GASTROENTEROLOGY 66:357-364, 1974 Cpyright 1974 by The Williams & Wilkins C, Vl. 66. N.3 Printed in U.S.A. RADIOIMMUNOASSAY FOR GASTRIC INIBITORY POLYPEPTIDE MARYANNE KUZIO, JILL R. DRYBURG, KATLEEN M. MALLOY, AND JON C. BROWN, P.D. Departments f Physilgy and Surgery, University f British Clumbia, Vancuver. British Clumbia. Canada A specific radiimmunassay fr gastric inhibitry plypeptide has been develped using guinea pig antisera raised t prcine gastric inhibitry plypeptide. ighly purified 125I-prcine gastric inhibitry plypeptide, further purified n Sephadex G-15 after labeling, was emplyed as a tracer. The sensitivity range is 25 t 25 pg. N crss reactivity culd be demnstrated with gastrin, glucagn, secretin, chlecystkinin-pancrezymin, mtilin, and vasactive intestinal peptide. Reprducibility f determinatins in different assays was demnstrated. The mean fasting serum cncentratin was 237 ± 14 pg per ml in 48 nrmal subjects. Serum cncentratins increased t abve 1,2 pg after a meal. Gastric inhibitry plypeptide (GIP) btained frm prcine dudenal mucsa extracts has been islated and purified, and the amin acid cmpsitin and sequence were determined.1-5 Physilgical studies have revealed that the plypeptide will inhibit acid secretin in the dg, stimulated by pentagastrin, insulin hypglycemia, and histamine. The ability f the plypeptide t inhibit histamine- and in- Received March 19, 1973. Accepted Octber 9, 1973. Address requests fr reprints t: Dr. Jhn C. Brwn, Department f Physilgy, University f Brit ish Clumbia, Vancuver 8, B. C., Canada. This wrk was supprted by grant MT 1972 frm the Medical Research Cuncil f Canada, and by a grant frm the Banting Research Fundatin t J. C. Brwn. The authrs wish t acknwledge the cntinuing supprt f Prfessr V. Mutt and Emeritus Prfessr E. Jrpes f the Karlinska Institutet, Stckhlm, Sweden, wh dnated pure natural secretin and chlecystkinin pancrezymin. rmnes were als supplied by Dr. E. Wunsch, Max Planck-Institut fur Eiweiss und Lederfrschung, Munich (synthetic glucagn, and synthetic human gastrin, 15 leucine substituted gastrin.) Drs. S. Blm and J. Plak supplied a small sample f vasactive intestinal peptide btained frm Dr. S. Said. 357 sulin hypglycemia-stimulated acid secretin shwed it t pssess gastric inhibitry prperties different frm secretin and chlecystkinin-pancrezymin (CCK PZ).6 Prerequisites fr the establishment f GIP as a hrmne have t include the demnstratin f the plypeptide in bld and tissues and a release frm tissues by physilgical mechanisms. In general, the gastrintestinal plypeptides have a multiplicity f actins, and quite ften different plypeptides will prduce similar respnses. Classical physilgical experiments using secretaggues in the dudenum, and measuring a physilgical respnse, have prved t be unsuitable t demnstrate the mechanisms prducing the release f GIP frm tissues int the bld. This study describes the prductin by immunizatin in guinea pigs f antibdies t prcine GIP and the develpment f a radiimmunassay f sensitivity suitable fr the detectin f GIP in sera f man. These antisera prduced by immunizatin f guinea pigs with relatively small amunts f prcine GIP have n measurable crss reactivity with natural secretin, synthetic glucagn, synthetic human gas-
358 KUZIO ETAL. Vl. 66, N.3 trin (15-Leu gastrin), pure prcine CCK-PZ, pure prcine mtilin, and pure prcine vasactive intestinal peptide. Materials and Methds Prductin f antisera. Tw preparatins f GIP have been used fr immunizatin. They were the EG stage lip and EG stage III which was further purified by rechrmatgraphy n Sephadex G 25 fine with.2 M acetic acid as eluting buffer. The preparatins were injected subcutaneusly in at least tw sites n the lwer abdmen;.4.umle f plypeptide was used per immunizatin. Initial immunizatins were perfrmed using an emulsin frmed with Freund's adjuvant r an emulsin frmed with Freund's adjuvant after cnjugatin f the ply peptide t bvine serum albumin. Cnjugates were prepared using the carbdiimide methd. 7 The degree f cnjugatin was assessed using high vltage electrphresis at p 6.5, after which the presence f free GIP culd nt be bserved. Bld was btained by cardiac puncture 8 t 12 days after immunizatin. Three f 6 guinea pigs had antibdies detectable at an tiserum dilutins f abut 1: 3 after eight t 11 immunizing dses f uncnjugated material. These animals were given ne immunizatin with cnjugated GIP, after which all demnstrated titers in excess f 1: 5,. After a secnd immunizatin with cnjugated material, 2 f these guinea pigs had titers f 1: 2,. Preparatin and purificatin f 1251-labeled GIP. Purified GIP (stage IV) has been established as a standard. Int several hundred ampules, 6.ug f purified GIP have been aliquted and these have been used in bth the labeling prcedure and fr standard curves. GIP was labeled with 125 1 by a minr mdificatin f the chlramine T technique. 8 One hundred micrliters f.4 M phsphate buffer, p 7.5, were added t an ampule cntaining 6.ug f GIP. Then rapidly, and in turn, were added 2 mc f 125 1 (2.uliters), 4.ug f chlramine T (1.uliters), and, after a 15-sec delay, 252.ug f sdium metabisulfite (2.uliters). The steps were perfrmed with air bubbling t ensure rapid and prper mixing. The 15-.uliter reactin vlume was added t a clumn f Sephadex G-15 (.6 by 35 cm). The clumn was eluted with buffer cntaining 2% plasma and 2% Trasyll (FBA Pharmaceuticals, Pinte Claire, Canada) in.2 M acetic acid. Apprximately 4 samples cntaining 3.uliters per tube were cllected (fig. 1). Sephadex G-15 was fund t be superir t G-25 r G-5. It can be seen frm figure 1 that the immunreactivity des nt share cmplete identity with the peak f radiactivity. It has been shwn, by plyacrylamide gel electrphresis, that GIP fragments in the presence f the xidizing agent chlramine T, and these 125 1 labeled fragments, because f smaller size, elute after the labeled GIP n Sephadex G-15 (unpublished bservatins). The fractins shwing the greatest immunre- 4 I ' 1.6 '" 3 " "'" =.r:;.: 'E ci a. 2 <I> 1 <: "... 1 2 Clumn Fractins - 1 pi Aliquts 3 4 1.2.8 a. '".r:; '" a..4 " FIG. 1. Separatin f 125I-prcine gastric inhibitry plypeptide (alp) frm free 125 1 n Sephadex G-15 (35 by.6 cm) with.2 M acetic acid. GIP was eluted in the first peak. Damage assays (e) and binding ability () f the aliquts are als shwn. B/F, bund-free.. mill.
March 1974 RADIOIMMUNOASSA Y FOR GIP 359.. Cl. '-" c: Cl..2.. 'if.6 \ \.4 \. Q. GIP Standard Lbel Tube # 6 /I /I #9 /I /I.---- mju. O.O+---------------r-------_r------------,, 2 3 4 5 GIP Cncentratin (pg./5 jjl. ) r, -------"r-------" 2 '25 I - GIP (C.PM. x1 3 ) FIG. 2. Standard curve t gastric inhibitry plypeptide (alp) (e---e) and cmparisn f fractins ns. 6 (--),9 (A--A), and 13 (Ll--Ll) frm labeling prcedure. Variatins f cunts per minute, frm 2 x 1' t 1.25 1', were added frm each fractin. The tracer cncentratin, 1 x 1' cunts per min. ffractin # 6 was fitted t the standard curve and ther bund-free '''I-prcine GIP fitted accrdingly. There is gd crrelatin between the standard prcine GIP and labeled GIP. activity and least damage were used in the assay. The prer immunreactivity f the fractins preceding the first peak culd be due t increased substitutins f 125 1 in the GIP mlecule. The percentage f radiactivity crrespnding t 1 25 I_GIP was calculated at apprximately 8%. The specific activity f 125I_GIP using this figure and neglecting damage was calculated t be 27 mc per mg. In figure 2, a curve, prduced when bund-free ratis frm dilutins flabeled and unlabeled prcine GIP were cmpared, is shwn. The 125I_GIP was almst as cmpletely identified by the antiserum as was unlabeled GIP. At high cncentratins f 1251_GIP, there was an apparent slight increase in bund-free ratis ver that fund frm unlabeled GIP. The fractins 6, 9, and 13 are equivalent t fractins 11, 14, and 18 f the chrmatgram shwn in figure 1. Cnditins f incubatin. The diluent buffer was made up as fllws:.4 M phsphate buffer (p 6.5), utdated human plasma (1%) extracted 18 hr with 1 % charcal at 4 C and shwing n detectable GIP n radiimmunassay and Trasyll (5%). Standard curves have been set up in which the cncentratin f Trasyll in the diluent buffer was, 2.5, 5., and 1%. Best results were btained with 5.% Trasyll and this cncentratin has been used in the diluent buffer thrughut the assay. The buffer was used in all dilutins and in crrecting the final incubatin vlume t 5 tlliters. The final vlume f the incubatin mixture fr the standard curves cnsisted f 1 tlliters f antiserum at an initial dilutin 1: 4, final dilutin 1: 2,, 1 tlliters f 1251_GIP cntaining 6 cunts per min, 1 tlliters f standard GIP. range 5 t 25 pg per 1 tlliters, and 2 tlliters f diluent buffer. Where test sera were being assayed, in place f 1 tlliters f GIP standard, 1 tlliters f serum were used, and diluent buffer was added t adjust the final vlume t 5 tlliters. An Oxfrd Pipettr was used thrughut. All assays were set up in triplicate, in silicnized glass culture tubes 1 by 75 mm, and then gently mixed n a Vrtex mixer. Incubatins were perfrmed at 4 C fr 48 hr r 72 hr befre separatin f the bund and free cmpnents. The 72-hr assay had a 24-hr preincubatin and a 48-hr pstincubatin after additin f the 125I_GIP. A 48-hr equilibrium assay at 4 C has been fund t be cmparable t the 72-hr assay. wever, incubatins f less than 48 hr were unsatisfactry, and "damage" was cnsidered excessive after incubatins which lasted mre than 72 hr. Separatin f bund and free GIP. Three
36 KUZIO ETAL. Vl. 66, N.3 separatin techniques have been tried using dixane, duble antibdy, and dextran-cated charcal. The dextran-cated charcal was superir t charcal alne, prducing the best separatin, and has been utilized in the assay. Dextran-cated charcal was prepared 24 hr befre use. The buffer (.4 M phsphate, p 6.5 with 2% human plasma and 1% Trasyll) was cled t 4 C prir t the additin f the dextran (T 7, Pharmacia Ltd, Uppsala, Sweden). Charcal (carbn declrizing neutral C-17, Fisher Scientific Ltd, Fair Lawn, N. J.) was added t the dextran slutin. The amunts added yielded a final cncentratin f 2.5 mg f charcal and.5 mg f dextran fr each 2 liters f buffer. The mixture was stirred vernight at 4 C. When separatin f the free frm the bund cmpnents was required, 2 liters f the charcal dextran suspensin was added t each tube and left fr 3 min. The tubes were then centrifuged at 2,8 rpm fr 15 min and the r NO GIP added.8 supernatant decanted. The tubes were crked and placed in plastic carrier tubes fr cunting. Bth the decanted prtin cntaining the bund plypeptide and the charcal dextran, t which was adsrbed the free plypeptide, were cunted in an autmatic l' cunter. Sensitivity and precisin f assay. The cncentratins f prcine GIP in the standard slutins ranged frm 5 t 25 r 5 pg. Antiserum frm guinea pig GP 76 was used at a 1: 2, final dilutin and was the antiserum used thrughut this study. The fall in bundfree rati prduced by lo pg was always much greater than when GIP was excluded. wever, fr assay purpses, the slpe between 25 and 25 pg was used (fig. 3). Nnspecific binding was measured in each assay and used in the calculatin f the amunt f immunlgically active GIP in the unknwn samples. Specificity f antisera. Cmparative immunreactivities f mtilin, natural prcine a...6 Q).4 c () I- a.. I x x \ '" (DIlL.2. I 5 1, I 25 1 25 GIP (pg) FIG. 3. Standard curve fr immunassay using antiserum GP 76 at a final dilutin f 1: 2,. The ttal vlume f the incubatin mixture was 5 ILliters.
March 1974 RADIOIMMUNOASSA Y FOR GIP 361.6 a.. "'".5.4 ;; ml"-.3 Glwcaqn... Secret... *-- CCK Mtilln.2.1. I r-,...--,----r--,-,----" 5 1 1 1 IO(x)() Weight f Peptides (pg) FIG. 4. Cmparative immunreactivities f prcine gastric inhibitry plypeptide (GIP), synthetic gastrin (15-Leu), synthetic glucagn, pure natural prcine secretin, and chlecystkinin-pancrezymin (CCK), and natural prcine mtilin. The ttal vlume f the incubatin mixture was 5 ILliters. secretin, synthetic glucagn, synthetic human gastrin (15-Leu), CCK-PZ, and 1-14 GIP in crss reactins versus 125I_GIP are shwn in figure 4. N significant crss reactivity culd be detected, when up t 1 ng f the peptides, ther than GIP, were added. Reprducibility. Reprducibility f the assay was checked by assaying serum samples frm tw experiments n different dates. The serum samples were kept frzen at 2 C between the assays. The results are shwn in figure 5. Only tw f the 14 duplicate bservatins differed by mre than 5%. The remainder were within 25%. Dilutin f serum with high GIP. A serum sample frm a patient (Gr), with a calculated serum GIP level f 4.5 ng per ml, was incubated at varius dilutins (with diluent buffer and charcal-extracted serum) and cmpared with a standard curve. The dilutin in diluent buffer at 5 Illiters per 5 Illiters was fitted t the standard curve and the ther dilutins pltted accrdingly. The results are shwn in figure 6. Results CCK-PZ preparatin (batch n. 26931) frm the Gastrintestinal rmne Research Labratry (Stckhlm, Sweden) has been assayed n 1 ccasins. The preparatin was fund t cntain 17 ± 5.9 pg f GIP per 1, pg. Each ampule (75 Ivy dg units with 3 JLg f preparatin) cntained apprximately 32 JLg f GIP. A recent batch f CCK-PZ (batch n. 27271)
362 KUZIO ETAL. Vl. 66, N.3 16 g 12. '" '"' <l '" :; 8 c 'e 1:: g 4.. '" Dates f Assay 1/11,2/5 1/22,2/5 4 8 12 16 GI P Cncentratin in Assay 2 (pg) FIG. 5. Reprducibility f determinatins n serum samples. The samples were assayed n different dates and stred at -2 C between assays. GIP, gastric inhibitry plypeptide. N '".<:; '" u Q..3.2.1 «> \ \ \ \ x \, e, r-, ----.16'--2'b---3T"b--4-b'-----,5b pg/5jji. GIP Standard 25 5 75 1 Gr Serum jji/5 jjl FIG. 6. Serum sample (Grl cntaining a high cncentratin f endgenus gastric inhibitry plypeptide (GIP), incubated at varius dilutins with diluent buffer (,,) r charcal-extracted serum (), and fitted t the standard curve (el at the dilutin in diluent buffer cntaining 5 ILliters per 5 ILliters. ElF, bund-free. has been shwn t cntain a much reduced GIP cntent, 25 pg per 1, pg f preparatin. Early mrning serum levels btained frm 48 subjects, after a 12-hr fast, have been determined at 237 ± 14 pg per ml (± SE), the range being 75 t 5 pg per ml. The subjects were within 1% f ideal bdy weight. Seven subjects were fasted vernight. Fasting serum samples were btained at 8: 45 AM. The subjects were then fed a standard breakfast between 9: 15 and 9: 55 AM. The breakfast cnsisted f 4 unces f range juice, 8 unces f whle milk, bacn and tw eggs with hash-brwn ptates, tast with cnserves, and cffee r tea. Serum samples were btained at 3-min intervals fr the fllwing 3 1/2 hr beginning 45 min after the start f the meal. The results are shwn in figure 7. Pure GIP (ne f the standards) was added t charcal-treated serum t yield a final cncentratin f 5 pg per ml. Ten determinatins n bth 1- and 2-JLliter samples yielded values f 54 ± 4 pg per 1 JLliters (mean ± SE) and 1 ± 1 pg per 2 JLliters (mean ± SE). Discussin Antisera t prcine GIP have been raised in guinea pigs by multiple injectins with bth uncnjugated and cnjugated plypeptide. The antisera have been used in the develpment f a sensitive radiim-
March 1974 RADIOIMMUNOASSA Y FOR GIP 363 munassay allwing the detectin f GIP in human sera bth in fasting and stimulated situatins. Prcine GIP has been labeled with 125 1 and a single clumn system using Sephadex G-15 has been used in islatin f labeled plypeptide frm the ther reactants and free 125I. This system wuld nt be expected t separate 125I_GIP frm GIP. wever, the dilutin curves prduced with labeled and unlabeled plypeptide are cmparable. Separatin f labeled and unlabeled GIP using carbxymethylcellulse has nt prduced better material. Dilutin f a patient's serum with either diluent buffer r charcal-extracted serum, and cmparisn with a standard curve, shws parallelism. It wuld appear then that the antiserum used in this study can identify labeled GIP as it des the unlabeled material, and that there is gd crss reactivity between human and prcine GIP. The mean serum level in 48 nrmal subjects after a 12-hr fast was 237 ± 14 pg per ml (mean ± SE). After feeding, the serum levels in 6 nrmal subjects demnstrated an increase abve 1,2 pg per ml within 45 min after the start f the meal. The levels remained elevated abve 1, pg per ml fr apprximately 2 hr, and significantly abve nrmal sme 4 hr later. During the develpment f the assay, the fasting GIP levels have been seen t fall as imprvements in labeling and separatin f bund and free fractins were intrduced. It had earlier been reprted 6 that the partially purified CCK-PZ preparatin (Gastrintestinal rmne Research Labratry) cntained between 1 and 2 p,g f GIP per ampule. This figure was arrived at frm physilgical studies used in fllwing the purificatin f GIP. Emplying the radiimmunassay, it is calculated that each ampule f CCK-PZ (batch n. 26931) cntains 32 p,g f GIP. A recent batch f CCK-PZ (batch n. 27271) has been fund t cntain substantially less GIP-8.3 p,g per ampule. The recent batches f CCK-PZ are prduced by a technique incrprating anther purificatin stage in which GIP appears t be remved. The ability f the antisera t crss-react with ther knwn gastrintestinal plypeptides and glucagn has been tested. N crss reactivity culd be detected when up t 1, pg f gastrin, glucagn, secretin, CCK-PZ, and mtilin were added. The same antisera used in the radiimmunassay fr GIP have recently been emplyed in lcalizatin f GIP cells in the dudenum and jejunum f dg and man, by the use fthe immunflurescence technique. 9 The cell has been tentatively identi- 16 12 I I!!! I - a. - 8 Q t') E., Cf) 4 i t Fasting -Meal 1 45 1 15 165 I f 225 Time in Minutes after cmmencing Meal FIG. 7. Serum gastric inhibitry plypeptide (GIP) levels frm 6 nrmal subjects after the ingestin f a standard meal (means ± SE). 285
364 KUZIO ETAL. Vl. 66, N. 3 fied by electrn micrscpy as the small granule cntaining Dl cell. 1 The cell is situated in the dudenum and jejunum and is distinct frm bth the S cell (secretin) and the L cell (enterglucagn). Prerequisites fr the establishment f a substance as a hrmne include the identificatin f the cell f rigin and a physilgical mechanism fr its release frm such a cell. It wuld appear that the cell f rigin fr GIP has been tentatively identified, and that changes in circulating GIP levels can be bserved by radiimmunassay, after ingestin f a mixed meal. GIP can be cnsidered a serius candidate fr hrmnal status. Current studies include attempts t identify the specific secretaggue fr the release f GIP frm the dudenum and jejunum by the use f radiimmunassay, investigatin int disease states in which GIP may playa rle, and attempts t crrelate bld changes in GIP with cncurrent physilgical changes. The latter is essential befre hrmnal status can be assigned t GIP. REFERENCES 1. Brwn JC Pedersn RA: A multiparameter study n the actin f preparatins cntaining chlecystkinin-pancrezymin. Scand J Gastrenterl 5:537-541, 197 2. Brwn JC, Pedersn RA, Jrpes JE, et al: Preparatin f highly active entergastrne. Can J Physil Pharmacl 47:113-114, 1969 3. Brwn JC, Mutt V, Pedersn RA: Further purificatin f a plypeptide demnstrating entergastrne activity. J Physil 29:57-64, 197 4. Brwn JC: A gastric inhibitry plypeptide. I. The amin acid cmpsitin and the tryptic peptides. Can J Bichem 49:255-261, 1971 5. Brwn JC, Dryburgh JR: A gastric inhibitry plypeptide II. The cmplete amin acid sequence. Can J Bichem 49:867-872, 1971 6. Pedersn RA, Brwn JC: Inhibitin f histamine-, pentagastrin-, and insulin-stimulated canine gastric secretin by pure "gastric inhibitry plypeptide". Gastrenterlgy 62:393-4, 1972 7. Parker CW: Nature f immunlgical respnses and antigen-antibdy interactin, Principles f cmpetitive prtein-binding assays. Edited by WD Odell, W Daughaday. Philadelphia, JB Lippinctt, 1971, p 25-48 8. unter WM, Greenwd FC: Preparatin f 1311_ labelled human grwth hrmne f high specific activity. Bichem J 89:114-123,1963 9. Plak JM, Blm SR, Kuzi M, et al: Cellular lcalizatin f gastric inhibitry plypeptide in the dudenum and jejunum. Gut 14:284-288, 1973 1. Vassall G, Capella C, Slcia E: Endcrine cells f the human gastric mucsa. Z Zellfrsch 118:49-67, 1971