e c t i n g n u c l e i c a c i d s . S i n g l e - s t r a n d e d n u c l e i c a c i d was i o d i n a t e d a n d m o n o c l o n a l a n t i b o d i e s (MCAs) s p e c i f i c f o r t h e m o d i f i e d b a s e w e r e p r o d u c e d . The MCAs were u s e d t o d e t e c t t h e m o d i f i e d b a s e i n ssRNA u s i n g b o t h an i n h i b i t i o n a s s a y i n s o l u t i o n and an i m r a u n o b l o t t i n g a s s a y on n i t r o c e l l u l o s e . The t e c h n i q u e has p r a c t i c a l a p p l i c a t i o n s i n p l a n t v i r o l o g y when i t i s d e s i r a b l e t o d e t e c t t h e p l a n t p a t h o g e n by a n u c l e i c a c i d h y b r i d i z a t i o n a s s a y . C u r r e n t l y , t h e s e a s s a y s a r e b e i n g done w i t h a r a d i o - l a b e l l e d m a r k e r , w h i c h r e q u i r e s t h e u s e o f e x p e n s i v e f a c i l i t i e s and s p e c i a l l y t r a i n e d t e c h n i c i a n s . A p r a c t i c a l c o l o r i m e t r i c a s s a y f o r d e t e c t i o n o f n u c l e i c a c i d h y b r i d i z a t i o n w o u l d a l l o w t h e s e a s s a y s t o be done i n t h e f i e l d i n much t h e same way as s e r o l o g i c a l d e t e c t i o n m ethods ( i e . E L I S A ) a r e c u r r e n t l y b e i n g u s e d . 80 i Summary of Research Results 1. S ingle -s t randed n u c l e i c ac id was iodinated by a procedure i n v o l v i n g modif ica t ions of Comraerford's (1971) r e a c t i o n . The react ion s p e c i f i c a l l y iodinates c y t i d i n e at the C-5 p o s i t i o n in s i n g l e - s t r a n d e d n u c l e i c a c i d . The product was s table and the react ion could be conducted reproducibly with large q u a n t i t i e s of substrate ( m i l l i g r a m s ) . 2. More than 2,000 MCAs were produced against the iodinated c y t i d i n e nucleoside conjugated to a prote in c a r r i e r . Only one of these (Rl) was found to be s p e c i f i c for i o d o c y t i d i n e . 3. MCA Rl could be used in a s o l i d phase ELISA to detect as l i t t l e as 50 ng of i o d o c y t i d i n e in c e l l u l a r RNA bound to n i t r o c e l l u l o s e (immunoblot assay) . 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A c i d s Res. 2:1189-1201. 90 Appendix L i s t o f A b b r e v i a t i o n s MCA monoclonal a n t i b o d y ELISA e n z y m e - l i n k e d immunosorbant a s s a y XRF X-ray f l u o r e s c e n c e UV u l t r a - v i o l e t BSA b o v i n e serum albumin HSA human serum albumin ova ovalbumin TLC t h i n - l a y e r chromatography TBE ' 10 mM T r i s , 100 mM b o r i c a c i d - s o d i u m b o r a t e , 10 mM EDTA pH 8.0 STE 100 mM NaCl, 50 mM T r i s - H C l , 1 mM Na 2EDTA pH 7.0 PBS 137 mM NaCl, 1.5 mM KH 2P0 4 , 8 mM NaHP0 4 . 7H 20, 3mM KC1 pH8. 0 PAGE 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 DMEM Dulbecco's M o d i f i e d E a g l e s Medium FCS f e t a l c a l f serum PEG p o l y e t h y l e n e g l y c o l HAT 10 mM h y p o x a n t h i n e , 0.04 mM a m i n o p t e r i n , 1.6 mM t h y m i d i n e HT 10 mM h y p o x a n t h i n e , 1.6 mM t h y m i d i n e OPD o-phenylenediamine 

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