UBC Theses and Dissertations

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UBC Theses and Dissertations

Immunochemical studies with natural gastrin Drummond, Peter Charles Patterson 1970

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IMMUNOCHEMIC AL STUDIES WITH NATURAL GASTRIN by PETER CHARLES PATTERSON DRUMMOND 3.Sc., U n i v e r s i t y o f B r i t i s h Columbia, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF RASTER OF SCIENCE i n the Department of PHYSIOLOGY We a c c e p t t h i s t h e s i s a s co n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September, 1970 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t 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 m a k e 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 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 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 V a n c o u v e r 8, C a n a d a D a t e c ^ c / /<7"7£ i ABSTRACT The a n t r a l hormone g a s t r i n has g e n e r a l l y been re g a r d e d as a non a n t i g e n i c m o l e c u l e n e c e s s i t a t i n g i t s c o n j u g a t i o n w i t h a l a r g e " c a r r i e r 1 1 t o be an e f f e c t i v e i n d u c e r of a n t i b o d y . Such c o n j u g a t i o n has n o r m a l l y t a k e n the form of c o v a l e n t b i n d i n g to a h i g h l y a n t i g e n i c m o l e c u l e or i o n i c b i n d i n g to an i n e r t p a r t i c l e . A l t e r n a t i v e l y , some s u c c e s s has been a c h i e v e d by the i n j e c t i o n of impure a n t r a l e x t r a c t s . T h i s r e p o r t d e s c r i b e s f o u r approaches t o the problem of the i n d u c t i o n of a n t i b o d i e s s p e c i f i c f o r g a s t r i n . I n i t i a l l y , n a t u r a l hog g a s t r i n was o b t a i n e d a f t e r the p r o -cedure of Gregory and Tracy (1964)* The pure a c t i v e hormone was m o d i f i e d by a l k a l i n e h y d r o l y s i s t o l i b e r a t e an N - t e r m i n a l amino group f o r c o v a l e n t c o n j u g a t i o n . The m o d i f i e d g a s t r i n , however, was not s p e c i f i c a l l y i d e n t i f i e d or i s o l a t e d i n q u a n t i t y . Consequently, pure and impure a n t r a l e x t r a c t s , hemocyanin-bound s y n t h e t i c human g a s t r i n and l a t e x - b o u n d g a s t r i n were a p p l i e d t o a v a r i e t y of a n i m a l s . P a s s i v e h e m a g g l u t i n a t i o n t e s t s i n ducks r e v e a l e d t i t r e s of 400 to 1600 t o b o t h the pure and impure ex-t r a c t s but a s e r i e s of f o u r i n j e c t i o n s of pure g a s t r i n i n t o an a n t r e c t o m i z e d r a b b i t f a i l e d to r a i s e d e t e c t & b l e a n t i b o d y . I n -j e c t i o n s of 3 mg« of SHG bound t o hemocyanin was u n s u c c e s s f u l ; a n t i b o d y t i t r e t o the c a r r i e r m olecule was h i g h but no s p e c i f i -c i t y appeared to be d i r e c t e d to the s y n t h e t i c hapten. The i m m u n i z a t i o n of c h i c k e n s w i t h l a t e x - b o u n d n a t u r a l g a s t r i n was more f r u i t f u l . A l t h o u g h a number of p r e c i p i t i n t e s t s e s t a b l i s h e d the presence of a n t i b o d i e s to g a s t r i n , i t was apparent t h a t the assay was i n a d e q u a t e as a s e n s i t i v e t e s t f o r the b i v a l e n t a n t i -gen. Furthermore, the a n t i b o d y t i t r e was not s u f f i c i e n t l y h i g h to be s u c c e s s f u l l y a p p l i e d to a radioimmunoassay. i i i TABLE OF CONTENTS Paj-e ABSTRACT i TABLE OF CONTENTS .. . i i i L IST OF TABLES v LIST OF FIGURES v i i i ACKNOWLEDGMENTS , ' i x INTRODUCTION 1 METHODS 6 1.. PREPARATION OF THE ANTIGEN 6 A. ISOLATION AND PURIFICATION OF HOG GASTRIN. 6 B. ISOLATION OF SALMON "GASTRIN" 10 2. ASSAY OF GASTRIN ' ' 10 A. ASSAY IN THE CAT.. 10 B. ASSAY IN THE DOG... 11 C. ASSAY I N THE BULLFROG 12 3. MODIFICATION OF GASTRIN 14 A. NaOH HYDROLYSIS OF GASTRIN V-\-E. NH.OH HYDROLYSIS OF GASTRIN 15 4 k. COUPLING OF GASTRIN • 16 A. COVALENT CONJUGATION OF HEMOCYANIN AND SYNTHETIC HUMAN GASTRIN 16 ' B. IONIC CONJUGATION OF LATEX AND NATURAL GASTRIN • 16 5. INDUCTION AND DEMONSTRATION OF ANTIBODIES TO GASTRIN 18 A. ANTIBODIES TO STAGES I AND I I I GASTRINS IS B. ANTIBODIES TO STAGE I I I GASTRINS... 21 i v Page C . A N T I B O D I E S TO A H E M O C Y A N I N - S Y N T H E T I C HUMAN G A S T R I N CONJUGATE 22 D , A N T I B O D I E S TO A L A T E X - S T A G E I I I G A S T R I N C O N J U G A T E . . • 22 R E S U L T S 28 1. I S O L A T I O N AND PURIFICATION OF G A S T R I N 28 A . PRODUCT A N A L Y S I S OF HOG G A S T R I N 2.8 B . PRODUCT A N A L Y S I S OF SALMON " G A S T R I N " % 2. RESPONSE OF B U L L F R O G F U N D I C MUCOSA TO G A S T R I N 37 3. M O D I F I C A T I O N OF G A S T R I N • 42 A . PRODUCT A N A L Y S I S OF A L K A L I N E H Y D R O L Y S I S OF G A S T R I N 42 i f . DEMONSTRATION OF A N T I B O D I E S TO G A S T R I N 44 A . A N T I B O D I E S TO STAGES I AND I I I G A S T R I N S 44 B . A N T I B O D I E S TO STAGE I I I G A S T R I N S 46 C . A N T I B O D I E S TO A H E M O C Y A N I N - S Y N T H E T I C HUMAN G A S T R I N CONJUGATE 46 D . A N T I B O D I E S TO A L A T E X - S T A G E I I I G A S T R I N CONJUGATE 48 D I S C U S S I O N 55 SUMMARY AND CONCLUSIONS 65 B I B L I O G R A P H Y • 67 V LIST OF TABLES Table P££XJ I COMPOSITIONS OF NUTRIENT AND MUCOSAL FLUIDS FOR A GASTRIN ASSAY ON THE FUNDIC MUCOSA OF THE BULLFROG Ran a c a t e s b i a n a 13 I I IMMUNIZATION SCHEDULE FOR THE INDUCTION OF ANTIBODIES TO STAGES I A N D . I l l GASTRINS IN TWO MALLARD DUCKS 20 I I I IMMUNIZATION SCHEDULE FOR THE.INDUCTION OF ANTIBODIES TO STAGE I I I GASTRINS IN AN ANTRECTOMIZED RABBIT 23 IV IMMUNIZATION SCHEDULE FOR THE INDUCTION OF ANTIBODIES TO HEMOCYANIN-SHG 2 - 1 ? IN THREE RABBITS 23 V IMMUNIZATION SCHEDULE FOR THE INDUCTION OF ANTIBODIES TO LATEX-GASTRIN IN THREE CHICKENS AND TWO RABBITS • 25 VI BLEEDING SCHEDULE AND TESTS USED FOR THE DETECTION OF ANTIBODIES TO A LATEX-GASTRIN CONJUGATE 26 V I I BIOASSAY FOR GASTRIN IN AN ANAESTHETIZED CAT FOLLOWING SEPHADEX G50 GEL FRACTIONATION 30 V I I I BIOASSAY FOR GASTRIN IN AN ANAESTHETIZED CAT FOLLOWING AMINOETHYL CELLULOSE FRACTIONATION 33 v i Table Page IX ELECTROPHORETIC MOBILITIES FOR GASTRINS GI AND G i l IN THREE SOLVENT SYSTEMS 35 X THIN LAYER CHROMATOGRAPHY REFERENCE DISTANCES FOR GASTRINS GI AND G i l IN TWO SOLVENT SYSTEMS 35 XI BIOASSAY FOR SALMON "GASTRIN" IN A CON-SCIOUS DOG FOLLOWING SEPHADEX G50 GEL FRACTIONATION ' , .. 39 X I I RESPONSE OF THE BULLFROG FUNDIC MUCOSA TO NATURAL GASTRIN 41 X I I I ELECTROPHORETIC MOBILITIES FOR NATURAL GASTRIN GI AND NaOH TREATED GI IN TWO SOLVENT SYSTEMS 43 XIV THIN LAYER CHROMATOGRAPHY REFERENCE DISTANCES FOR NATURAL GASTRIN GI AND NaOH TREATED GI IN THREE SOLVENT SYSTEMS 43 XV DESCENDING PAPER CHROMATOGRAPHY REFERENCE DISTANCES OF SAMPLES FROM THE SERIAL HYDROLY-SIS OF GASTRIN GI BY 1 M AMMONIA SOLUTION WITH NINI-IYDRIN STAINING INTENSITIES 45 XVI SEMIQUANTITATIVE PASSIVE HEMAGGLUTINATION TESTS FOR ANTIBODIES TO STAGES I AND I I I GASTRINS ON SERUM DRAWN FROM A SINGLE DUCK 47 XVII SEMIQUANTITATIVE RING TESTS FOR ANTIBODIES TO HEMOCYANIN AND GASTRIN 49 Table X V I I I SEMIQUANTITATIVE PASSIVE HEMAGGLUTINATION TESTS FOR ANTIBODIES TO HEMOCYANIN MID GASTRIN ON POOLED SERUMS FROM THREE RABBITS. XIX ASSAYS FOR ANTIBODIES TO GASTRIN BY GEL DIFFUSION AND BIOASSAY TECHNIQUES XX RESULTS FROM A BLAIR-TYPE GASTRIN ASSAY" FOR ANTIBODY. . ' v i i i LIST OF FIGURES F i g u r e Page 1 FRACTIONATION' OF HOG GASTRIN ON SEPHADEX G-50 GEL 29 2 FRACTIONATION OF HOG GASTRIN ON AMINO-ETHYL CELLULOSE POV/DER ' 32 3 FRACTIONATION OF SALMON "GASTRIN" ON SEPHADEX G50 GEL 38 A RESPONSE OF THE BULLFROG FUNDIC MUCOSA TO NATURAL GASTRIN AO 5 BLAIR-TYPE GASTRIN ASSAY FOR ANTIBODY IN AN ANAESTHETIZED CAT 53 ACKNOWLEDGMENT i x I w i s h t o extend my s i n c e r e g r a t i t u d e t o Dr. J . C. Brown and Dr. J . A. Pearson f o r t h e i r a s s i s t a n c e throughout the p r e p a r a t i o n o f t h i s work. 1 INTRODUCTION The term g a s t r i n was f i r s t proposed by E d k i n s i n 1905. I t was a p p l i e d t o the a c t i v e f r a c t i o n of an aqueous e x t r a c t from the a n t r a l mucosa, which on i n t r a v e n o u s i n f u s i o n produced s t i m u -l a t i o n o f a c i d and p e p s i n s e c r e t i o n from the c a t f u n d i c mucosa ( S d k i n s , 1906). Acceptance was prompt and g a s t r i n f o l l o w i n g s e c r e t i n became the second hormone known t o e x i s t . The g a s t r i n c oncept, however, was t o remain on u n c e r t a i n ground f o r many y e a r s as f u r t h e r i n v e s t i g a t i o n s i n d i c a t e d t h a t the presence of h i s t a m i n e i n the e x t r a c t c o u l d account f o r i t s g a s t r i c a c t i v i t y ( B a r g e r and D a l e , 1910-11; Dale and L a i d l a w , 1910-11). T h i r t y y e a r s l a t e r , Komarov (1938) succeeded i n p r e p a r i n g a h i s t a m i n e - f r e e a n t r a l e x t r a c t p o s s e s s i n g a l l o f the a c t i v i t y of the E d k i n p r o d u c t . T h i s work d i d much t o r e s t o r e i n t e r e s t i n the concept of g a s t r i n as a s p e c i f i c hormone and was i n s t r u m e n t a l i n e s t a b l i s h i n g p r o o f f o r the e x i s t e n c e of g a s t r i n and i t s impor-tance i n the c o n t r o l of g a s t r i c s e c r e t i o n . The c u r r e n t phase of r e s e a r c h , concerned 'with the c h e m i c a l n a t u r e of g a s t r i n , may be c r e d i t e d l a r g e l y t o the work of Gregory and h i s a s s o c i a t e s . T h i s group was the f i r s t t o p r epare a v i r t u a l l y pure n a t u r a l g a s t r i n and d e s c r i b e i t s p r o p e r t i e s on subcutaneous i n j e c t i o n s i n t o c o n s c i o u s dogs and humans (Gregory and Tracy, 1961). I n more r e c e n t p u b l i c a t i o n s (Gregory and T r a c y , 1964) these workers a c h i e v e d the i s o l a t i o n s of two hog g a s t r i n s . I n the same year the s e q u e n c i n g was determined (Gregory e t a l . , 1964) a n d the s y n t h e s i s of the h e p t a d e c a p e p t i d e was completed (Anderson e t a l . , 1964)• The s y n t h e t i c and n a t u r a l l y o c c u r r i n g g a s t r i n s were 2 d e s i g n a t e d g a s t r i n I and g a s t r i n I I , d i f f e r i n g o n l y by the s u l -f a t i o n of the t y r o s y l r e s i d u e i n the l a t t e r . F u r t h e r work (Gre-gory, 1966) i n d i c a t e d t h a t the g a s t r i n m o l e c u l e from A s p e c i e s of a n i m a l s v a r i e d by no more than two r e s i d u e s ( p o s i t i o n s 5 and/ or 6). A more s i g n i f i c a n t f i n d i n g , however, was t h e i r e a r l i e r o b s e r v a t i o n t h a t the C - t e r m i n a l t e t r a p e p t i d e amide e x h i b i t e d the e n t i r e range of p h y s i o l o g i c a l p r o p e r t i e s of the pa r e n t hormone. I n the p a s t decade a gre a t d e a l of i n t e r e s t has e v o l v e d i n the p r o d u c t i o n of a n t i b o d i e s t o g a s t r i n . The p r i n c i p l e aim of the i m m u n o l o g i c a l approach has been the development of an immuno-assay f o r the measurement of g a s t r i n i n serum and t i s s u e . ' A secondary o b j e c t i v e concerned the i d e n t i f i c a t i o n o f the g a s t r i n r e l e a s i n g c e l l w i t h f l u o r e s c e i n tagged a n t i b o d y . I n 1961, Waddell e t a l . were abjre t o o b t a i n a n t i b o d i e s t o a crude a n t r a l e x t r a c t and demonstrate a r e d u c t i o n of g a s t r i c s t i m u l a t i o n i n a c o n s c i o u s dog f o l l o w i n g i n c u b a t i o n w i t h g a s t r i n . S c h n e i d e r et a l . (1967) advanced t h i s work by d e m o n s t r a t i n g the s p e c i f i c i t y of a n t i s e r u m t o a crude a n t r a l e x t r a c t f o r n a t u r a l g a s t r i n and the C - t e r m i n a l t e t r a - and p e p t a p e p t i d e s . The c l i n i c a l s i g n i f i -cance of the g a s t r i n assay became e v i d e n t w i t h the d i s c o v e r y t h a t the g a s t r i c agent p r e s e n t i n the Z o l l i n g e r - E l l i s o n syndrome ( Z o l l i n g e r and E l l i s o n , 1955) bore an' • i d e n t i c a l c o m p o s i t i o n t o t h a t of g a s t r i n (Gregory e t a l . , 1967 > 1969). T h i s f i n d i n g prompted a number of workers t o implement a d i a g n o s t i c t e s t f o r the d e t e c t i o n of b l o o d g a s t r i n . U n t i l r e c e n t l y , the d e f i n i t i v e d i a g n o s i s of the syndrome r e l i e d p r i m a r i l y on c l i n i c a l f i n d i n g s w i t h subsequent s u r g i c a l c o n f i r m a t i o n . I n the b e l i e f t h a t pure g a s t r i n was non a n t i g e n i c Stremple and Meade (1968) u t i l i z e d terpene-bound s y n t h e t i c human g a s t r i n t o r a i s e a n t i b o d i e s t o g a s t r i n and advanced the f i r s t but crude radioimmunoassay. The assay, a l t h o u g h more s e n s i t i v e t h a n c o n v e n t i o n a l c a t ( B l a i r et a l . , 1968) or r a t (Stremple and Meade, 1968) b i o a s s a y s , was un-a b l e t o d e t e c t g a s t r i n a t c o n c e n t r a t i o n s l e s s than 1 ng. per ml. serum. Nor was s p e c i f i c i t y of the radioimmunoassay s u f f i c i e n t l y p r e c i s e t o excl u d e the i n t e r f e r e n c e of c h o l e c y s t o k i n i n - p a n c r e o z y -min which b e a r s an i d e n t i c a l C - t e r m i n a l p e n t a p e p t i d e (Mutt and J o r p e s , 1968). T h i s work was f o l l o w e d s h o r t l y by a double a n t i b o d y radioimmunoassay which p e r m i t t e d the q u a n t i t a t i o n of g a s t r i n i n l e s s than 10 pg. per ml. serum (McGuigan, 1968a). The a v i d i t y of c h o l e c y s t o k i n i n - p a n c r e o z y m i n f o r a n t i s e r u m t o a gastrin-bovixre serum albumin c o n j u g a t e , proved t o be s i g n i f i c a n t l y l e s s than t h a t t o g a s t r i n ('McGuigan, 1969) • F u r t h e r a p p l i c a t i o n of these t e c h n i q u e s employed f l u o r e s c e i n tagged a n t i b o d y and l e d t o the i d e n t i f i c a t i o n of the g a s t r i n r e l e a s i n g c e l l (McGui-gan, 1968c). Recent improvements of the radioimmunoassay have i n c r e a s e d both the s e n s i t i v i t y (McGuigan, 1970) and s p e c i f i c i t y (Yalow and Berson, 1970) t o g a s t r i n . The i n i t i a l o b j e c t i v e o f t h i s study was d i r e c t e d toward the development of an i n e x p e n s i v e and manageable radioimmunoassay f o r b l o o d g a s t r i n . I t had a l s o been a n t i c i p a t e d t h a t f l u o r e s c e i n tagged a n t i b o d y would be u s e f u l i n the i d e n t i f i c a t i o n and l o c a l -i z a t i o n of the g a s t r i n r e l e a s i n g c e l l i n a v a r i e t y of a n i m a l s w h i l e the u l t r a s t r u c t u r e of the c e l l , p r e s e n t l y unknown, c o u l d be e l u c i d a t e d by f e r r i t i n tagged a n t i b o d y t e c h n i q u e s ( S i n g e r , if 1959). Although a n t i b o d i e s to g a s t r i n have been induced by a number of methods, no attempt has been made with the pure na-t u r a l a n t i g e n . Furthermore, the s e l e c t i v e cleavage of the c y c l i -zed glutamyl r e s i d u e of the molecule by enzyme or a l k a l i n e hy-d r o l y s i s and i t s subsequent covalent b i n d i n g to a p r o t e i n has not been accomplished. The d i f f i c u l t i e s i n r a i s i n g a n t i b o d i e s to g a s t r i n are i n -herent i n i t s s i z e and s t r u c t u r e . The molecular weight of the s u l f a t e d molecule ( G i l = 2195) i s c o n s i d e r a b l y l e s s than that g e n e r a l l y r e q u i r e d to be an e f f e c t i v e i n ducer of antibody f o r -mation and e f f o r t s to o b t a i n antiserum to pure g a s t r i n have f a i l e d (Maklhouf, 196k)• Antiserum r a i s e d by the e a r l i e r wor-ke r s (Waddell et a l . , 1961; Schneider et a l . , 1967) appeared to l a c k the s p e c i f i c i t y necessary f o r a s u i t a b l e immunoassay. O d e l l et a l . (1968) have emphasized t h i s need of a high degree of s p e c i f i c i t y i n immunoassay systems. Consequently, g a s t r i n was bound to f o r e i g n p r o t e i n s (McGuigan, 1967, 1968a, 1968b, 1969, 1970) or terpenes ( S t r e m p l e ; e t a l . , 1967; Stremple and Meade, 1968) to render a s p e c i f i c a n t i g e n i c complex. McGuigan found that s y n t h e t i c human g a s t r i n 2 - 17 or i t s C-terminal t e t r a -peptide amide, when c o v a l e n t l y bound by the f r e e v N - t e r m i n a l amino group to bovine gamma g l o b u l i n or bovine serum albumin, was an e f f e c t i v e i n ducer of a n t i b o d i e s s p e c i f i c f o r g a s t r i n d e r i v a -t i v e s . The n a t u r a l hormone l a c k s the f r e e amine s i n c e the N-ter m i n a l p o s i t i o n of the molecule i s blocked by a pyroglutamyl r e s i d u e . The exposure of an amine by enzyme or a l k a l i n e hydroly-s i s i s then necessary f o r covalent c o n j u g a t i o n . I o n i c b i n d i n g 5 of n a t u r a l g a s t r i n t o charged p a r t i c l e s i s an a t t r a c t i v e a l t e r -n a t i v e t o c o v a l e n t c o n j u g a t i o n . T h i s approach t o the r a d i o -immunoassay was i n i t i a l l y employed t o couple the s y n t h e t i c mole-c u l e t o p o s i t i v e l y charged p o l y m e t h y l m e t h a c r y l a t e l a t e x p a r t i c l e s ( S t r e m p l e e t a l . , 1967, Stremple and Meade, 1968) and i s attemp-te d i n t h i s study w i t h the n a t u r a l g a s t r i n . T h i s r e p o r t d e s c r i b e s the p r e p a r a t i o n of the a n t i g e n , the i n d u c t i o n and d e m o n s t r a t i o n . o f a n t i b o d i e s to g a s t r i n . While the p r i n c i p l e aim concerned the i n d u c t i o n of "pure" a n t i b o d y , an attempt has a l s o been d i r e c t e d t o o b t a i n a n t i s e r u m t o p a r t i a l l y p u r i f i e d a n t r a l e x t r a c t s . Both p u r i t i e s o f the n a t u r a l hormone were p r e p a r e d by the method of Gregory and Tracy (1964)« The i n d u c t i o n of a n t i b o d i e s t o pure n a t u r a l hog g a s t r i n i s c a t e g o r i z e d i n t o (1) the i s o l a t i o n and p u r i f i c a t i o n of g a s t r i n , (2) N-t e r m i n a l m o d i f i c a t i o n of the m o l e c u l e , (3) c o v a l e n t or i o n i c c o u p l i n g of g a s t r i n , and (4) the d e m o n s t r a t i o n of a n t i b o d i e s t o g a s t r i n . 6 METHODS 1. PREPARATION OF THE. ANTIGEN A. ISOLATION AND PURIFICATION OF HOG GASTRIN The procedure employed f o r the i s o l a t i o n and p u r i f i c a t i o n o f hog g a s t r i n was drawn e s s e n t i a l l y from the method of Gregory and Tracy (1964). Hog antrums were c o l l e c t e d i n b a t c h e s of 300 w i t h i n 30 min. of the a n i m a l s b e i n g s l a u g h t e r e d . They were opened, washed g e n t l y i n c o l d t a p water and b o i l e d i n 60 1. o f tap water f o r AO min. The s o l u t i o n was c o o l e d t o 25°C w i t h the a d d i t i o n of i c e and a l l o w e d t o stand o v e r n i g h t . The antrums were removed and the l i q u o r was f i l t e r e d t h r o ugh 12 l a y e r s of f i n e cheese c l o t h . A f t e r the a d d i t i o n of 150 g. of d i e t h y l a m i n o e t h y l c e l l u l o s e powder (Whatman DEAE 11), the l i q u o r was s t i r r e d by . p r o p e l l o r f o r 3 hours. The " f l o e " was c o l l e c t e d by f i l t r a t i o n t h r o u gh A l a y e r s of f i n e n e t t i n g (organza) and washed t h o r o u g h l y w i t h c o l d t a p water. Adsorbed g a s t r i n and i n e r t p r o t e i n were e l u t e d by s t i r r i n g the " f l o e " i n t o , 2./+ 1. of 0.1 M NaOH f o r 30 min. and f i l t e r i n g i t through an organza covered Buchner f u n n e l . A f t e r b r i n g i n g the f i l t r a t e t o pH 7.0 w i t h g l a c i a l a c e t i c a c i d , i t was c o o l e d t o 10°C, brought to pH A.O a g a i n w i t h g l a c i a l ace-t i c a c i d and r e f r i g e r a t e d o v e r n i g h t a t 6°C. The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n . i n 1 1 . b u c k e t s (1500 x g. f o r 13 min. at 0°C) and s t o r e d a t -20°C u n t i l r e q u i r e d . An amount of p r e c i p i t a t e e q u i v a l e n t to 600 antrums was d i s s o l v e d i n water, brought to pH 10.0 w i t h I 4 . 8 N ammonia s o l u -t i o n and the volume made up t o 600 ml. w i t h d i s t i l l e d water. 7 While m a i n t a i n i n g the temperature a t 15 t o 20°C, 300 g. of HPO^ was d i s s o l v e d i n the s o l u t i o n and 2+80 ml. of p e r o x i d e - f r e e i s o p r o p a n o l was s l o w l y added w i t h v i g o r o u s s t i r r i n g . A f t e r f u r -t h e r s t i r r i n g f o r 30 min., the m i x t u r e was c e n t r i f u g e d i n 1 1. bu c k e t s (1500 x g. f o r 15 min. a t 0°C) and the upper aqueous phase c o l l e c t e d by s i p h o n i n g . The aqueous e x t r a c t was r e c o v e r e d by thorough s h a k i n g w i t h 2 volumes of p e r o x i d e - f r e e e t h y l e t h e r * and 50 ml. o f d i s t i l l e d water i n a s e p a r a t o r y f u n n e l . F o l l o w i n g s e p a r a t i o n of the two phases, the lo w e r aqueous l a y e r was d r a i n e d o f f and e x t r a c t e d t w i c e a g a i n w i t h 2 volumes of ether, and 50 ml. of water. The r e s i d u a l e t h e r was removed by vacuum a e r a t i o n f o r 30 min. a t room t e m p e r a t u r e , and the aqueous phase ( a t 10°C) was brought t o pl-l 4.0 w i t h g l a c i a l a c e t i c a c i d and r e f r i g e r a t e d o v e r n i g h t a t 6°C. The r e s u l t i n g p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n i n 50 s i l . t u b e s (8000 x g. f o r 10 min. a t 0°C) and r e d i s s o l v e d i n 50 ml. of c o l d d i s t i l l e d water w i t h the a d d i -t i o n o f 1 m l . o f 14.8 N ammonia s o l u t i o n . T h i s s o l u t i o n was r e f e r r e d t o as Stage I g a s t r i n . P r e c i p i t a t i o n w i t h g l a c i a l a c e t i c a c i d a t pH 4.0 was r e -peated. F o l l o w i n g r e f r i g e r a t i o n f o r 2 hou r s , t h e p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n (8000 x g. f o r 10 min. a t 0°C) and d i s s o l v e d i n 3 "to 5 n i l . of d i s t i l l e d water w i t h 2 drops of ammonia s o l u t i o n . The s o l u t i o n was then passed through a c o l -umn. (5 x 100 cm.) of Sephadex 050 g e l (medium grade, Pharmacia) " K"Peroxide-free e t h y l e t h e r was pr e p a r e d by s h a k i n g 1 1. o f e t h y l e t h e r w i t h 60 g. FeSO^, 6 ml. HpSO, C. and 110 ml. of d i s t i l l e d w ater. ' ^ 8 packed i n 0.OA M NH^HCO^ (pH 8.0). The e f f l u e n t flow r a t e was held constant at 80 ml. per hour by tube clamps. F r a c t i o n s of 10 ml. were c o l l e c t e d on a LKB U l t r o r a c f r a c t i o n c o l l e c t o r (type 7000) and absorbance was recorded at 280 m jx over a 1 cm. l i g h t path i n a s i n g l e beam p r e c i s i o n spectrophotometer (Bausch and Lomb). Randomly s e l e c t e d tubes were assayed f o r g a s t r i c a c t i v i t y ( H + output) i n an an a e s t h e t i z e d cat or conscious dog and the a c t i v e f r a c t i o n s " pooled and l y o p h i l i z e d to constant weight. T h i s product was r e f e r r e d to as Stage I I g a s t r i n . When r e q u i r e d , the Stage I I product was d i s s o l v e d i n a few ml. of 0.02 M NH, HC0 7 (pH 7.8) and adsorbed onto aminoethyl c e l l u l o s e powder (Whatman AS 11) i n a column (1 x 20 cm.) packed i n the bicarbonate s o l u t i o n . The two hog g a s t r i n s , GI and GII, were e l u t e d * from the column u s i n g a grad i e n t c o n c e n t r a t i o n of 0.02 to 0.2 M NH^HCO^ at constant pH. F r a c t i o n s of L ml. were c o l l e c t e d a t a flow r a t e of 8 ml. per hour and absorbance was r e -corded at 280 m p. by spectrophotometer. G a s t r i c a c t i v i t y a:s ex-pressed by H. output was assayed i n an an a e s t h e t i z e d c at and the two a c t i v e - f r a c t i o n s , GI and GII were pooled s e p a r a t e l y and each l y o p h i l i z e d to constant weight. Although f u r t h e r p u r i f i c a t i o n by i o n exchange chromatography with g e n t l e r g r a d i e n t s (0.05 to 0.2 M and 0.06 to 0.2 M f o r GI and GII r e s p e c t i v e l y ) was attempted, t h i s step was not r o u t i n e l y employed. The f i n a l product was r e f e r r e d to as Stage I I I g a s t r i n s GI and GII. •^Before the gr a d i e n t e l u t i o n was begun, the adsorbed product was washed f r e e of excess s a l t s by e q u i l i b r a t i o n with 0.02 M NH. HCO,. 9 The p u r i t y of the Stage I I I product was determined by zone e l e c t r o p h o r e s i s (low v o l t a g e ) , t h i n l a y e r chromatography, de-sc e n d i n g paper chromatography and amino a c i d a n a l y s i s . E l e c t r o -p h o r e t i c a n a l y s i s of the two g a s t r i n s was executed by narrow band a p p l i c a t i o n of 100 /ag. of GI and G i l on Whatman 3 MM paper s t r i p s ( 1 x 6 i n s . ) . The s t r i p s were e l e c t r o p h o r e s e d * s i m u l t a n e o u s l y i n 0.1 N b o r a t e b u f f e r (pH 8.5) p y r i d i n e : g l a c i a l a c e t i c a c i d : w a t e r (10:0.4:90 v/v, pH 6.6) and 13% g l a c i a l a c e t i c a c i d (pH 2.0) a t 36 v, per cm. f o r 45 min. A f t e r d r y i n g , the paper s t r i p s were developed s u c c e s s i v e l y i n n i n h y d r i n spray ( W a l d i , 1965) and c h l o r i n e - t o l u i d i n e r e a g e n t s ( W a l d i , I965K Ascending t h i n l a y e r chromatograms were prepa r e d by spot a p p l i c a t i o n of 5 t o 100 yag. of GI and G i l t o s i l i c a g e l p l a t e s (Eastman No. 6060). The saraft p i e s were a l l o w e d t o ascend s i m u l t a n e o u s l y f o r 7 hours i n n-pro-p a n o l : g l a c i a l a c e t i c a c i d : w a t e r (100:10:30 v/v, pH 3*2) and i n n-propanol:water (70:30 v/v, pH 7.4)• The p l a t e s were then de-ve l o p e d by the n i n h y d r i n and c h l o r i n e - t o l u i d i n e methods. Bescen-d i n g paper chromatographic a n a l y s i s was conducted i n n - b u t a n o l : g l a c i a l a c e t i c a c i d : w a t e r (40:10:50 v/v, pH 3.2) o n l y . GI and G i l were s p o t t e d (75 /ig«) on Whatman 3 MM paper s h e e t s (19 x 54 cm.) and the s o l v e n t was a l l o w e d t o m i g r a t e s i m u l t a n e o u s l y i n a s e a l e d j a r f o r 14 hours. The sh e e t s were d r i e d and developed un-der P a u l y s p r a y ( E a s l e y , 1965) and n i n h y d r i n r e a g e n t s r e s p e c t i v e l y . Amino a c i d a n a l y s i s of the Stage I I I p r o d u c t was c a r r i e d out on a B i o c a l Amino A c i d A n a l y s e r . I n i t i a l l y , 10 n moles of g a s t r i n G i l was h y d r o l y z e d f o r 16 hours i n 6 N HC1 a t 1 1 0 ° C i n a vacuum s e a l e d *Gelman Delux Model w i t h 500 v. powerpack. 10 tube a f t e r which time the a c i d was removed by l y o p h i l i z a t i o n . The h y d r o l y s a t e was d i s s o l v e d i n 0.2 M sodium c i t r a t e (pH 2.25), a p p l i e d t o a 50 cm. column of A-6 r e s i n and e l u t i o n c a r r i e d out w i t h a two b u f f e r system. A f t e r m i x i n g w i t h n i n h y d r i n and h e a t i n g t o 100°C the absorbance of the e l u a t e was r e c o r d e d con-t i n u o u s l y a t wavelengths of i+AO and 570 m jx over a 7 m^. l i g h t p a t h and mole i n t e g e r s of each amino a c i d were c a l c u l a t e d a c c o r d i n g t o s t a n d a r d methodology. B. ISOLATION OF SALMON "GASTRIN" The attempted i s o l a t i o n o f salmon ( P a c i f i c Coho) g a s t r i n employed the same e x t r a c t i o n procedure as d e s c r i b e d f o r hog gas-t r i n (Gregory and Tracy, 1964). The stomachs of f r e s h l y k i l l e d salmon were s e c t i o n e d a t the p y l o r u s and c e n t r a l i n f l e c t i o n . I n i t i a l l y , A00 of the antrums were opened, washed g e n t l y w i t h c o l d t a p water and f r o z e n o v e r n i g h t . The e x t r a c t i o n was c a r r i e d through t o p u r i f i c a t i o n on Sephadex G50 g e l and g a s t r i n a c t i v i t y assay i n a c o n s c i o u s dog. 2. ASSAY OF GASTRIN A. ASSAY IN THE CAT A n a e s t h e s i a was in d u c e d i n a s t a r v e d c a t w i t h a m i x t u r e of 95% oxygen and 5% f l u o t h a n e passed v i a an e n d o t r a c h e a l tube i n t o a c a t box. A n a e s t h e s i a was m a i n t a i n e d w i t h 80 mg. of </. -c h l o r a l o s e per kg. i n f u s e d i n t o the saphenous v e i n . The t r a c h e a •was c a n n u l a t e d and r e s p i r a t i o n was supported v/ith a Harvard 11 r e s p i r a t o r y pump a t a r a t e of 17 r e s p i r a t i o n s per rnin. and a s t r o k e volume o f 25 c c . per kg. The v a g i v/ere s e c t i o n e d i n the neck and b i l a t e r a l splanchnectomy was performed t h r o u g h f l a n k i n c i s i o n s . A f t e r p y l o r i c l i g a t i o n , a stomach tube was i n s e r t e d t h r ough an esophageal i n c i s i o n . The temperature of the a n i m a l was m a i n t a i n e d a t 38.5°C throughout the assay w i t h a h e a t i n g pad c o n t r o l l e d by a r e c t a l thermometer ( t e l e - t h e r m o m e t e r ) . F o l l o w i n g r e p e a t e d washouts t o remove stomach c o n t e n t s , the a n i -mal was covered and l e f t u n d i s t u r b e d f o r ' 2 hours. A f t e r e q u i l i b r a t i o n , 100 ml. stomach washouts (2 x 50 ml.) .were c a r r i e d out every 10 min. F r a c t i o n s of 10 ml. from each washout were t i t r a t e d t o pH 7.0 w i t h 0.01 M NaOH u s i n g an automa t i c t i t r a t i o n assembly (Radiometer T i l ) and pH meter (Ra-diometer SE 2 5 ) . T o t a l H + output f o r 2 or 5 washout p e r i o d s was dete r m i n e d . When b a s a l output appeared c o n s t a n t , i n j e c t i o n s of 1.0 ml. of 0 .9% s a l i n e ( c o n t r o l ) or 0.2 ml. from randomly s e l e c t e l u a t e tubes i n 0.8 ml. of 0.9% s a l i n e were i n f u s e d over a 30 sec. p e r i o d . H output was then r e c o r d e d f o r 2 or 3 washout p e r i o d s f o l l o w i n g each i n j e c t i o n . B. ASSAY IN THE DOG Ch r o n i c dogs were prepar e d w i t h a l a r g e bore g a s t r i c c a n n u l a d r a i n i n g a v a g a l l y and s y m p a t h e t i c a l l y d e n e r v a t e d f u n d i c pouch ( B i c k e l ) . W h i l e r e s t r a i n e d i n a P a v l o v s t a n d , the a n i m a l s v/ere g i v e n c o n t r o l i n f u s i o n s o f 20 ml. s a l i n e over a 10 min. p e r i o d v i a the r a d i a l v e i n . Pouch washouts (2 x 25 ml.) were conducted every 12 min. and 10 ml. f r a c t i o n s of each were 1 2 t i t r a t e d t o pH 7 . 0 w i t h 0 . 0 1 M NaOH. When b a s a l a c i d s e c r e t i o n was e s t a b l i s h e d , * 0.5 ml. f r a c t i o n s from randomly s e l e c t e d e l u a t e tubes i n 1 9 . 5 ml. 0 . 9 % s a l i n e were i n f u s e d over a 1 0 min. p e r i o d . H and p e p s i n o u t p u t s i n yaeq. and mg. of t y r o s i n e r e s p e c t i v e l y were determined f o r J>& min. or 3 washout p e r i o d s f o l l o w i n g the onset of i n f u s i o n . C. ASSAY IN THE BULLFROG The stomach mucosa of the b u l l f r o g Rana c a t e s b i a n a was i s o l a t e d by c a r e f u l l y s t r i p p i n g the t i s s u e f r e e of mu s c u l a t u r e w h i l e immersed i n an u n b u f f e r e d , oxygenated n u t r i e n t s o l u t i o n . * * A s u p p o r t i n g g l a s s tube b e a r i n g a c i r c u l a r f l a n g e was i n s e r t e d i n t o the t i s s u e and the ends were t i e d o f f t o form a pouch of f u n d i c mucosa. The p r e p a r a t i o n was p l a c e d i n a s m a l l c y l i n d e r c o n t a i n i n g 1 0 ml. of b u f f e r e d n u t r i e n t f l u i d ( T a b l e I ) . E i g h t ml. washouts ( 2 x L ml.) of mucosal f l u i d were c a r -r i e d out a t 1 5 min. i n t e r v a l s and the e n t i r e volume was t i t r a t e d t o pH 7 . 0 w i t h 0 . 0 1 M NaOH. A f t e r c o n s t a n t b a s a l s e c r e t i o n was e s t a b l i s h e d , a s t a n d a r d assay was p r e p a r e d by r e p l a c i n g the nu-t r i e n t b u f f e r w i t h 4 . 5 x 1 0 ~ ^ t o i f . 5 x 10"-' M c o n c e n t r a t i o n s of Stage I I I hog g a s t r i n s p r e d i s s o l v e d i n the same b u f f e r . The ac-t i v e s o l u t i o n was washed out and r e p l a c e d a f t e r 1 5 rain, w i t h n u t r i e n t b u f f e r and the H + output was determined f o r a 3 0 min. * F o l l o w i n g 3 c o n s e c u t i v e t i t r a t i o n s l e s s than 1 0 0 ueq. per 1 2 min. p e r i o d and v a r y i n g l e s s than 1 0 % . * * H e r e a f t e r r e f e r r e d t o as mucosal f l u i d ( T a b l e I ) . 13 TABLE I COMPOSITION'S OF NUTRIENT AND MUCOSAL FLUIDS* FOR A GASTRIN ASSAY ON THE FUNDIC MUCOSA OF THE BULLFROG Rana c a t e s b i a n a Compound N u t r i e n t F l u i d Mucosal F l u i d NaCl 85.3 ra M/L 85.3 m M/L K C l 3.4 3.4 C a C l 2 1.8 1.8 MgSO^ 0.8 0 KH oP0. 2 4 0.8 0 NaHCO^ 17.8 0 Glucose 11.0 „ 11.0 Oxygen 100 /o 0 % *The pH's f o r the n u t r i e n t and mucosal f l u i d s v/ere 7.80 and 6.20 r e s p e c t i v e l y . p e r i o d . 3. MODIFICATION OF GASTRIN A. NaOH HYDROLYSIS OF GASTRIN Stage I I I hog g a s t r i n GI ( 7 .3 mg.) was d i s s o l v e d i n 0 .5 ml. of 1.0 M NaOH and a l l o w e d to stand a t room temperature i n a g l a s s s t o p p e r e d tube. A f t e r AO hours of i n c u b a t i o n , 0.9 ml. of g l a c i a l a c e t i c a c i d was added t o p a r t i a l l y n e u t r a l i z e the hydro-l y s a t e (pH 8.0). The product was d e s a l t e d by f i l t r a t i o n t h r o ugh a column (1 x 20 cm.) of Sephadex G10 g e l (medium grade, Pharma-c i a ) packed i n 0.04 M NH^HCO^ (pH 8 . 0 ) . F r a c t i o n s of A ml. of e l u a t e were c o l l e c t e d a t a f l o w r a t e o f 10 ml. per hour and absorbance was r e c o r d e d a t 280 m ji. The f r a c t i o n s w i t h i n the s i n g l e absorbance peak i n the v o i d volume, were p o o l e d and l y o p h i l i z e d t o c o n s t a n t weight. P r o d u c t a n a l y s i s was ac c o m p l i s h e d by zone e l e c t r o p h o r e s i s (low v o l t a g e ) , a s c e n d i n g t h i n l a y e r chromatography and column chromatography. E l e c t r o p h o r e s i s s t r i p s (Whatman 3 MM paper) were prepare d by narrow band a p p l i c a t i o n of 100 jig. g a s t r i n GI, NaOH t r e a t e d GI and Stage I I g a s t r i n s . The bands were a l l o w e d t o mi g r a t e at 36 v. per cm. f o r 2 hours i n 0.1 N b o r a t e b u f f e r (pH 8.5) and i n 15% g l a c i a l a c e t i c a c i d (pH 2 . 0 ) . The s t r i p s were d r i e d and developed s u c c e s s i v e l y w i t h n i n h y d r i n and c h l o r i n e -t o l u i d i n e methods. T h i n l a y e r chromatograms were prepar e d by s p o t - a p p l i c a t i o n s of 100 yag. of GI, NaOH t r e a t e d GI and Stage I I g a s t r i n s onto s i l i c a g e l p l a t e s (Eastman, No. 6060 ) . The p l a t e s 15 were a l l o w e d to stand f o r 7 hours i n n-propanol:water (70:50 v/v, pH 7-k), n - p r o p a n o l : g l a c i a l a c e t i c a c i d : w a t e r (100:10:50 v/v, pH 3 .2) and i n n - b u t a n o l : g l a c i a l a c e t i c a c i d : w a t e r (40 : 1 0 : 5 0 v/v, pH 3 . 2 ) . The p l a t e s were" a g a i n developed by the n i n h y d r i n and c h l o r i n e - t o l u i d i n e methods. The remainder of the NaOH h y d r o l y -s a t e (5-3 rag.) was d i s s o l v e d i n 20 ml. ' 0 . 0 1 M ammonia s o l u t i o n , brought t o pH 3«4 w i t h f o r m i c a c i d and adsorbed onto a column (1 x 20 cm.) of s u l f o e t h y l Sephadex beads (SE Sephadex, Pharma-c i a ) . The r e a c t i o n p r o d u c t s were e l u t e d from the column u s i n g a g r a d i e n t c o n c e n t r a t i o n of 0.01 to 0.2 H ammonium.formate. F r a c t i o n s of 4 ml. were c o l l e c t e d - at- a f l o w r a t e of 8 ml. per hour and were r e a d a t an absorbance of 280 m ji. B. NH. OH HYDROLYSIS OF GASTRIN 4 A s m a l l amount of g a s t r i n G i l ( 2 .2 mg.) was d i s s o l v e d i n 0.6 ml. of 1.0 M ammonia s o l u t i o n and a l l o w e d t o stand a t room temperature i n a g l a s s s t o p p e r e d tube. At 6 hour i n t e r v a l s , 0.1 ml. of the r e a c t i o n m i x t u r e was removed and n e u t r a l i z e d w i t h 0.2 ml. of 0 .5 M g l a c i a l a c e t i c a c i d . Each r e a c t i o n p r o d u c t was l y o -p h i l y z e d t o c o n s t a n t weight and r e d i s s o l v e d i n 0.1 ml. of d i s -t i l l e d water. Spot a p p l i c a t i o n s of 75 Jig- of the h y d r o l y s a t e s were a p p l i e d t o a 19 x 54. cm. paper sheet (Whatman 3 MM) and a l l o w e d t o m i g r a t e i n n - b u t a n o l : g l a c i a l a c e t i c a c i d : w a t e r (40: 10:50 v/v, pH 3 .2 ) f o r 14 hours i n a s e a l e d j a r . The sheet was d r i e d and developed s u c c e s s i v e l y i n P a u l y and n i n h y d r i n r e a g e n t s r e s p e c t i v e l y . 16 L. COUPLING OF GASTRIN A. C'OVALENT CONJUGATION OF HEMOCYANIN AND SYNTHETIC HUMAN GASTRIN 2 - 1 7 The c o n j u g a t i o n of hemocyanin and s y n t h e t i c human g a s t r i n 2 - 1 7 (SHG 2 - 17, I.C.I.) was attempted through a m o d i f i c a t i o n of the method of S c h i c k and S i n g e r (1961). Hemocyanin from the P a c i f i c K eyhole l i m p e t was prepared a c c o r d i n g t o the procedure of Campbell e t a l . (1964). I n a s i n g l e s y n t h e s i s of the c o n j u g a t e , 10 mg. of SHG 2 - 17 was d i s s o l v e d i n 1.0 ml. of 0.01 M p o t a s -sium phosphate b u f f e r (pH 7.4)• A f t e r the s o l u t i o n was c o o l e d to 4°C, 100 jil. of t o l u e n e - 2 , A - d i i s o c y a n a t e (TDC) was added and the m i x t u r e s t i r r e d on an i c e b a t h f o r 45 min. I n s o l u b l e TDC was then removed by c e n t r i f u g a t i o n and the s u p e r n a t a n t f l u i d added t o 25 rag. of prepare d hemocyanin p r e d i s s o l v e d i n 1.5 n i l . of 50% s a t u r a t e d sodium t e t r a b o r a t e . The r e a c t i o n m i x t u r e was s t i r r e d a t 45°C i n a water b a t h f o r 2 hours, a f t e r which time i t was d i a l y z e d a g a i n s t 2 1. of 0.15 M NaCl i n 0.01 M potassiu m phosphate b u f f e r f o r 4 hours a t 6°C. The p r e c i p i t a t e -which r e -mained a f t e r d i a l y s i s was removed by c e n t r i f u g a t i o n (10,000 x g. f o r 10 min. a t 0°C). B. IONIC CONJUGATION OF LATEX AND NATURAL HOG GASTRIN The c o n j u g a t i o n of l a t e x and Stage I I I g a s t r i n s was ac h i e v e d by a m o d i f i c a t i o n of the method of StEemple and Meade (1968). An e q u a l volume of 2% hexadecyltrimethylammonium b r o -mide was added t o 11% n e u t r a l l y charged p o l y m e t h y l m e t h a c r y l a t e 17 l a t e x p a r t i c l e s (0.5 )x d i a m e t e r , B o f o r s ) . The p a r t i c l e s v/ere p o s i t i v e l y charged by d i a l y z i n g the s u s p e n s i o n f o r 12. hours a g a i n s t d i s t i l l e d water, 24 hours a g a i n s t 0.2% hexadecyltrimethylammonium bromide and a g a i n f o r 24 hours i n d i s t i l l e d water. The l a t e x was shown to bear a p o s i t i v e charge by m i c r o s c o p i c e x a m i n a t i o n on the a d d i t i o n of sodium l a u r y l s u l f a t e . * A 0.1% g a s t r i n s o l u t i o n was pr e p a r e d by d i s s o l v i n g the Stage I I I p r o d u c t i n a s m a l l volume of 0.15 M sodium phosphate b u f f e r (pH 7.5). One h a l f o f the charged d i a l y s a t e was c a r e f u l l y suspended i n the g a s t r i n s o l u t i o n i n a r a t i o of 5:2 to g i v e a f i n a l c o n c e n t r a t i o n bf 1 rng. g a s t r i n per 1.5 ml. a n t i g e n i c m i x t u r e . C o n t r o l suspen-s i o n s o f the phosphate b u f f e r and the charged l a t e x were p r e -pared i n the same manner and b o t h were i n c u b a t e d a t 37°C i n a water b a t h f o r 1 hour w i t h o c c a s i o n a l s t i r r i n g . The g a s t r i n was shown to be bound to the l a t e x p a r t i c l e s u s i n g a s t a n d a r d c a t b i o a s s a y . The co n j u g a t e d p a r t i c l e s were f r e e d of unbound g a s -t r i n by r e p e a t e d washings i n the phosphate b u f f e r . Coupled and c o n t r o l s u s p e n s i o n s o f the l a t e x were c e n t r i f u g e d (1000 x g. f o r 10 min. a t 23°C) and the s u p e r n a t a n t decanted each time through 5 washings. H output i n response to. i n t r a v e n o u s i n f u s i o n s of the c o n j u g a t e d and c o n t r o l s u s p e n s i o n s i n t o an a n a e s t h e t i z e d c a t was determined as p r e v i o u s l y d e s c r i b e d . *A n e g a t i v e l y charged d e t e r g e n t which r a p i d l y a g g r e g ates the p o l y a c r y l a t e p a r t i c l e s . Io 5. INDUCTION AND DEMONSTRATION OF ANTIBODIES TO GASTRIN A. ANTIBODIES TO STAGES I AND I I I GASTRINS I n j e c t i o n s o f Stages I and I I I hog g a s t r i n s i n 1.0 ml. of d i s t i l l e d w a t e r* and e m u l s i f i e d w i t h an e q u a l volume of Freunds complete a d j u v e n t ( D i f c o ) were g i v e n to 2 randomly s e l -e c t e d M a l l a r d ducks. The i m m u n i z a t i o n schedule i s l i s t e d i n Table I I . * * The b i r d s were b l e d from the b r a c h i a l v e i n on days 35j 65 and 165* C o n t r o l serum was o b t a i n e d • from 2 n o n - I n j e c t e d ducks a t each b l e e d i n g . L i p i d - f r e e serum was g e n e r a l l y a c q u i r e d by s t a r v a t i o n of the ducks Zk hours p r i o r to b l e e d i n g ; however, i n some ca s e s i t was n e c e s s a r y t h a t the l i p i d s were s e p a r a t e d by h i g h speed c e n t r i f u g a t i o n (25>000 x g. f o r 30 min. a t 0°C)'. A n t i b o d y to g a s t r i n was d e t e c t e d by p a s s i v e h e m a g g l u t i n a t i o n and/ or p r e c i p i t i n r i n g ( i n t e r f a c i a l ) t e s t s (Campbell et a l . , 196A). For the p a s s i v e h e m a g g l u t i n a t i o n t e s t s , sheep b l o o d was c o l l e c t e d i n an e q u a l volume of A l s e v e r s s o l u t i o n ( D i f c o ) and a l l o w e d t o e q u i l i b r a t e a t 6°C f o r a t l e a s t one week b e f o r e use. F i v e ml. of. the b l o o d s u s p e n s i o n and 8-ml. of 0.9% s a l i n e were mixed t h o r o u g h l y i n a 15 ml. Corex tube and c e n t r i f u g e d (500 o x g. f o r 5 min. a t 23>C). A f t e r the su p e r n a t a n t was decanted the c e l l s were c a r e f u l l y resuspended i n 10 ml. s a l i n e by P a s t e u r p i p e t t e and the s u s p e n s i o n was a g a i n c e n t r i f u g e d . The washing *Both d i s s o l v e d stages'.of the gast r i n ' s were, passed t h r o u g h a m i l l i p o r e f i l t e r (M.F.C.) under a s p i r a t o r s u c t i o n , p r i o r to e m u l s i f i c a t i o n . * * I n j e c t i o n s were a d m i n i s t e r e d a t 2 t h i g h l o c a t i o n s . 19 was r e p e a t e d 3 t i m e s a f t e r which 0.5 n i l . of the packed e r y t h r o -c y t e s were added t o 20 ml. of phosphate b u f f e r e d s a l i n e (pH 7.2) and a g a i n resuspended. The c e l l s were tanned by m i x i n g an eq u a l volume o f the s u s p e n s i o n w i t h f r e s h l y p r e p a r e d 0.005% t a n n i c a c i d (3 ml.) and i n c u b a t i n g the m i x t u r e i n a water b a t h a t 37°C f o r 10 min. A f t e r i n c u b a t i o n , the c e l l s were washed t w i c e a g a i n i n 3 ml. .of phosphate b u f f e r e d s a l i n e (pH 7.2) and resuspended i n 3 ml. o f the same b u f f e r . To c o a t the c e l l s , 1 ml. of the tanned s u s p e n s i o n was mixed w i t h A ml. of phosphate b u f f e r e d s a l i n e (pH 6.4) and 1 ml. of 0.025% a n t i g e n (Stages I or I I I g a s t r i n s ) i n 0.9% s a l i n e . C o n t r o l c e l l s were prepared by the s u b s t i t u t i o n o f s a l i n e f o r the a n t i g e n . A f t e r a l l o w i n g the tubes t o stand a t room temperature f o r 10 min., the s u s p e n s i o n s were c e n t r i f u g e d (500 x g. f o r 5 min. a t 23°C), the s u p e r n a t a n t s decanted and the c e l l s resuspended i n 2 ml. o f i n a c t i v a t e d nor-mal duck serum d i l u e n t * (NDS). C e n t r i f u g a t i o n and r e s u s p e n s i o n i n 1 ml. NDS was r e p e a t e d . H e m a g g l u t i n a t i o n tubes (13 x 100 mm.) were p r e p a r e d by m i x i n g 0.05 ml. of the a n t i g e n coated c e l l s w i t h 0.5 ml. of t w o - f o l d s e r i a l d i l u t i o n s of i n a c t i v a t e d d i l u t e d a n t i s e r u m (1:100 i n NDS). C o n t r o l s were p r e p a r e d by m i x i n g (1) 0.05 ml. of the coated c e l l s w i t h 0.5 ml. NDS and (2) 0.05 ml. o f the c o n t r o l c e l l s w i t h 0.5 ml. o f a n t i s e r u m d i l u e n t . The tube r a c k was shaken t o suspend the c e l l s and the tubes v/ere s e a l e d w i t h p a r a f i l m . The tubes were th e n a l l o w e d t o stand a t room temperature f o r 12 hours a f t e r which time h e m a g g l u t i n a t i o n ^ I n a c t i v a t e d a t 56°C f o r 30 min. and d i l u t e d 1:100 i n 0.9% s a l i n e . TABLE I I . IMMUNIZATION SCHEDULE FOR THE INDUCTION OF ANTIBODIES TO STAGES I AND I I I GASTRINS IN TWO MALLARD DUCKS Day A n t i g e n 0 2 5 mg. Stage I G a s t r i n s g I! If II 1 6 " " " 2 5 it n it 5 8 " " " 1 0 2 1 mg. Stage I I I G a s t r i n s 1 2 1 .» " " 1 5 5 11 11 11 \ 21 of the c e l l s was s c o r e d a c c o r d i n g t o C a r p e n t e r (1968). When the r i n g t e s t was t o be used t o e s t a b l i s h the zone of e q u i v a l e n c e , u n d i l u t e d a n t i s e r u m was p l a c e d by P a s t e u r p i p e t t e i n t o a s e r i e s of Kimax p r e c i p i t i n tubes (6 x 50 '(am.) t o a depth of 10 mm. Decimal d i l u t i o n s of a n t i g e n (Stages I and I I I gas-t r i n s ) from an i n i t i a l c o n c e n t r a t i o n of 0.25 mg. <• per ml. s a l i n e , was c a r e f u l l y p l a c e d over the a n t i s e r u m to form a sharp i n t e r -f a c e . A c o n t r o l s e r i e s of NDS w i t h the a n t i g e n d i l u t i o n s were a l s o p r e p a r e d . The tubes were examined f o r i n t e r f a c e p r e c i p i t a -t i o n a f t e r 3 hours a t room temperature and a g a i n f o l l o w i n g o v e r -n i g h t r e f r i g e r a t i o n a t 6°C. The p r e c i p i t i n r e a c t i o n was graded a c c o r d i n g t o C a r p e n t e r (1968) and the t i t r e i n d i c a t e d by the r e c i p r o c a l o f the h i g h e s t a n t i g e n d i l u t i o n which gave a d e t e c -t a b l e p r e c i p i t i n l i n e . B. ANTIBODIES TO STAGE I I I GASTRINS Stage I I I g a s t r i n s i n e q u a l volumes of d i s t i l l e d water and Freunds complete a d j u v e n t ( l ml. each) were g i v e n by i n t r a -muscular i n j e c t i o n s to a s i n g l e randomly s e l e c t e d , a n t r e c t o m i z e d w h i t e r a b b i t . The i m m u n i z a t i o n s c h e d u l e i s l i s t e d i n Table I I I . F o l l o w i n g ear v e i n b l e e d i n g on days 17 and 29, t h e serum was assayed f o r the presence of a n t i b o d y by p a s s i v e h e m a g g l u t i n a t i o n as p r e v i o u s l y d e s c r i b e d . 2 2 C. ANTIBODIES TO A HEMOCYANIN-SYNTHETIC HUMAN GASTRIN 2 - 1 7 CONJUGATE The d i a l y z e d hemocyanin-SHG 2 - 1 7 c o n j u g a t e was emul-s i f i e d w i t h an e q u a l volume of Freunds complete a d j u v e n t and i n -j e c t e d i n t o 3 randomly s e l e c t e d w h i t e r a b b i t s a c c o r d i n g t o the sc h e d u l e g i v e n i n Table IV. A l l a n i m a l s were b l e d by the ear v e i n on days 1 9 and 3 8 . R a b b i t s RSR 1 and RSR 2 o n l y , were b l e d on day 6 0 and the s i n g l e r e m a i n i n g r a b b i t a g a i n on day 1 8 7 . Assays f o r a n t i b o d i e s t o hemocyanin and Stage I I I g a s t r i n s were conducted on the p o o l e d serums a t each b l e e d i n g by p a s s i v e h e m a g g l u t i n a t i o n and/or p r e c i p i t i n r i n g t e s t . D. ANTIBODIES TO A LATEX-STATE I I I GASTRIN CONJUGATE Ga s t r i n - b o u n d l a t e x s u s p e n s i o n s were g i v e n i n i t i a l l y t o 3 randomly s e l e c t e d 1 0 - w e e k - o l d c h i c k e n s by b r a c h i a l wing v e i n and t o 2 5 - w e e k - o l d r a b b i t s by m a r g i n a l ear v e i n i n j e c t i o n s . C o n t r o l a n i m a l s ( 2 c h i c k e n s and 2 r a b b i t s ) were i n j e c t e d w i t h unbound l a t e x s u s p e n s i o n s . The i m m u n i z a t i o n f o r the 2 s p e c i e s of a n i m a l s i s l i s t e d i n Table V. The a n i m a l s were bleep from the b r a c h i a l wing v e i n or m a r g i n a l ear v e i n and the serums were a s s a y e d ' f o r a n t i b o d y t o g a s t r i n by the r i n g t e s t , g e l d i f f u s i o n t e s t s and u l t i m a t e l y by c a t b i o a s s a y . The s e r i e s of t e s t s over a 7 month p e r i o d i s l i s t e d i n Table V I . P r e c i p i t i n r i n g t e s t s were c a r r i e d out w i t h the r a c t i o n m i x t u r e i n the f i r s t tube c o n t a i n i n g 0 . 2 ml. a n t i s e r u m o r c o n t r o l serum o v e r l a i n w i t h 1 0 0 , 7 2 , or 6 4 jig. of the t e s t a n t i g e n ( S t a g e f - I I I g a s t r i n s or SHG-22y 1 7 ) .Sinv-Q.5':.mlv."-.0.15 M sodium phosphate b u f f e r (pH 7 - 5 ) and TABLE I I I IMMUNIZATION SCHEDULE FOR THE INDUCTION OF ANTIBODIES TO STAGE I I I GASTRINS IN AN ANTRECTOMIZED RABBIT Day A n t i g e n 0 1 mg. Stage I I I G a s t r i n s 7 18 25 II it TABLE 'IV IMMUNIZATION SCHEDULE FOR THE INDUCTION OF ANTIBODIES TO HEMOCYANIN-SHG 2-17 IN THREE RABBITS* Day A n t i g e n Route 0 0.5 ml. Footpad 21 0.4 ml. I n t r a m u s c u l a r *A maximum of 0.2 ml. a n t i g e n , c o r r e s p o n d i n g to about 700 yag. SHG 2-17, was i n j e c t e d a t any one s i t e . 24 0.5 ml. 0 .9% s a l i n e . The r i n g t i t r e s were r e a d a f t e r i n c u b a t i o n for. 5 hours i n a 37°C water b a t h and 12 and 48 hours a t 6°C. Agar g e l d i f f u s i o n t e c h n i q u e s were c a r r i e d out a f t e r the methods of Campbell et a l . . ( l 9 6 4 ) . Oudin g e l d i f f u s i o n tubes were p r e -pared, by m i x i n g an e q u a l volume of l i q u i d agar ( l o n a g a r No. 2, 1.7% i n b o r a t e b u f f e r , pH 8.4) w i t h a n t i s e r u m o r c o n t r o l serum and a l l o w e d t o s o l i d i f y i n Kimax p r e c i p i t i n t u b e s (6 x 50 mm.). D o u b l i n g d i l u t i o n s of the t e s t a n t i g e n v/ere p l a c e d above the agar. I n the double d i f f u s i o n F r e e r system, and eq u a l volume of 0.85% agar was l a y e r e d between the a n t i s e r u m and the upper d i l u -t i o n s of the t e s t a n t i g e n . An O u c h t e r l o n y p l a t e was pr e p a r e d w i t h c h i c k e n a n t i s e r u m i n the c e n t e r w e l l and a n t i g e n d i l u t i o n s near the susp e c t e d p o i n t of e q u i v a l e n c e ( l to 8 yag. Stage I I I g a s t r i n s per ml.) i n the p e r i p h e r a l w e i l s . A l l agar g e l d i f f u -s i o n p r e p a r a t i o n s were s e a l e d and i n c u b a t e d a t room temperature. Readings were c a r r i e d out d a i l y f o r 10 days and the p r e c i p i t i n bands s c o r e d as a s i n g l e +. A f t e r c a r r y i n g . o u t the procedure d e s c r i b e d f o r the p r e c i -p i t i n r i n g t e s t , the tubes were c e n t r i f u g e d (1500 x g. f o r L Q min. a t 0°C) t o remove p r e c i p i t a t e and t h e s u p e r n a t a n t of s e l e c -t e d tubes were assayed f o r g a s t r i n i n an a n a e s t h e t i z e d c a t . The c a t b i o a s s a y was pr e p a r e d as p r e v i o u s l y d e s c r i b e d . The i n j e c -t i o n p r o c e d u r e , however, was conducted a f t e r the method of B l a i r e t a l . ( 1 968 ) . I n two such p r e p a r a t i o n s , the a n i m a l was brought t o s e c r e t e a background H + output of l e s s t h a n 400 yieq. p e r 15 min. by the i n j e c t i o n of a s t a n d a r d g a s t r i n (0.05 t o 0.35 ug. SHG 1 - 17 i n 1 ml. 0 .9% s a l i n e g i v e n over 45 s e c ) . When TABLE \J IMMUNIZATION SCHEDULE FOR THE INDUCTION OF ANTIBODIES TO LATEX-GASTRIN IN THREE CHICKENS AND TWO RABBITS* Day A n t i g e n Route No. C h i c k e n s I n j e c t e d No. R a b b i t s I n j e c t e d 0 1 rag. I n t r a v e n o u s 3 2 11 1 mg. • I n t r a v e n o u s ' -3 2 20 1 mg. I n t r a v e n o u s 3 2 31 1 mg. I n t r a m u s c u l a r 3 2 70 3-25 -mg. I n t r a v e n o u s 1 0 92 3.25 mg. I n t r a v e n o u s 1 0 137 3.25 mg. I n t r a v e n o u s 1 0 The i n t r a m u s c u l a r i n j e c t i o n was i n i t i a l l y e m u l s i f i e d w i t h an equ a l volume of Freunds complete a d j u v e n t and g i v e n a t 3 t h i g h l o c a t i o n s . TABLE VI/' BLEEDING SCHEDULE AND TESTS USED FOR THE DETECTION OF ANTIBODIES TO A LATEX-GASTRIN CONJUGATE No. No. C h i c k e n s R a b b i t s Day Te s t Employed B l e d B l e d 37 R i n g , Oudin 3 • 2 46 R i n g , Oudin 3 2 55 R i n g , Oudin 3 2 88 R i n g , Oudin, P r e e r , O u c h t e r l o n y 3 2 149 R i n g , O u din, P r e e r 1 0 200 R i n g , Oudin, P r e e r 1 0 240 R i n g , Oudin, P r e e r , Bioasse.y 1 0 27 a c o n s t a n t l e v e l o f s e c r e t i o n was e s t a b l i s h e d , f o r a l e a s t 3 washout p e r i o d s (if5 m i n . ) , a f r a c t i o n * of the a n t i s e r u m or con-t r o l serum, i n c u b a t e d w i t h SHG 1 - 1 7 and near the suspected p o i n t of e q u i v a l e n c e , was s u b s t i t u t e d f o r the s t a n d a r d . The s e c r e t o r y response of the "unknowns" was determined f o r a 30 min. p e r i o d f o l l o w i n g i n j e c t i o n and e x p r e s s e d as a percentage of the p r e d i c t e d response had t h e r e been no s u b s t i t u t i o n . Two s t a n d a r d i n j e c t i o n s between each unknown a l l o w e d f o r the e s t i -m a t ion of the changing response to g a s t r i n . *The f r a c t i o n of the "unknown" was d i l u t e d i n 0.9% s a l i n e t o b r i n g the c o n c e n t r a t i o n of SHG 1 - 1 7 near to t h a t of the back-ground i n j e c t i o n s . 28 RESULTS 1. ISOLATION AND PURIFICATION OF GASTRIN A. PRODUCT ANALYSIS OF HOG GASTRIN Hog g a s t r i n was prepared e s s e n t i a l l y by the method of Gregory and Tracy (1964). In the f i n a l stages of p u r i f i c a t i o n , b i oassays f o r g a s t r i n a c t i v i t y were preformed on a l i q u o t s from Sephadex G50 and AE column f r a c t i o n a t i o n . F r a c t i o n a t i o n on Sephadex G50 A t y p i c a l e l u t i o n curve f o r the f r a c t i o n a t i o n of g a s t r i n on Sephadex G50 g e l u s i n g 0.0A M NH^HCO^ (pH 8 . 0 ) , i s i l l u s t r a t e d i n F i g u r e 1. The absorbance of each tube f r a c t i o n (10 ml.) was measured over a 1.0 cm. l i g h t path at a wave l e n g t h of 280 rn p.. The shaded area (tubes 120-150) contained the e n t i r e amount of g a s t r i n a c t i v i t y as measured from the response of an ana e s t h e t i z e d cat to 0 . 2 ml. i n j e c t i o n s of randomly s e l e c t e d e l u a t e tubes. The p r e c i s e data from the bioassay- i s l i s t e d i n Table V I I i n -d i c a t i n g the tube f r a c t i o n assayed, the i n i t i a l washout pH and the H + output i n yaeq. per 20 min. f o l l o w i n g i n j e c t i o n . The maximal response, a 2/+ f o l d i n c r e a s e over b a s a l H output, f a l l s midway through the a c t i v e peak. Repeat i n j e c t i o n s to the same f r a c t i o n i n d i c a t e d that H + output d e v i a t e d < 0% over a 12 hour experimental p e r i o d . The assay, however, i s only semiquantita-t i v e i n nature since i t does not as s e s s changing responsiveness throughout the course of i n j e c t i o n . -Eluate--tube f r a c t i o n s con-t a i n i n g g a s t r i n a c t i v i t y were pooled and l y o p h i l i z e d to constant 0 . 5 O - | ^ 0 . 4 0 A CO CVJ 0 . 3 0 -I O 0 . 2 0 H ID •o o . i o H -i 1 1 ^ — i r o . o H 1 — 2 0 4 0 6 0 8O IOO I 2 0 1 4 0 160 1 8 0 Fraction No. FIGURE 1: FRACTIONATION OF HOG GASTRIN ON SEPHADEX G50 GEL TABLE V I I BIOASSAY FOR GASTRIN IN AN ANAESTHETIZED CAT FOLLOWING SEPHADEX G50 GEL FRACTIONATION F r a c t i o n H Output Number Washout pH (yj.eq./20 min.) S a l i n e C o n t r o l 6.4 20.0 56 • 6.2 20.1 70 6.2 26.1 90 6.0 25.0 110 5.9 30.1 120 3.4 106.3 125 2.8 418.2 130 • 2.7 479.8 135 2.7 • 424.9 140 2.8 337.2 150 3.1 146.3 160 5.4 50.2 170 t 6.2 25-3 3 1 weight. F r a c t i o n a t i o n on AE 1 1 I n a t y p i c a l p u r i f i c a t i o n , LO mg. of the Stage I I p r o d u c t from Sephadex f r a c t i o n a t i o n was adsorbed onto a column of amino-e t h y l c e l l u l o s e (Whatman AE 1 1 ) and e l u t e d w i t h a g r a d i e n t con-c e n t r a t i o n of 0 . 0 2 - 0 . 2 M NH^HCO^ (pH 8 . 0 ) . The e l u t i o n c u r v e , ' r e c o r d e d a t a wavelength of 2 8 0 rn p., i s i l l u s t r a t e d i n F i g u r e 2 . C o n d u c t i v i t y throughout the r u n was found to be e x a c t l y . .• - 2 l i n e a r from the onset of g r a d i e n t e l u t i o n ( s l o p e 1 . 7 5 x 1 0 m i l l i m h o s per m l . ) . Two w e l l s e p a r a t e d peaks c o r r e s p o n d i n g t o the g a s t r i n s GI and G I I d e s i g n a t e d by Gregory and Tracy ( 1 9 6 4 ) ? are a pparent. A s t a n d a r d g a s t r i n a ssay i n an a n a e s t h e t i z e d c a t from randomly s e l e c t e d tube f r a c t i o n s ( 0 . 2 ml.) c o n f i r m e d t h a t b o t h absorbance peaks c o n t a i n e d a l l of the a c t i v i t y ( t u b e s 6 0 -1 0 0 ) . T able V I I I l i s t s the complete d a t a from the b i o a s s a y i n d i -c a t i n g the tube assayed f o r a c t i v i t y , i n i t i a l washout pH and H + output i n yaeq. over a 3 0 min. p e r i o d , f o l l o w i n g i n j e c t i o n . A maximal response of a 5 2 f o l d i n c r e a s e over b a s a l H + output was r e c o r d e d i n the suspected GII f r a c t i o n . Both absorbance peaks (tub e s 6 0 - 7 8 and 7 9 - 9 5 ) were pool e d s e p a r a t e l y and l y o p h i l -i z e d t o c o n s t a n t weight, y i e l d i n g 9 . 0 mg. of GI and 1 2 . 0 mg. G I I from 6 0 0 hog antrums. W h i l e the y i e l d r a t i o of the two g a s t r i n s ( 3 : 4 ) corresponded e x a c t l y to t h a t r e c o r d e d by Gregory and Tracy ( 1 9 6 4 ) , the t o t a l y i e l d of b o t h the Stage I I (AO mg.) and Stage I I I ( 2 1 mg.) p r o d u c t s was c o n s i d e r a b l y l e s s than the p u b l i s h e d v a l u e s ( 5 4 and 3 9 mg. r e s p e c t i v e l y ) . A t o t a l of 9 1 5 0 32 i o - , no Fraction No. i i : FIGURE 2: FRACTIONATION OF HOG GASTRIN ON AMINOETHYL CELLULOSE POWDER TABLE fVITI BIOASSAY FOR GASTRIN IN AN ANAESTHETIZED CAT FOLLOWING AMINOETHYL CELLULOSE FRACTIONATION Number Washout pH (/aeq./30 min.) S a l i n e C o n t r o l 5.0 25.3 40 3.8 52.5 50 3.5 90.6 56 . 3.5 80.8 66 2.8 • 574.6 71 2.7 794.8 74 2.7 618.6 78 2.9 400.5 82 2.7 613.8 86 2.4 1301.6 89 2.7 670.4 97 3.2 219.5 100 3.1 197.7 3k hog antrums i n 15 e x t r a c t i o n s were p r o c e s s e d f o r g a s t r i n f o l l o w -i n g the d e s c r i b e d p r o c e d u r e . A n a l y s i s of G a s t r i n s GI'and GII i E l e c t r o p h o r e s i s : Stage I I I g a s t r i n s GI and GII e l e c t r o - -phoresed a t low v o l t a g e (360 v.) i n 0.1 N b o r a t e b u f f e r (pH 8.5)> p y r i d i n e : g l a c i a l a c e t i c a c i d : w a t e r ( 1 0 : 0 . 4 : 90 v/v, pH 6.6) and 15% g l a c i a l a c e t i c a c i d (pH 2 .0 ) were shown t o c o n t a i n no d e t e c -t a b l e i m p u r i t i e s . Both g a s t r i n s e l e c t r o p h o r e s e d as s i n g l e com-pact s p o t s as shown by s t a i n i n g w i t h the c h l o r i n e - t o l u i d i n e r e -age n t s . Moreover, the s p o t s d i d not r e a c t t o a n i n h y d r i n s t a i n i n g . The e l e c t r o p h o r e t i c m o b i l i t i e s * of the p r o d u c t s i n t h r e e s o l v e n t systems are. g i v e n i n Table I X . Th i n ' L a y e r Chromatography: Both g a s t r i n s GI and GII s t a i n e d as s i n g l e compact s p o t s on s i l i c a g e l p l a t e s a f t e r r u n s i n n-p r o p a n o l : g l a c i a l a c e t i c a c i d : w a t e r ( 100 :10 :30 v/v, pH 3 .2) and n-propanol:water (70 :30 v/v, pH 7 . 4 ) . Amounts t o as l i t t l e as 5 yag per a p p l i c a t i o n c o u l d be d e t e c t e d w i t h c h l o r i n e - t o l u i d i n e s t a i n i n g w h i l e no r e a c t i o n t o n i n h y d r i n c o u l d be observed even-i n h e a v i e r a p p l i c a t i o n s (100 y a g . ) . T able X - l i s t s the r e f e r e n c e d i s t a n c e s of the g a s t r i n s i n the two s o l v e n t systems.** *The r a t i o of the d i s t a n c e of m i g r a t i o n s of the sample t o the b l u e component of the s t a n d a r d RBY dye (Gelman). **The . r a t i o of the d i s t a n c e of m i g r a t i o n o f the sample t o t h a t of the s o l v e n t f r o n t . •35-' TABLE '--IX ELECTROPHORETIC MOBILITIES FOR GASTRINS GI AND"GII IN THREE SOLVENT SYSTEMS Bo r a t e P y r i d i n e : A c e t i c A c i d : 15% A c e t i c S o l v e n t B u f f e r Water ( 1 0 : 0 . 4 : 90 v/v) A c i d GI 0.91 + 0.05 0.57 + O.Oif OJ+A + 0.02* GII 0.98 + 0.05 0.71 + 0.0A O.Zf4 + 0.04 TABLE X THIN LAYER CHROMATOGRAPHY REFERENCE DISTANCES FOR GASTRINS GI AND GII IN TWO SOLVENT SYSTEMS P r o p a n o l : A c e t i c A c i d : Propanol:Water S o l v e n t Water ( 100 :10 :30 v/v) (70:30 v/v) GI 0.72 + 0.04 0.64 ± 0.05 G I I 0.71 + 0.04 0.62 + 0.05 36 Descending Paper Chromatography: Both g a s t r i n s GI and GII run s i m u l t a n e o u s l y i n n - b u t a n o l : g l a c i a l a c e t i c a c i d : w a t e r (40: 10:30 v/v, pH 3-2) s t a i n e d as s i n g l e s p o t s w i t h the Pa u l y r e -agent. No r e a c t i o n was e v i d e n t on the a p p l i c a t i o n of n i n h y d r i n s p r a y . The r e f e r e n c e d i s t a n c e s f o r b o t h g a s t r i n s was 0.31 + 0.05 and v a r y i n g the amount of a p p l i e d sample from 10 to 100 yag. f a i l e d t o d i s t i n g u i s h between the m o b i l i t i e s of the two p r o d u c t s . Amino A c i d A n a l y s i s : Amino a c i d a n a l y s i s of a 10 n mole g a s t r i n GII h y d r o l y s a t e c o n f i r m e d t h a t the Stage I I I p r o d u c t was the v i r t u a l l y pure hormone. A n a l y s i s of the readout absorbance peaks, c o r r e c t e d t o the n e a r e s t i n t e g e r , i n d i c a t e d t h a t the p r o -duct bore an i d e n t i c a l c o n s t i t u t i o n t o t h a t r e p o r t e d by Gregory and Tracy (1964). I t was apparent t h a t the h y d r o l y s i s (110°C i n 6 N HC1 f o r 16 hours) was i n c o m p l e t e . A g l u t a m y l g l u t a m i c a c i d d i p e p t i d e was e l u t e d from the h y d r o l y s a t e which may have accoun-ted e n t i r e l y f o r the low g l u t a m i c a c i d c o n s t i t u t i o n . * I n par-t i c u l a r , the h y d r o l y s a t e was shown to be f r e e of c y s t i n e , i s o -l e u c i n e , l e u c i n e , l y s i n e , s e r i n e , t h r e o n i n e and v a l i n e . T r y p t o -phan, n o r m a l l y d e s t r o y e d under the s p e c i f i e d c o n d i t i o n s , was not e s t i m a t e d . B. PRODUCT ANALYSIS OF SALMON "GASTRIN" The i s o l a t i o n of-salmon " g a s t r i n " was attempted employing * C a l c u l a t e d to be 3.85* S i x g l u t a m y l residue.s a r e p r e s e n t i n each m o l e c u l e . an i d e n t i c a l procedure as used f o r the e x t r a c t i o n of hog g a s t r i n (Gregory and Tracy, 196A). F i g u r e 3 i l l u s t r a t e s one o f two c o l -umn f r a c t i o n a t i o n s of an e x t r a c t from 2+00 salmon antrums. The pro d u c t was e l u t e d from the g e l (Sephadex G50) u s i n g 0.02+ M NH^HCO^ (pH 8.0) and r e a d a t a wavelength of 280 ra p.. The shaded a r e a ( t u b e s 57 - 62) i n d i c a t e s t h a t , u n l i k e the e l u t i o n of hog g a s t r i n from the g e l , the m a j o r i t y of g a s t r i c a c t i v i t y i s c o n t a i n e d w i t h i n r a t h e r than beyond the l e a d i n g p r o t e i n peak. B i o a s s a y r e s u l t s were o b t a i n e d , i n t h i s case, from a c o n s c i o u s dog p r e p a r a t i o n . Table X I : l i s t s the p r e c i s e d a t a from the b i o a s s a y d e s i g n a t i n g the tube number assayed, the i n i t i a l wash-out pH and the t o t a l H output e x p r e s s e d i n y i e q . per 36 min. f o l l o w i n g i n j e c t i o n (0.5 m l . ) . A maximal response of a 3.5 f o l d i n c r e a s e over b a s a l H out p u t o c c u r s i n the b r e a k t h r o u g h peak w h i l e some r e s i d u a l a c t i v i t y s t i l l appears i n the d e s c e n d i n g l i m b of the e l u t i o n c u r v e . 2. RESPONSE OF THE BULLFROG FUNDIC MUCOSA TO GASTRIN The r e s p o n s i v e n e s s of a b u l l f r o g (Rana c a t e s b i a n a ) f u n d i c mucosa t o d e c i m a l d i l u t i o n s of Stage I I I hog g a s t r i n i s i l l u s -t r a t e d i n F i g u r e L. The d a t a from the same b i o a s s a y i s l i s t e d i n Table X I I i n d i c a t i n g the i n i t i a l washout pH and H + output ex-p r e s s e d i n yaeq. per 30 min. f o l l o w i n g s u b s t i t u t i o n w i t h each 10 f o l d d i l u t i o n of a c t i v e b u f f e r . T h r e s h o l d response t o the ex-t r a c t e d g a s t r i n o c c u r s a t about A.5 x 10 ^ M (90 ng.) w h i l e a maximal response of 2..5 f o l d i n c r e a s e over b a s a l H + output i s g i v e n t o 4.5 x 10" 6 M (90 yug.). FIGURE y. FRACTIONATION OF SALMON "GASTRIN" ON SEPHADEX G50 GEL TABLE XI BIOASSAY FOR SALMON "GASTRIN" IN A CONSCIOUS DOG FOLLOWING SEPHADEX G50 GEL FRACTIONATION F r a c t i o n H1" Output Number Washout pH (yaeq./36 min.) S a l i n e C o n t r o l 3.3 101.9 59 2.5 346.A 69 3.0 127.3 83 3.0 191.4 97 • • 3.0 139.7 113 2.8 190.1 Control I O - 9 I O - 8 I O - 7 I O - 6 I O - 5 Control Molarity of Gastrin x 4.5 FIGURE L: RESPONSE OF THE BULLFROG FUNDIC MUCOSA TO NATURAL GASTRIN 0 TABLE X I I RESPONSE OF THE BULLFROG FUNDIC MUCOSA TO NATURAL GASTRIN M o l a r i t y H Output of G a s t r i n Washout pH (yueq./^O B u f f e r C o n t r o l . 4 .8 2.05 4 .5 x I O " 9 4.6- 2.70 4 .5 x I O " 8 4 .2 3.62 4 . 5 x 10~. 7 4.1 5.02 4 . 5 x 1 0 ~ 6 4.1 5.12 4 .5 x I O - 5 4 .5 4 .55 2+2 3. MODIFICATION OF GASTRIN A. PRODUCT ANALYSIS OF ALKALINE HYDROLYSIS OF GASTRIN NaOH H y d r o l y s i s : Stage I I I hog g a s t r i n GI (7-3 mg.), d i s -s o l v e d i n 0.5 ml. 1.0 M NaOH and i n c u b a t e d f o r 2+0 hours a t room temperature, was a p p l i e d t o a column of Sephadex G10 g e l . The t r e a t e d p r o d u c t was d e s a l t e d w i t h 0.02+ M NH^HCO^ (pH 8.0). Pr o -duct a n a l y s i s of NaOH t r e a t e d and n a t u r a l g a s t r i n (GI) by e l e c -t r o p h o r e s i s i s g i v e n i n Table X I I I . The a p p l i e d samples (100 yag were e l e c t r o p h o r e s e d i n b o r a t e b u f f e r (pH 8.5) and 15% a c e t i c a c i d (pH 2.0) f o r 2 hours each and s t a i n e d by n i n h y d r i n and c h l o r i n e - t o l u i d i n e methods. Each t a b l e d v a l u e i n d i c a t e s the e l e c t r o p h o r e t i c m o b i l i t y of the a p p l i e d p r o d u c t ( s ) w i t h r e s p e c t to the b l u e component of the s t a n d a r d RBY dye (Gelman). A n i n -h y d r i n p o s i t i v e component was o b s e r v a b l e a f t e r r u n s i n both s o l -vent systems. T h i s component was e v i d e n t as the t r a i l i n g spot i n the b o r a t e system but appeared unseparated from the l e a d i n g f r a c t i o n a t the l o w e r pH (15% g l a c i a l a c e t i c a c i d ) . T h i n l a y e r s e p a r a t i o n of the p r o d u c t s (100 yag.) was c a r r i e d out on s i l i c a g e l p l a t e s . R^ v a l u e s f o r NaOH t r e a t e d and n a t u r a l g a s t r i n GI i n 3 s o l v e n t systems are g i v e n i n Table XIV. . A l l v a l u e s were ob t a i n e d from s p o t s s t a i n e d by the c h l o r i n e - t o l u i d i n e t e c h n i q u e but i n each case a t r a i l i n g peak appeared which r e a c t e d t o the a p p l i c a t i o n of the n i n h y d r i n r e a g e n t . A bulk, s e p a r a t i o n o f the h y d r o l y z e d and n a t u r a l g a s t r i n s was attempted by s u l f o e t h y l Sephadex (SE Sephadex) . f r a c t i o n a t i o n . The remainder o f the NaOH r e a c t i o n p r o d u c t (5.3 mg.) was a p p l i e d t o the beads and e l u t e d TABLE X I I I ELECTROPHORETIC MOBILITIES FOR NATURAL GASTRIN GI AND NaOH TREATED GI IN TWO SOLVENT SYSTEMS S o l v e n t B o r a t e B u f f e r 15% A c e t i c A c i d GI NaOH T r e a t e d GI 0.91 + 0.05 0.91 + 0.05 0.30 + 0.05 0.38 + 0.04 0.38 + O.OA TABLE vXIV THIN LAYER CHROMATOGRAPHY REFERENCE DISTANCES FOR NATURAL GASTRIN GI AND NaOH TREATED GI IN THREE SOLVENT SYSTEMS S o l v e n t B u t a n o l : A c e t i c A c i d : Water (4-0: 10:50 v/v) P r o p a n o l : A c e t i c A c i d : Water (100: 10:30 v/v) Propanol:Water (70:30 v/v) GI NaOH T r e a t e d GI 0.22 + 0.03 0.21 + 0.03 0.74 + 0.05 0.71 + 0.05 O.58 + 0.05 0.61 + 0.05 0.64 + 0.06 0.30 + 0.04 4.4 w i t h 0.01 to 0.2 M ammonium formate. Absorbance r e c o r d e d a t a wave l e n g t h of 280 m ji i n d i c a t e d the f a i l u r e of the p r o d u c t s t o se p a r a t e under the g i v e n c o n d i t i o n s of i o n exchange, e l u t i o n . NH/GH H y d r o l y s i s : Table XV l i s t s a s e r i e s of v a l u e s of p r o d u c t s o b t a i n e d from the h y d r o l y s i s of g a s t r i n GII under a l k a l i n e c o n d i t i o n s (1.0 M NH^ i n w a t e r ) . Samples of 75 yj-g V e r • a p p l i c a t i o n from time s t a g g e r e d h y d r o l y s e s were a l l o w e d t o r u n on a de s c e n d i n g paper chromatogram i n n - b u t a n o l : g l a c i a l a c e t i c a c i d : w a t e r (2+0:10:50 v/v, pH 3.2). The t a b l e i n c l u d e s an a r b i -t r a r y s c a l e of n i n h y d r i n s t a i n i n g i n t e n s i t y a t each R f v a l u e s i n c e n i n h y d r i n and P a u l y s t a i n s f o r the i n c u b a t i o n s appeared t o c o i n c i d e . 2+. DEMONSTRATION OF ANTIBODIES TO GASTRIN A. ANTIBODIES TO STAGE I AND I I I GASTRINS The i m m u n i z a t i o n s c h e d u l e f o r two M a l l a r d ducks i s l i s t e d i n Table I I . E i g h t i n t r a m u s c u l a r i n j e c t i o n s c o n t a i n i n g a t o t a l of 125 mg. Stage I ' g a s t r i n and 3 mg. Stage I I I g a s t r i n were g i v e n t o 2 b i r d s over a peasiod of 23 weeks. S e m i q u a n t i t a -t i v e r e s u l t s from p a s s i v e h e m a g g l u t i n a t i o n ' t e s t s (Campbell et a l . , 1962+) on serum o b t a i n e d from one of two ducks i n j e c t e d w i t h the a n t r a l e x t r a c t s are g i v e n i n Table XVI. Normal duck serum (NDS) and non s p e c i f i c c o n t r o l s (NS) were i n c u b a t e d s i m u l t a n e o u s l y w i t h the a n t i s e r u m d i l u t i o n s of 1:100 to 1:25,600 i n ND S - s a l i n e . Stages I and I I I e x t r a c t s were employed as the d e t e c t o r a n t i g e n 45. . TABLE XV' . DESCENDING PAPER CHROMATOGRAPHY REFERENCE DISTANCES OF SAMPLES FROM THE SERIAL HYDROLYSIS OF GASTRIN GI BY A 1 M AMMONIA SOLUTION WITH NINHYDRIN STAINING INTENSITIES* I n c u b a t i o n Time (Hours) R e f e r e n c e D i s t a n c e N i n h y d r i n I n t e n s i t y 0 0.30 + 0.05 -12 0.31 + 0.05 -r 18 •- ' 0 .30 + 0.05 + + 24 0.30 + 0.05 + + 30 0.31 + 0.05 + + + 36 0.31 + 0.05 +++ -Graded from no r e a c t i o n (-) to a s t r o n g n i n h y d r i n r e a c t i o n (+++). /-,.6 (250 yjg. per ml.) throughout the s e r i e s of t e s t s . The second duck proved to be l e s s r e s p o n s i v e t o i m m u n i z a t i o n , r e s u l t i n g i n c o n s i s t e n t l y l ower t i t r e s than the f i r s t (1+00:1600). B. ANTIBODIES TO STAGE I I I GASTRINS The i m m u n i z a t i o n schedule f o r a s i n g l e w h i t e r a b b i t to i n t r a m u s c u l a r i n j e c t i o n s of pure n a t u r a l g a s t r i n i s l i s t e d i n Table I I I . A t o t a l of A mg. of the e x t r a c t I n Freund's complete a d j u v e n t was i n j e c t e d over a p e r i o d of 25 days. A n t i b o d i e s , how ever, c o u l d not be d e t e c t e d by p a s s i v e h e m a g g l u t i n a t i o n a f t e r the s e r i e s of A i n j e c t i o n s and the ani m a l d i e d b e f o r e f u r t h e r serum a n a l y s i s was p o s s i b l e . C. ANTIBODIES TO A HEMOCYANIN-SYNTHETIC HUMAN GASTRIN CONJUGATE S y n t h e t i c human g a s t r i n 2 - 1 7 (SHG 2 - 1 7 , 10 mg.) bound w i t h l i m p e t hemocyanin were i n j e c t e d i n t o 3 white r a b b i t s i n a p e r i o d of 21 days. The amount of a n t i g e n and. r o u t e of i n -j e c t i o n employed i s g i v e n i n Table IV. The r e s u l t s from a s e r i e of 3 r i n g t e s t s ( C a r p e n t e r , 1968) a p p l i e d t o the serum drawn from the 3 r a b b i t s 19 days a f t e r p r i m a r y i n j e c t i o n are g i v e n i n Table XVII i n d i c a t i n g a h i g h a n t i b o d y t i t r e t o hemocyanin (1000 to 10j000) but no d e t e c t a b l e t i t r e t o the Stage I I I m o l e c u l e . Hemocyanin and g a s t r i n d e t e c t o r a n t i g e n s were used a t an i n i t i a l c o n c e n t r a t i o n of 70 and 0.25 mg. r e s p e c t i v e l y . A s e r i e s of p a s s i v e h e m a g g l u t i n a t i o n t e s t s f o r a n t i b o d i e s on p o o l e d r a b b i t serum i s l i s t e d i n Table X V I I I . Assays a t day TABLE XVI.;" SEMIQUANTITATIVE PASSIVE HEMAGGLUTINATION TESTS FOR ANTIBODIES TO STAGES I AND I I I GASTRINS ON SERUM DRAWN FROM A SINGLE DUCK D e t e c t o r A n t i g e n A n t i s e r u m D i l u t i o n Day (250 yj.g./ml.) 1:100 1:400 1:1600 1: 64OO 1:25,600 -"Ni )S. NS 35 Stage I G a s t r i n s ++ + + .+ + + + 65 Stage I G a s t r i n s +++ ++ + + + + + 165 Stage I G a s t r i n s +4-+ ++ + + + 35 Stage I I I G a s t r i n s . ++ + + + + + + 65 Stage I I I G a s t r i n s ++•+ ++ ++ + + + JL 165 Stage I I I G a s t r i n s +++ + + + -1. + + 43 f o l l o w i n g i n i t i a l i n j e c t i o n were conducted on serum p o o l e d from the b l e e d i n g of a l l 3 r a b b i t s w h i l e t e s t s on days 60 and 18? were c a r r i e d out oh the serums of 2 a n i m a l s . V i s u a l o b s e r v a t i o n r e -v e a l e d a h i g h t i t r e of a n t i b o d i e s t o the hemocyanin moiety (25,600 to 51 j200) but t h a t the serums were a p p a r e n t l y l a c k i n g any a n t i b o d y to the hapten. Normal r a b b i t serum (NRS) and non s p e c i f i c (NS) c o n t r o l s gave v i r t u a l l y no a g g l u t i n a t i o n of the c e l l s . D. ANTIBODIES TO A LATEX-STAGE I I I GASTRIN CONJUGATE Table .V l i s t s the i m m u n i z a t i o n s c h e d u l e of l a t e x bound g a s t r i n i n 3 c h i c k e n s and 2 r a b b i t s . The a n t i g e n was g i v e n by i n t r a v e n o u s and i n t r a m u s c u l a r i n j e c t i o n s over a 10 week p e r i o d . The r e s u l t s of g a s t r i n a n t i b o d y assay from a v a r i e t y of ,agar g e l d i f f u s i o n t e s t s and 'cat b i o a s s a y are l i s t e d i n Table XIX. The r e s u l t s of the- s i n g l e O u c h t e r l o n y system attempted, not shown i n the T a b l e , was n e g a t i v e . A l l t e s t s were conducted a t l e a s t 6 days a f t e r i n j e c t i o n of the l a t e x - b o u n d g a s t r i n . The Table i n -d i c a t e s the day o f serum w i t h d r a w a l and the p o i n t of e q u i v a l e n c e of the d e t e c t o r a n t i g e n i n jig. per ml. f o r each t e s t . Stage I I I hog g a s t r i n was employed i n t e s t s up to and i n c l u d i n g those c a r r i e d out on day 1A9 w h i l e the s y n t h e t i c hormone (SHG 2 - 1 7 ) was used t h e r e a f t e r . A l l a s s a y s a f t e r day 55 were d u p l i c a t e d . I t was e v i d e n t t h a t i n most cases a n t i b o d i e s to g a s t r i n were p r e s e n t , however i n low t i t r e s . The r i n g t e s t , e s p e c i a l l y , appeared p o o r l y s u i t e d f o r the d e t e c t i o n of the p r e c i p i t a t i n g system. TABLE XVII -SEMIQUANTITATIVE RING TESTS FOR ANTIBODIES TO HEMOCYANIN AND GASTRIN IN RABBITS Assays were conducted on serum drawn from t h r e e a n i m a l s 19 days a f t e r a prim a r y i n j e c t i o n of a, hemocyanin-SHG 2-17 c o n j u g a t e . R a b b i t D e t e c t o r A n t i g e n A n t i gen D i l u t i o n 1:1 1:10 X 1:10 1:10 3 1:10^ 5 b l : K r 1:10° 1:10 7 S a l i n e C o n t r o l RSR 1 Hemocyanin +++ +++ ++ + + _ - -RSR 2 Heniocyanin +++ +++ ++ ++ + + - -RSR 3 ' .Hemocyanin +++ ++ ++ + + _ - -RSR 1 Stage I I I G a s t r i n s - - - - - - -RSR 2 Stage I I I G a s t r i n s _ _ _ _ — RSR 3 Stage I I I G a s t r i n s TABLE X V I I I SEMIQUANTITATIVE PASSIVE HEMAGGLUTINATION TESTS FOR ANTIBODIES TO HEMOCYANIN AND GASTRIN ON POOLED SER WIS FROM THREE RABBITS. A l l 'tests were conducted a f t e r t h e second of two i n j e c t i o n s of a hemocyanin-SHG 2-17 c o n j u g a t e . D e t e c t o r A ,. A n t i g e n D i l u t i o n Day A n t i g e n 1:100 1:A00 1:1600 1:64-00 1:25,600 1:51,200 NES NS 58 Hemocyanin +++ +++ +++ ++ + + JL. 60 Hemocyanin +++ ++-+ +++ +++ + + 187 Hemocyanin +++ +++ ++ ++ + + -33 Stage I I I G a s t r i n s 60 Stage I I I G a s t r i n s 187 Stage I I I G a s t r i n s 51 A c a t bioassay. s u b s t a n t i a t e d the f i n d i n g s of the g e l d i f -f u s i o n - p r e c i p i t i n t e s t s . F i g u r e 5 i l l u s t r a t e s a t y p i c a l "gas-t r i n a n t i b o d y assay" r e c o r d i n an a n a e s t h e t i z e d c a t a f t e r the method, of B l a i r et a l . (1968). The F i g u r e i n d i c a t e s s t a n d a r d g a s t r i n (0.35 yj-g- of SHG 1 - 1 7 i n 1 ml. s a l i n e ) o r "unknown" i n j e c t i o n s a t 15 min. i n t e r v a l s . The l a t t e r r e f e r s t o a n t i s e r u m or c o n t r o l serum i n c u b a t e d w i t h SHG 1 - 1 7 i n a c c o r d w i t h the r i n g t e s t , b o t h of which were i n j e c t e d a t SHG 1 - 17 c o n c e n t r a -t i o n s a p p r o x i m a t i n g t h a t of the s t a n d a r d . The s o l i d l i n e denotes the s e c r e t o r y . r e s p o n s e w h i l e the dashed l i n e e s t i m a t e s the p r e -d i c t e d response from the background i n j e c t i o n s . The s e c r e t o r y response of the unknowns i s r e c o r d e d i n yueq. o f H per j>0 min. p e r i o d f o l l o w i n g i n j e c t i o n and i s then expressed as a percentage of the p r e d i c t e d response had t h e r e been no s u b s t i t u t i o n . The r e s u l t s of 3 unknowns and t h e i r c o r r e s p o n d i n g c o n t r o l s ( l a t e x i n j e c t e d c h i c k e n serum) a re g i v e n in. Table XX. While the con-t r o l serums a re somewhat h i g h e r than the p r e d i c t e d response (100%), i t i s c l e a r t h a t the c o r r e s p o n d i n g e x p e r i m e n t a l serums at 0.39 and O.78 yj.g. per ml. do po s s e s s some i n a c t i v a t i n g a n t i -body. No i n a c t i v a t i o n i s e v i d e n t a t a h i g h e r a n t i g e n concen-t r a t i o n (3-12 yig. per m l . ) . TABLE XIX ASSAYS FOR ANTIBODIES TO GASTRIN BY GEL DIFFUSION AND BIOASSAY TECHNIQUES T i t r e s a r e g i v e n I n yug. per ml. a t the observed p o i n t of e q u i v a l e n c e . Day R i n g . T e s t Oudin T e s t s F r e e r Test Cat B i o a s s a y 37 0 0 46 0 4.5 55 0 4.5 88 1 4 2 149 1 0 200 0 2 2 249 0 0.39 - 0.78 53 Injection of Standard 4 0 0 - i Collection Period No. t t Injection of Unknown FIGURE 5: BLAIR-TYPE GASTRIN ASSAY FOR ANTIBODY IN AN ANAESTHETIZED CAT RESULTS FROM A BLAIR-TYPE GASTRIN ASSAY FOR ANTIBODY An t i s e r u m s were i n c u b a t e d w i t h d i l u t i o n s of s y n t h e t i c a n t i g e n (SHG 1-17) and a s s e s s e d f o r g a s t r i c a c t i v i t y a s percentage of a. background H' output i n an a n a e s t h e t i z e d c a t . A n t i g e n C o n c e n t r a t i o n % o f P r e d i c t e d Response G a s t r i n I n j e c t e d C h i c k e n C o n t r o l C h i c k e n 0.39 86.0 154-4 0.7 8 93.8 145.8 3.12 102.2 102.6 55-DISCUSSION. W i t h i n the p a s t decade radioiraraunologic t e c h n i q u e s have been developed f o r serum and t i s s u e measurements o f such hormones as i n s u l i n (Yalow and Berson, i960), glucagon (Unger e t a l . , 1961), growth hormone (Hunter and Greenwood, 1962) and even the non a n t i g e n i c p o l y p e p t i d e b r a d y k i n i n (Spragg, Austen and Haber, 1966) t o name o n l y a few. More r e c e n t l y s e v e r a l groups have extended the radioimmunoassay t e c h n i q u e to the he p t a d e c a p e p t i d e g a s t r i n . A n t i b o d i e s t o g a s t r i n , n e c e s s a r y f o r the radioimmuno-assay have been induced by the i n j e c t i o n of c o u p l e d (McGuigan, 1967, 1968a, 1968b; Stremple and Meade, 1968) or uncoupled ( O d e l l e t a l . , 1968; l a l o w and Berson, 1970) a n t i g e n . I t i s apparent from the h i g h degree o f p r e c i s i o n and s p e c i f i c i t y o f the r a d i o -immunoassays t h a t e i t h e r approach i s adequate i n s u p p l y i n g s u i t -a b l e a n t i b o d y t i t r e s . I t was i n p a r t the purpose o f the p r e s e n t study t o de v e l o p a radioimmunoassay f o r the d e t e c t i o n of g a s t r i n i n serum and t i s s u e and w h i l e the pure hormone i s d o u b t f u l l y a n t i g e n i c , a v a r i e t y of approaches were made f o r the procurement of a n t i b o d i e s . Most i m m u n i z a t i o n s were u n s u c c e s s f u l . As i s o f t e n the case i n im m u n i z a t i o n the s m a l l q u a n t i t y of crude and pure a n t r a l e x t r a c t s l i m i t e d i n j e c t i o n t o a few randomly s e l e c t e d a n i m a l s i n each s c h e d u l e . I n a l l but one case, the s u c c e s s o f i m m u n i z a t i o n r e l i e d g r e a t l y on the e x t r a c t i o n procedure taken e s s e n t i a l l y from the r e v i s e d methods of Gregory and Trac y (19ok)• The f r a c t i o n a t i o n of an a n t r a l e x t r a c t (from 600 hogs) on Sephadex G50 g e l i n F i g u r e 1, c l e a r l y shows the s e p a r a t i o n of a l a r g e i n e r t p r o t e i n peak f.f'ora a s m a l l e r f r a c t i o n o f merging peaks, b o t h of which c o n t a i n c o n s i d e r a b l e g a s t r i n a c t i v i t y (Table V I I ) . I t i s p r o b a b l e t h a t a t t h i s stage of p u r i f i c a t i o n , i n s p i t e of a c l o s e s i m i l a r i t y of the g a s t r i n s GI and GII i n s i z e and s t r u c t u r e , some s e p a r a t i o n of the two e x i s t s . S i x of 10 Sephadex r u n s gave a p a r t i a l s e p a r a t i o n of the g a s t r i n s which c o u l d b e s t Toe a t t r i b u t e d to a Sephadex a d s o r p t i o n phenomenon (Eaker and P o r a t h , 1967). The r e p r o d u c i b i l i t y of the e l u t i o n pat t e r n was s u f f i c i e n t t o omit the assay f o r a c t i v i t y f o r most r u n s and n o r m a l l y " a c t i v e " peaks were p o o l e d f o r i o n exchange sep-a r a t i o n . L i n e a r c o n c e n t r a t i o n g r a d i e n t e l u t i o n from AE c e l l u -l o s e y i e l d e d an almost complete absorbance s e p a r a t i o n ( F i g u r e 2) but assay a n a l y s i s r e v e a l e d t h a t a minimum of a 16 f o l d i n -c r e a s e over b a s a l H output s t i l l remained between the two peaks (Table V I I I ) . The a c t i v e peaks, a l t h o u g h l e s s dense than those r e c o r d e d by Gregory and Tracy (absorbance of 9-0 v s . 2.2 a t 280 m p.)} "were i d e n t i c a l i n n a t u r e and s e p a r a t i o n and were assumed to be g a s t r i n s GI and GII as d e s i g n a t e d by these a u t h o r s . G I I , p o s s e s s i n g the s u l f a t e d t y r o s y l r e s i d u e , appears to be r e t a i n e d and i s e l u t e d beyond the GI peak. B o t h p o s s e s s comparable gas-t r i n a c t i v i t i e s i n the a n a e s t h e t i z e d c a t . - The s e l e c t i o n of the a n a e s t h e t i z e d c a t or c o n s c i o u s dog f o r use i n the assay of a n t r a l e x t r a c t s was a r b i t r a r y a l t h o u g h the r e s u l t s suggested t h a t the c a t p r e p a r a t i o n was much the s u p e r i o r o f the two. B a s a l s e c r e t i o n r a t e s were g e n e r a l l y more c o n s t a n t i n the c a t than the dog. Moreover, the c a t as w e l l as b e i n g the 5? more r e s p o n s i v e t o e l u a t e i n j e c t i o n s , showed no troublesome " d u p l e x " e f f e c t i l l u s t r a t e d by the i n h i b i t i o n of II output a t l a r g e r doses. T h i s e f f e c t , noted by Gregory and Tracy (1964)} l i m i t s the use of the dog i n the assay of g a s t r i n u n l e s s s m a l l doses are adhered t o . I t must be emphasized t h a t q u a n t i t a t i v e l y , the g a s t r i n a s s a y i n the a n a e s t h e t i z e d c a t i s a crude t e c h n i q u e w h i l e i t n e g l e c t s the changing r e s p o n s i v e n e s s of the p r e p a r a t i o n d u r i n g the course of the experiment. The method of B l a i r et a l . ' , used i n t h i s r e p o r t f o r the assessment of a n t i b o d y to g a s t r i n , e s t i m a t e s t h i s change and i s p r e f e r a b l e i n the q u a n t i t a t i o n of unknowns. The d e c i s i o n t o for e g o f u r t h e r p u r i f i c a t i o n of the Stage I I I p r o d u c t was based l a r g e l y on t h e • f i n d i n g s o f low v o l t a g e e l e c t r o p h o r e s i s , t h i n l a y e r chromatography, d e s c e n d i n g paper chromatography and amino a c i d a n a l y s i s . I n a l l s e p a r a t i o n t e c h -n i q u e s GI and GII i n v a r i a b l y m i g r a t e d as w e l l d e f i n e d s p o t s w i t h o u t e vidence of i m p u r i t i e s ( T a b l e s IX and X ) . S o l v e n t pH changes c o n f i r m e d the homogeneity of the pr o d u c t and a l l s p o t s appeared t o be n i n h y d r i n n e g a t i v e . The l a t t e r o b s e r v a t i o n i s i n a c c o r d w i t h the f i n d i n g s o f Gregory and Tracy (1964) t h a t b o t h g a s t r i n s l a c k a f r e e ©<-ann.no group. E l e c t r o p h o r e t i c m o b i l i t i e s and d e s c e n d i n g and t h i n l a y e r chromatography r e f e r e n c e d i s t a n c e s i n t h i s r e p o r t a re g i v e n o n l y as an i n d i c a t i o n of homo-g e n e i t y of the a p p l i e d sample and must be i n t e r p r e t e d w i t h c a r e . N e v e r t h e l e s s , i t was c o n f i r m e d t h a t the n a t u r a l g a s t r i n s e l e c t r o -phoresed a t a l k a l i n e and s l i g h t l y a c i d i c pH's (pH 6.6) bore a net n e g a t i v e charge w h i l e some s u p p r e s s i o n of i o n i z a t i o n was 5 8 e v i d e n t a t the lower pH ( T a b l e I X ) . The e l e c t r o p h o r e t i c m o b i l i t y of GII exceeded t h a t of GI; an o b s e r v a t i o n which i s i n agreement w i t h the r e s u l t s of Gregory and Tracy (1964). The g a s t r i n s were i n s e p a r a b l e by t h i n l a y e r chromatography (Table X) a l t h o u g h the m i g r a t e d p r o d u c t s gave the b e s t r e s o l u t i o n of the t e c h n i q u e s attempted. That the R f v a l u e s are somewhat h i g h e r i n the s o l -vent system o f l o w e s t pH c o r r e s p o n d s to the e x p e c t a t i o n t h a t s u p p r e s s i o n of i o n i z a t i o n o f the a c i d i c r e s i d u e s i n c r e a s e s p o l y -p e p t i d e s o l u b i l i t y i n the m o b i l e phase. The m i g r a t i o n of GI and GII on d e s c e n d i n g paper chromatograms a l s o i n d i c a t e s the d i f f i -c u l t y i n s e p a r a t i n g two m o l e c u l e s of i d e n t i c a l net charge and s i m i l a r m o l e c u l a r weight. Amino a c i d a n a l y s i s c o n f i r m e d t h a t the Stage I I I product was v i r t u a l l y pure. Tryptophan, a p p a r e n t l y d e s t r o y e d , was not e s t i m a t e d but the h i g h absorbancy of the ex-t r a c t i n s o l u t i o n a t 2 8 0 m jx i n d i c a t e d i t s presence i n the m o l e c u l e . S a l m o n ' " g a s t r i n " u n l i k e hog g a s t r i n was not i d e n t i f i a b l e as a d i s t i n c t absorbance peak i n the Sephadex e l u a t e ( F i g u r e 3 ) a l -though some g a s t r i c a c t i v i t y was -present ( 3 * 5 f o l d i n c r e a s e over b a s a l c o n d i t i o n s ) . While s l i g h t r e s i d u a l a c t i v i t y i s con-t a i n e d i n the d e s c e n d i n g l i m b (Table X I ) , the e l u t i o n of the major . p o r t i o n of a c t i v i t y i n the v o i d volume argues a g a i n s t any s i z e s i m i l a r i t y of hog and salmon g a s t r i n m o l e c u l e s . The a c t i -v i t y beyond the b r e a k t h r o u g h peak may r e f l e c t the e l u t i o n of breakdown p r o d u c t s of the n a t u r a l salmon "hormone." F u r t h e r ex-t r a c t i o n would seem n e c e s s a r y to c o n f i r m t h a t salmon " g a s t r i n " does i n f a c t e x i s t and i s l o c a t e d i n the s o - c a l l e d antrum of the -'--59-stomach. A l t h o u g h the two v a r i a t i o n s of the s t a n d a r d c a t b i o a s s a y f o r g a s t r i n i n t h i s r e p o r t proved t o be p r e c i s e and r e p r o d u c i b l e , the p r e p a r a t i o n i s r e l a t i v e l y e x p e n s i v e and time consuming. I t was a n t i c i p a t e d t h a t the use o f the i n v i t r o b u l l f r o g mucosa would p e r m i t a more r a p i d y e t s e n s i t i v e assessment f o r g a s t r i n a c t i v i t y . A s i m i l a r p r e p a r a t i o n t o the one d e s c r i b e d h e r e i n has been shown t o be r e s p o n s i v e t o c o n c e n t r a t i o n s as low as 2.5 x I O - 9 M G I I (Davidson e t a l . , 1 9 6 7 ) . These a u t h o r s .found, however, t h a t the response was s e a s o n a l dependent and t h a t f a l l •and w i n t e r mucosae were not as r e c e p t i v e t o GII as were those i n the s p r i n g and summer months. I n t h i s study a t o t a l of 10 m u c o s a l . p r e p a r a t i o n s were attempted ( F e b r u a r y and August) of which o n l y 2 responded f a v o u r a b l y t o the n a t u r a l hormone. Both were August p r e p a r a t i o n s . The a p p l i c a t i o n o f thes e p r e p a r a t i o n s was c o n s e q u e n t l y r e s t r i c t e d t o a few t r i a l s w i t h the pure h o r -mone and was not used as an assay f o r "unknowns." T h r e s h o l d f o r g a s t r i n a c t i v i t y appeared t o be comparable t o the c o n c e n t r a t i o n of 2 .5 x I O - 9 M i n d i c a t e d by Davidson e t a l . (1967) ( F i g u r e A, Table X I I ) . Maximal response o c c u r r e d a t a h i g h e r c o n c e n t r a t i o n —6 —7s o f the a c t i v e b u f f e r ( 4«5 x 10 v s . 2 .5 x 10 ) and was l e s s i n t e n s e ( A 0 % ) . The m o d i f i c a t i o n of a p e p t i d e b e a r i n g a p y r o g l u t a m y l r e s i -due by e x p o s i n g an o< -amino group, i s not unique. Dekker e t a l . (I9A9) were a b l e t o open the b l o c k i n g r e s i d u e o f the t r i p e p t i d e f a s t i g i a t i n under m i l d a l k a l i n e c o n d i t i o n s . More r e c e n t l y , Brdmback and D o o l i t t l e (1963) employed NaOH h y d r o l y s i s t o 6 0 l i b e r a t e the N - t e r m i n a l amino group i n f i b r i n o p e p t i d e B. How-ever, s i n c e these t e c h n i q u e s were used f o r the purpose of pep-t i d e s e q u e n c i n g , the su c c e s s of s e l e c t i v e a l k a l i n e h y d r o l y s i s of g a s t r i n i n h i g h y i e l d was not a s s u r e d . Under i d e a l c o n d i t i o n s i t would be a n t i c i p a t e d t h a t maximal u n c y c l i z a t i o n s h o u l d o c c u r •with minimal p e p t i d e bond h y d r o l y s i s . E l e c t r o p h o r e t i c a n a l y s i s of the NaOH h y d r o l y s a t e r e v e a l e d t h a t a s p e c i f i c h y d r o l y s i s d i d i n f a c t take p l a c e ( T a b l e X I I I ) . At s l i g h t l y a l k a l i n e pH's (pH 8.5) a t r a i l i n g component appeared on h y d r o l y s i s which c o i n c i d e d w i t h the e x p e c t a t i o n s of an u n c y c l i z e d N - t e r m i n a l r e s i d u e of GI. No s e p a r a t i o n o f the p r o d u c t s was apparent a t the low e r pH (pH 2.0) a l t h o u g h i t might have been expected. T h i n l a y e r chromato-graphy of NaOH t r e a t e d GI y i e l d e d f u r t h e r e v i d e n c e t h a t s p e c i f i c h y d r o l y s i s had o c c u r r e d (Table X I V ) . Two s p o t s were d e t e c t e d ; the one of low e r v a l u e r e a c t i n g t o n i n h y d r i n . At s l i g h t l y a l k a l i n e and a c i d i c pH*s the m o d i f i e d g a s t r i n m o l e c u l e would be expected t o be l e s s m o b i l e than the n a t u r a l g a s t r i n by v i r t u e o f the a d d i t i o n of two i o n i z a b l e groups. No s e p a r a t i o n o f the p r o d u c t s was e v i d e n t w i t h a b u t a n o l s o l v e n t system but ru n s i n n - p r o p a n o l : a c e t i c a c i d : w a t e r (pH 3-2) i n d i c a t e d the s e p a r a t i o n of a t r a i l i n g f r a c t i o n , presumably the m o d i f i e d m o l e c u l e . B u l k s e p a r a t i o n of the p r o d u c t s by s u l f o e t h y l Sephadex f r a c t i o n a t i o n a t an a c i d pH was u n s u c c e s s f u l . T h i s f a i l u r e would i n d i c a t e t h e r e e x i s t s no p d s i t i v e charge d i f f e r e n c e between the m o d i f i e d and n a t u r a l g a s t r i n s a t pH 3-2. H y d r o l y s i s w i t h 1 M ammonia s o l u t i o n c a r r i e s the advantage o f a s a l t t h a t i s e a s i l y separ-a b l e from the p r o d u c t ( s ) . Table XV r e v e a l s the p r o g r e s s i v e 61 l i b e r a t i o n o f a f r e e oi -amino group by the s e r i a l h y d r o l y s i s of g a s t r i n . The r e f e r e n c e d i s t a n c e s of b o t h the m o d i f i e d and na-t u r a l g a s t r i n s appeared i d e n t i c a l , s u g g e s t i n g t h a t no p e p t i d e bond h y d r o l y s i s had o c c u r r e d . S p e c i f i c -enzyme h y d r o l y s i s of the N - t e r m i n a l p y r o g l u t a m y l r e s i d u e appeared t o be an a t t r a c t i v e a l t e r n a t i v e t o a l k a l i n e m o d i f i c a t i o n . The enzyme p y r r o l i d o n y l p e p t i d a s e , i s o l a t e d from a s t r a i n of Pseudomonas b a c t e r i a has been found t o be h i g h l y s p e c i f i c f o r the c l e a v a g e of the c y c l i z e d r e s i d u e ( D o o l i t t l e and Armentrout, 1 9 6 8 ) . As a r e s u l t of i t s apparent a p p l i c a t i o n t o t h i s s t u d y , the enzyme was I s o l a t e d from an a v a i l a b l e s t r a i n °f 2* f l u o r e s c e n s . The e l u t i o n curve from Sephadex G200 g e l d i f f e r e d o n l y from the p u b l i s h e d r e c o r d by a 1 2 f o l d i n c r e a s e i n absorbance a t 2 8 0 m jx a t t r i b u t e d i n p a r t to a 50% i n c r e a s e i n a p p l i e d sample. A l t h o u g h the e x t r a c t was c a r r i e d through t o a p a r t i a l l y p u r i f i e d s t a t e , a s u i t a b l e p y r o g l u t a r n y l d i p e p t i d e c o u l d not be o b t a i n e d f o r the assay of p e p t i d a s e a c t i v i t y . The removal of the b l o c k i n g r e s i d u e of g a s t r i n by enzyme h y d r o l y s i s i s y e t to be a c c o m p l i s h e d . I n the p a s t , r a b b i t s have been a common c h o i c e f o r the p r o d u c t i o n o f a n t i b o d i e s to g a s t r i n (Waddell et a l . , 1 9 o l ; Schneider et a l . , 1 9 6 7 ; McGuigan, 1967, 1 9 6 8 a , 1 9 6 8 b , 1 9 6 9 ; O d e l l et a l . , 1 9 6 7 ) but the use of b i r d s , some of which are h i g h l y r e s p o n s i v e t o p r o t e i n i m m u n i z a t i o n , has been r e s t r i c t e d ( S t r e m p l e and Meade, 1 9 6 8 ) . Chickens have bee.n shown t o be the most r e l i a b l e p r o d u c e r s of a n t i b o d y w h i l e clucks are l e s s r e s p o n -s i v e (Wolfe and Dilks, 1 9 4 8 ; Schmidt and Wolf, 1 9 5 3 ) . I t i s 62 r e l e v a n t t h a t i n these s t u d i e s a g r e a t d e a l o f v a r i a t i o n i n a n t i g e n i c r e s p o n s i v e n e s s e x i s t s i n a g i v e n s p e c i e s . I n t h i s r e -gard i t cannot be a c c u r a t e l y p r e d i c t e d how a b i r d w i l l respond to i n j e c t i o n . T h i s r e p o r t shows t h a t two d i d respond to immuni-z a t i o n w i t h the Stage I and I I I a n t r a l e x t r a c t s ( T a b l e X V I ) . One duck i n d i c a t e d a t i t r e of 1600 to bo t h d e t e c t o r a n t i g e n s a f t e r L i n j e c t i o n s of the crude e x t r a c t and was not i n c r e a s e d w i t h 3 subsequent i n j e c t i o n s of the pure hormone. T h i s o b s e r v a -t i o n i s i n c o n t r a s t t o t h a t of Schneider et a l . (1967) whose r a b b i t s r e q u i r e d r e p e a t e d i n j e c t i o n s o f crude g a s t r i n and the use of goat a n t i - g l o b u l i n t o o b t a i n comparable t i t r e s . A l t h o u g h i t was o r i g i n a l l y supposed t h a t pure g a s t r i n was non a n t i g e n i c (Maklhouf e t a l . , 1964) & s i n g l e attempt was made i n t h i s study on an a n t r e c t o m i z e d r a b b i t i n the b e l i e f t h a t the removal of the endogenous a n t i g e n would i n c r e a s e p r e c i p i t a t i n g a n t i b o d y t i t r e . A f t e r a s e r i e s of L i n j e c t i o n s , however, a n t i b o d i e s t o the pure hormone c o u l d not be d e t e c t e d by p a s s i v e hemagglu-t i n a t i o n . I t i s w e l l known t h a t hemocyanin i s a h i g h l y a n t i g e n i c p r o -t e i n and bound, to SHG 2 - 1 7 would appear as a p r o m i s i n g t e c h -nique f o r the i n d u c t i o n of a n t i b o d i e s t o the hapten. I n t h i s study a l t h o u g h an immense t i t r e was ac h i e v e d to the c a r r i e r mol-e c u l e i n each of 3 r a b b i t s , t h e r e was no evidence of e i t h e r p r e -c i p i t a t i o n or h e m a g g l u t i n a t i o n w i t h the pure a n t i g e n ( T a b l e s XVII and X V I I I ) . While comparable Immunizations by McGuigan (1967,, 1968a, 1968b, 1969) w i t h SHG 2 - 1 7 bound albumi n o r g l o b u l i n produced s u f f i c i e n t a n t i b o d y t i t r e t o the hapten, i t may be presumed t h a t the 3 mg, i n j e c t e d i n t o each r a b b i t was l a r g e l y u nconjugated. No e f f o r t was made h e r e i n t o determine the ex-t e n t o f c o n j u g a t i o n . Subsequent t o t h i s a t t empt, the use of l a t e x - b o u n d g a s t r i n was a l o g i c a l s t e p s i n c e the c o u p l i n g o f the charged p a r t i c l e t o the hapten i s a s i m p l e p r o c e d u r e , f r e e o f the c o m p l e x i t i e s of c o v a l e n t c o n j u g a t i o n . A number of assays f o r a n t i b o d i e s t o g a s t r i n i n a c h i c k e n were c a r r i e d out i n the course of i n j e c t i o n , a l m o s t a l l of which r e l i e d on the p r e c i p i t a -t i n g a n t i g e n - a n t i b o d y complex (Table XIX). The s i n g l e d i f f u s i o n Oudin t e s t proved t o be the more c o n s i s t e n t i n d e t e c t i n g p r e c i -p i t i n l i n e s . The h i g h e s t t i t r e r e c o r d e d from Oudin p r e c i p i t a -t i o n was a c h i e v e d a f t e r h i n j e c t i o n s (A mg.) and was not i n -c r e a s e d by f u r t h e r i m m u n i z a t i o n . Double d i f f u s i o n P r e e r t e s t s used s p a r i n g l y , gave s i m i l a r t i t r e s w h i l e the r i n g t e s t proved i n c o n s i s t e n t . The i n c o n s i s t e n c y i s not s u r p r i s i n g s i n c e the hapten would appear t o p o s s e s s a maximum o f two a n t i g e n i c de-te r m i n a n t s i t e s . Thus w h i l e l a t t i c e p r e c i p i t a t i o n has been used h e r e i n f o r the d e t e c t i o n of a n t i b o d i e s t o g a s t r i n by v i r t u e o f i t s s i m p l i c i t y , i t i s not w e l l s u i t e d f o r such an a n t i g e n - . a n t i b o d y system. McGuigan (1968) has observed t h a t the a v i d i t y of a n t i b o d i e s t o SHG 2 - 1 7 - a l b u m i n f o r SHG- 1 - 13 i s s l i g h t and concluded t h a t the C - t e r m i n a l t e t r a p e p t i d e amide of g a s t r i n i s p r o b a b l y the s i n g l e a n t i g e n i c d e t e r m i n a n t s i t e on the hapten. Stremple and Meade (1968) and the p r e s e n t study n e v e r t h e l e s s , have r e v e a l e d some- s u c c e s s w i t h the r i n g t e s t and g e l d i f f u s i o n t e c h n i q u e s i n d i c a t i n g a minimum o f two a n t i g e n i c s i t e s . I t would' appear t h e n , from these s t u d i e s t h a t the i o n i c b i n d i n g o f Gii-l a t e x a l l o w s the N-terminus of the hapten to f u n c t i o n as a sec-ond a n t i g e n i c s i t e s i n c e i t i s p r o b a b l y the c e n t r a l l y l o c a t e d f r e e c a r b o x y l groups which p r o v i d e the l i n k t o the charged l a -t e x . I t f o l l o w s t h a t l a t e x - b o u n d g a s t r i n produces a t l e a s t a b i v a l e n t a n t i b o d y 'while the p r o t e i n bound m o l e c u l e does not. A B l a i r - t y p e c a t b i o a s s a y supported the f i n d i n g s of p r e c i -p i t i n t e s t s t h a t a l t h o u g h i n low t i t r e , a n t i b o d y d i d e x i s t ( F i g u r e 5, Table XX). I t a l s o l e n d s f u r t h e r e vidence t h a t the . C - t e r m i n a l t e t r a p e p t i d e amide of the m o l e c u l e i s a t l e a s t a p o r t i o n of the a n t i g e n i c determinant s i t e . Furthermore, i t was e v i d e n t from the combined r e s u l t s t h a t a n t i b o d i e s were not p r e -sent i n s u f f i c i e n t l y h i g h t i t r e s to warrant t h e i r a p p l i c a t i o n t o a radioimmunoassay. SUMMARY AND CONCLUSIONS 1. A. Two n a t u r a l hog g a s t r i n s were i s o l a t e d and p u r i f i e d a f t e r the p rocedure of Gregory and Tracy (±%L). The p u r i t y o f the e x t r a c t s was d e termined by low v o l t a g e e l e c t r o p h o r e s i s , t h i n l a y e r chromatography, d e s c e n d i n g paper chromatography and amino a c i d a n a l y s i s . I t was apparent t h a t f o l l o w i n g a s i n g l e a n i o n exchange p u r i f i c a t i o n , the p r o d u c t s c o n t a i n e d no d e t e c t a b l e im-p u r i t i e s . Amino a c i d a n a l y s i s o f a g a s t r i n GII h y d r o l y s a t e i n -d i c a t e d an i d e n t i c a l c o m p o s i t i o n t o t h a t o f p u b l i s h e d f i n d i n g s . B. An a n t r a l e x t r a c t from salmon possessed some g a s t r i c a c t i v i t y but on the b a s i s of two p r e l i m i n a r y e x t r a c t i o n s , i t i s suggested t h a t the s i z e of the agent i s somewhat l a r g e r than hog g a s t r i n . 2. A. N a t u r a l hog g a s t r i n was h y d r o l y z e d by NaOH i n t o two d i s t i n c t components. I t i s suggested t h a t from s e p a r a t i o n s on e l e c t r o p h o r e s i s and t h i n l a y e r chromatography t h a t the t r a i l i n g component i n each case i s a ..modified g a s t r i n b e a r i n g an un-c y c i i z e d N - t e r m i n a l g l u t a m y l r e s i d u e . B. S e r i a l h y d r o l y s i s of n a t u r a l hog g a s t r i n by an ammonia s o l u t i o n r e v e a l e d the p r o g r e s s i v e l i b e r a t i o n o f a f r e e amine. I t i s p r o b a b l e t h a t the m o d i f i e d component i s the u n c y c l i z e d N-terminus of the h e p t a d e c a p e p t i d e . 3.- A. The i n j e c t i o n of impure and pure hog a n t r a l e x t r a c t s i n t o two ducks i n d u c e d a n t i b o d y t i t r e s of A00 and 1600 t o b o t h a n t i g e n s u s i n g the p a s s i v e h e m a g g l u t i n a t i o n t e s t . B. A n t i b o d i e s t o the pure n a t u r a l g a s t r i n were u n d e t e c t e d i n an a n t r e c t o m i z e d r a b b i t a f t e r a s e r i e s of A i n j e c t i o n s . T h i s f i n d i n g i s i n a c c o r d w i t h e a r l i e r work which i n d i c a t e d t h a t the pure hormone was non a n t i g e n i c i n humans. C. The i n d u c t i o n of a n t i b o d i e s t o a hemocyanin-SHG 2 - 17 c o n j u g a t e i n 3 r a b b i t s r e v e a l e d a h i g h t i t r e t o the c a r r i e r m o l e c u l e but no a n t i b o d y s p e c i f i c i t y d i r e c t e d toward the g a s t r i n hapten. D. The i m m u n i z a t i o n o f c h i c k e n s t o a l a t e x - g a s t r i n c o n j u -gate produced a low but d e t e c t a b l e t i t r e of a n t i b o d y t o g a s t r i n . I t was c o n c l u d e d t h a t the p r e c i p i t i n r e a c t i o n i s p o o r l y s u i t e d f o r t h e d e t e c t i o n o f the g a s t r i n a n t i b o d y system. N e v e r t h e l e s s , the r e s u l t s gave ev i d e n c e t h a t the a n t i b o d y t o g a s t r i n was not p r e s e n t i n s u f f i c i e n t t i t r e f o r i t s a p p l i c a t i o n t o a r a d i o -immunoassay. BIBLIOGRAPHY ANDERSON, J.C., BARTON, M.A., GREGORY, R.A., HARDY, P.M., KENNER, G.W., MacLEOD, J.K., PRESTON, J . , SHEPPARD, R.C., AND MORLEY, J.S. (1964) The a n t r a l hormone g a s t r i n . S y n t h e s i s o f G a s t r i n . Nature 204, 933-934• BARGER, G. AND DALE, H.H. (1910-11) - i m i n a z o l y l e t h y l a m i n e A d e p r e s s o r c o n s t i t u e n t of I n t e s t i n a l mucosa. J . P h y s i o l . A l , 499-503. BLAIR, E.L., KEENLYSIDE, E.M., NEWELL, D.J., REED, J.D., AND RICHARDSON, D.D.. (1968) Assay o f g a s t r i n by means of i t s g a s t r i c a c i d s t i m u l a t i n g a c t i v i t y . J . P h v s i o l . 198, 613-626. BLOMBACK, B. 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(1964) The a n t r a l hormone g a s t r i n . S t r u c t u r e of g a s t r i n . Nature 20A, 9 3 1 - 9 3 3 . GREGORY, R.A. AND TRACY, H.J. (1961) The p r e p a r a t i o n and p r o -p e r t i e s o f g a s t r i n . J . P h y s i o l . 136, 5 2 3 - 5 4 3 . GREGORY, R.A. AND TRACY, H.J. (1964) The c o n s t i t u t i o n and p r o p e r t i e s of two g a s t r i n s e x t r a c t e d from hog a n t r a l mucosa. Gut. 5 , 103-114* GREGORY, R.A. (1966) Memorial l e c t u r e : The i s o l a t i o n and c h e m i s t r y of g a s t r i n . G a s t r o e n t e r o l o g y 5_1> 9-53-959. GREGORY, R.A. GROSSMAN, M.I., TRACY, H.J., AND BENTLY, P.H. ( 1 9 6 7 ; Nature of the g a s t r i c secretagogue i n Z o l l i n g e r -E l l i s o n tumours. The L a n c e t 2 , 5 4 3 - § 4 4 . GREGORY, R.A.. TRACY, H.J., AGARWAL, K.L., AND GROSSMAN, M.I. (1969) Aminoacid c o n s t i t u t i o n o f two g a s t r i n s i s o l a t e d from Z o l l i n g e r - E l l i s o n tumour t i s s u e . 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(1955) P r i m a r y p e p t i c u l c e r -a t i o n s of the jejunum a s s o c i a t e d w i t h i s l e t c e l l tumors of the pan c r e a s . Ann. Surg. 14^2, 709-728. 

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