The antigenicity of pig insulin

Diabetologia, Feb 1970

Torsten Deckert, Else Grundahl

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The antigenicity of pig insulin

The Antigenicity of Pig Insulin* TORSTt~N DECKERT a n d ELSE GRUNDAHL 0 1 0 Die Antigen- 1 Medical Department , Blegdamshospital, Copenhagen , Niels Steensen's Hospital, Gentofte and State Hospital , Nykobing Sjaelland Summary. Treatment of h u m a n subjects with a neutral solution of pure pig insulin crystals does not lead to the formation of insulin antibodies. Thirty-six non-diabetics with psychiatric diseases were treated with a neutral solution of crystalline pig insulin for a m a x i m u m period of 104 days, using a 24-h dosage of up to 208 i.u. I n patients who had previously not received insulin t r e a t m e n t (24 patients), insulin antibodies could not be demonstrated after the termination of the insulin treatment. However, in patients who had previously received t r e a t m e n t with acid solutions of insulin consisting of pig and ox insulin (12 patients), it was possible in almost all cases to demonstrate insulin antibodies after the termination of the insulin treatment. I n addition, 10 pigs received t r e a t m e n t with solutions of pure pig insulin. Insulin antibodies could be demonstrated in only 2 pigs, both treated with acid solutions of recrystallized pig insulin, whereas antibodies could not be demonstrated in pigs treated with neutral solutions of recrystallized pig insulin. The reason why pure preparations of pig insulin are in most cases also antigenic to man, is presumably t h a t the pig insulin preparations are injected as suspensions (zinc insulins, N P H insulin) or as is the case with acid solutions of insulin, converted to suspensions after injection, since insulin precipitates when the acid insulin solution is neutralized by tissue fluid. - Rdsumd. Le t r a i t e m e n t de sujets humains avec tme solution neutre de cristaux d'insuline pure de pore ne provoque pus la formation d'anticorps insuliniques. Trente-six sujets non-diab6tiques, atteints de maladies psychiatriques, ont dt6 traitds avec une solution neutre d'insuline cristallisde de pore, p e n d a n t 104 jours au maxim m n , en utilisant une dose quotidierme allant jusqu'~ 208 U. Chez les patients qui n ' a v a i e n t pus dt6 traitds a u p a r a v a n t par l'insuline (24 patients), on ne pouvait pas ddmontrer la prdsenee d'anticorps insuliniques apr~s la fin du traitement ~ l'insuline. Par contre, chez les patients (12) qui avaient dt6 traitds a u p a r a v a n t par des solutions aeides d'insuline (insuline de pore et insuline de boeuf), il dtait possible duns presque t o u s l e s eas de ddmontrer la prdsence d'anticorps insuliniques apr6s la fin du traitem e n t par l'insuline. - - E n outre, 10 pores ont re~u u n raitement avee des solutions d'insuline pure de pore. On a p u trouver des anticorps insuliniques seulement chez 2 pores, traitds avec des solutions aeides d'insuline de pore recristallisde, tandis qu'on n ' a pus pu trouver d'anticorps ehez les pores traitds avec des solutions neutres d'insuline de pore recristallisge. La raison pour laquelle les prdparations pures d'insuline de pore sont duns la plupart des cas dgalement antigdniques chez l'homme, est probablement que les prdparations d'insuline de pore sont injeetdes en suspensions (insuline-zinc,NPH-insuline), ou que, eomme e'est le cas pour les solutions acides d'insuline, elles se transforment en suspensions apr~s l'injeetion, puisque l'insuline pr4cipite quand la solution acide d'insuline est neutralistic par les liquides tissulaires. Zusammenfassung. Behandlung von 3~Iensehen mit neutralen LSsungen yon reinen Schweineinsulinkristallen fiihrte nicht zur Bildung yon Insulinantik6rpern. 36 Patienten mit psychiatrisehen K r a n k h e i t e n ohne Diabetes wurden maximal 104 Tage lung t/iglich mit einer neutralen LSsung yon reinen Schweineinsulinkristallen gespritzt. Die hbchste Tagesdosis war 208 E. Bei Patienten, die friiher niemals eine Insulinkur durchgemacht h a t t e n (24 Patienten), k o n n t e n naeh der K u r mit neutralem Sehweineinsulin keine Insulinantik6rper naehgewiesen werden. Bei Patienten, die friiher einmal I n s u l i n k u r e n mit Sehweine-Rinderinsulinkristallen,gel6st in verdiinnter HC1, durchgemacht h a t t e n (12 Patienten), k o n n t e n jedoch in beinahe allen F~llen naeh der K u r mit neutralem Sehweineinsulin, Insulinantik6rper nachgewiesen werden. - - Von 10 Sehweinen, die mit Sehweineinsulin gespritzt wurden, k o n n t e n Insulinantik6rper nur bei Schweinen nachgewiesen werden, die mit Sehweineinsulin in saurer L6sung gespritzt waren. Es wird angenommen, dal3 die Ursache zur Antigenizit/it vieler Schweineinsulinpr/iparate Mensehen gegeniiber daran liegt, dab sic als Suspensionen gespritzt werden (Zink-Insuline, N P H - I n s u l i n ) oder -- wie es mit den sauren Insulin-LSsungen der Fall ist -- naeh der Injektion zu Suspensionen umgewandelt werden, indem Insulin pr/~eipitiert, wenn die sauren L6sungen yon der Gewebefliissigkeit neutralisiert werden. Key-words: Insulin antibodies, pig insulin, isoimmunization, insulin dose. H u m o r a l i n s u l i n a n t i b o d i e s can be d e m o n s t r a t e d i n a l m o s t all subjects who h a v e b e e n t r e a t e d w i t h insu. O ' D o n o v a n , 1966). This is p r e s u m a b l y because ox i n s u l i n differs more i n s t r u c t u r e from h u m a n i n s u l i n lin (Deckert, 1967). I n m a n , however, the a n t i g e n i c i t y t h a n pig i n s u l i n does. of p r e p a r a t i o n s of pig i n s u l i n seems to be less pro. n o u n c e d t h a n the a n t i g e n i c i t y of p r e p a r a t i o n s of pure ox i n s u l i n or m i x t u r e s of pig a n d ox i n s u l i n (Devlin a n d The sole s t r u c t u r a l difference b e t w e e n pig i n s u l i n a n d h u m a n i n s u l i n is the C - t e r m i n a l a m i n o acid i n the B - c h a i n , which is a l a n i n e i n pig i n s u l i n a n d t h r e o n i n e i n h u m a n i n s u l i n (Table 1). I t has, nevertheless , b e e n f o u n d t h a t a n t i b o d y f o r m a t i o n i n diabetic subjects c a n n o t be avoided, e v e n t h o u g h these p a t i e n t s are p a r e d e x c l u s i v e l y from pig insulin (Berson a n d Yalow, 1963; D e c k e r t , 1964; L o c k w o o d a n d P r o u t , 1965) . T h e r e a s o n for t h e a n t i g e n i c i t y of pig insulin in m a n has n o t been clarified. I t can h a r d l y be due to t h e s t r u c t u r a l difference existing b e t w e e n pig insulin a n d h u m a n insulin, since i t has n o t been possible to d e m o n s t r a f e a n y i m m u n o l o g i c a l difference b e t w e e n p i g insulin a n d h u m a n insulin ( D e c k e r t a n d J c r g e n s e n , 1966) , n o r b e t w e e n pig insulin a n d d e s a l a n i n e - p i g insulin (Berson a n d Y&low, 1963), a p r e p a r a t i o n of p i g insulin from which t h e C - t e r m i n a l a m i n o acid, alanine, h a s b e e n split off. F u r t h e r , t h e s u p p o s i t i o n t h a t t h e s t r u c t u r e of circulating endogenous h u m a n insulin is different from t h a t of c r y s t a l l i z e d pig insulin or h u m a n insulin a f t e r p r e p a r a t o r y purification, m u s t be r e g a r d e d as unlikely, since i t can be shown t h a t p l a s m a insulin from n o r m a l s u b j e c t s as well as o v e r w e i g h t s u b j e c t s a n d d i a b e t i c s does n o t differ i m m u n o l o g i c a l l y from c r y s t a l l i n e p i g insulin or h u m a n insulin (Deekert a n d J r 1966) . The following i n v e s t i g a t i o n s were m a d e in a n attempt to determine the reason for the antigenicity of l o n g - a c t i n g pig i n s u l i n p r e p a r a t i o n s . Material and Methods T w e n t y - f o u r p a t i e n t s w i t h p s y c h i a t r i c diseases who were n o n - d i a b e t i c s a n d who h a d n o t p r e v i o u s l y received t r e a t m e n t w i t h insulin, were g i v e n a n i n t r a m u s c u l a r i n j e c t i o n of a n e u t r a l solution of r e c r y s t a l l i z e d p i g insulin n e a r l y e v e r y d a y for p e r i o d s u p to 104 d a y s . The size of t h e insulin dose v a r i e d from p a t i e n t t o p a t i e n t a n d from d a y t o d a y , d e p e n d i n g on t h e a i m a n d effect of t h e t r e a t m e n t . T a b l e 2 shows t h e d u r a t i o n of t h e t r e a t m e n t a n d t h e m a x i m u m dose of insulin p e r 24 h. A similar t r e a t m e n t was given t o 12 p a t i e n t s w i t h p s y c h i a t r i c diseases who were n o n - d i a b e t i c , b u t who h a d p r e v i o u s l y , some m o n t h s t o several y e a r s before, been t r e a t e d w i t h acid solutions of r e c r y s t a l l i z e d p i g - o x insulin for a s h o r t e r p e r i o d (Table 3). B l o o d samples were t a k e n from t h e c u b i t a l vein on t h e d a y prior to t h e c o m m e n c e m e n t of t h e insulin t r e a t m e n t , a n d 2 d a y s a f t e r t h e t e r m i n a t i o n of t h e insulin t r e a t m e n t . S e r u m for insulin a n t i b o d y i n v e s t i g a t i o n was s t o r e d a t - - 20 ~C. Name A . B . H . B.1VLC. H.M. I.S. J.S. G.N. L . P . J . J . H . G . L . J . E . P . A.M. L . R . K . B . J . B . P . O.J. I . B . M . A . H . B . I . N . E . R . J . L.N. E . T . B . H . J . O . J . B . Name V . J . K . I . N . E.1VI.J. T.L. E . L . H . E . N . V.A. K. 1-I.P. N . E . M . E . T . A . H . B.N. F o r i s o i m m u n i z a t i o n tests, 12 pigs were used weighing b e t w e e n 22 a n d 28 kg, 11 of t h e m f r o m one l i t t e r ( B r u n f e l d t a n d D e c k e r t , 1964a) . Six pigs were t r e a t e d w i t h r e c r y s t a l l i z e d p i g insulin dissolved in p h o s p h a t e buffer ( p t I 7.3), free from antiseptics. F o u r pigs were t r e a t e d w i t h r e c r y s t a l l i z e d pig insulin dissolved in d i l u t e d h y d r o c h l o r i c acid ( p H 3.2) t o which g l y c e r o l a n d a n a n t i s e p t i c h a d been a d d e d . Two pigs were u s e d as controls (Table 4). The biological s t r e n g t h of t h e insulin p r e p a r a t i o n s was 2 4 - - 2 6 i . u . / m g . I n s u l i n w i t h o u t t h e a d d i t i o n of a d j u v a n t s was i n j e c t e d subc u t a n e o u s l y daffy for 8 7 - - 9 0 days. Nine of t h e a n i m a l s received a total of 2250 i.u. insulin each, beginning with 15 i.u. daily, later with increasing doses. One animal (Brunfeldt and Deekert, 1964 a) received a total of 1050 i. u. insulin. The d a y prior to a n d two d a y s after the t e r m i n a t i o n of the insulin t r e a t m e n t , blood samples were taken and serum for antibody investigation was stored at -- 20 ~ C. The human sera were examined for insulin antibodies by a modification of the double antibody technique described b y S k o m a n d Talmage (1958). 100 ~1 of serum to which 2.5 ~1 heparin L E O was a d d e d (5000 units/ml) and 100 ~1 of blind (0.04 M p h o s p h a t e buffer, pI-I 7.4 with 5o/o h u m a n albumin) were b o t h i n c u b a t e d for 4 days at 4~ with 100 ~1 of an 0.04 M p h o s p h a t e buffer, p H 7.4, containing 0.5% h u m a n alb u m i n a n d 0.9% NaC1 t o g e t h e r with n o t quite 0.2 ixg l~SI-pig insulin/ml. The specific r a d i o a c t i v i t y of the iodinated insulin was a b o u t 60 mCi/mg. After s t a n d i n g for 4 days, 50 F1 of the i n c u b a t i o n m i x t u r e was diluted with 5000 ~zl 0.04 M p h o s p h a t e buffer, p i t 7.4, containing 0.25% h u m a n albumin. Two h u n d r e d F1 of this dilution was i n c u b a t e d for 24 h with 100 ~l of a dilution of serum f r o m rabbits i m m u n i z e d with h u m a n g a m m a globulin. The dilution of the r a b b i t a n t i h u m a n g a m m a globulin was m a d e with 0.04 M p h o s p h a t e buffer, pI-I 7.4, a n d was so adjusted t h a t 100 ~1 of the dilution could completely precipitate the a m o u n t of g a m m a globulin present in the reaction mixture. After the incubation, the precipitate formed was filtered off on oxoid filters. The precipitate was rinsed twice with 0.04 M p h o s p h a t e buffer containing 0.5% h u m a n albumin, a n d the r a d i o a c t i v i t y t h e n m e a s u r e d in a Wellcounter (Philips). The "insulin-antibody c o n c e n t r a t i o n " was expressed as the a m o u n t of radioactive insulin b o u n d to the immunoglobulins, as a percentage of the t o t a l radioactivity, i.e. the r a d i o a c t i v i t y of the sample on the filter minus the r a d i o a c t i v i t y of the blind sample on the filter, as a percentage of the total activity. The total activity corresponded to the overall radioactivity in the second incubation mixture (corresponding to approximately 2 mFg 125I-insulin). All determinations were performed as double tests. SEM (double determinations) = 0.19%. The 1~'sI-piginsulin was supplied by Nordisk Insulinlaboratorinm. The rabbit anti-human gamma-globulin was supplied by Dansk Svovlsyre and Supcrphosphat Fabrik A/S, and the freeze-dried human albumin was supplied by Statens Seruminstitut. Using the same method, the insulin antibodies were examined for their ability to discriminate between pig insulin and ox insulin, after varying amounts of recrystallized pig insulin and recrystallized ox insulin, respectively, had previously been added to the serum. The pig and ox insulin crystalline powders were supplied by Nordisk Insulinlaboratorinm. After the addition of 13~I-pig insulin, the sera from the pigs were examined by means of immunoelectrophoresis and autoradiography as previously described (Brunfeldt and Deckert, 1964a) . The specific activity of the isotope-labelled insulin was 140--180 mCi/mg. It was used in a concentration of approximately 0.04 Fg lalI-insulin/ml serum. Results Patients who h a d n o t previously been t r e a t e d with insulin h a d an "insulin-antibody c o n c e n t r a t i o n " of < 1.2%, n a m e l y 0.46 ~: 0.34. E l e v a t e d insulin-antibody concentration could n o t be d e m o n s t r a t e d in a n y of the patients or the animals, prior to the c o m m e n c e m e n t of the insulin t r e a t m e n t . 1816 o-----_oy~__.~__~x o o ox insulin x pig insulin -5o c o ~ o x x 5 0 0.01 0.05 0.1 0.5 1 .u,g insulin/m[ serum I n o n l y one (O.J.) of the 24 patients who had n o t previously been t r e a t e d with insulin, could insulin antibodies be d e m o n s t r a t e d after the t e r m i n a t i o n of the insulin t r e a t m e n t (Table 2). However, the eoncenafter the termination of the insulin treatment. However, 2 of the 4 pigs t h a t had been treated with recrystallized pig insulin in acid solution showed insulin antibodies after the termination of the insulin t r e a t m e n t (Fig. 2, Table 4). tration of insulin antibody was very slight, being only significantly higher t h a n in non-insulin-treated patients at the 5 ~ level, but not at the 1% level. I n contrast to the absence of antibody formation in the patients not previously treated with insulin, 10 out of the 12 patients who had previously received insulin t r e a t m e n t with mixed pig-ox insulin preparations showed insulin antibodies following the renewed insulin treatment (Table 4). I n one case, the insulin antibodies formed were examined for their ability to discriminate between pig and ox insulin. No discrimination was found (Fig. 1). None of the 6 pigs treated with recrystMlized pig insulin in neutral solution showed insulin antibodies D i s c u s s i o n The absence of insulin antibody formation after t r e a t m e n t with solutions of neutral pig insulin in patients not previously treated with insulin, is not due to the t r e a t m e n t being too low in intensity, since a corresponding intensity of t r e a t m e n t with acid pig-ox insulin solutions resulted in the formation of insulin antibodies (Table 5) in almost all cases - - as previously demonstrated (Deckert, 1964) . I t is remarkable t h a t the t r e a t m e n t of h u m a n subjects with large doses of pig insulin in neutral somtion did not lead to a demonstrable formation of antibodies. This is in contrast to the findings in patients treated with longacting pig insulin preparations, since it had already been reported (Deckert, 1964) t h a t circulating insulin antibodies could be demonstrated in 11 out of 12 patients after t r e a t m e n t with N P H - p i g insulin (Table 5). I t thus appears as if the v e r y slight antigenicity of pig insulin in h u m a n subjects is potentiated when the insulin is injected subcutaneously as a suspension of crystals with a long-acting effect. There are a n u m b e r of factors which suggest t h a t a suspension of insulin particles constitutes a greater stimulus to the reticuloendotheliM system of the organism around the site of injection t h a n a neutral solution, which is rapidly reabsorbed. Thus, experiments with pigs show t h a t the injection of acid insulin preparations, which following injection are transformed into a suspension as a result of isoelectric precipitation at the site of injection,can likewise lead to the formation of insulin antibodies, whereas this is not the case when the insulin is injected as a solution whose p t I corresponds to the p H of the tissue fluid. Frankhauser and Morell (1968) were only rarely able to demonstrate insulin antibody formation in patients treated with neutral solutions of pig insulin, whereas in patients treated exclusively with semilente suspensions, which are almost pure pig insulin, Devlin and Duggan (1968) found insulin antibodies in all eases. The antigenicity of pig insulin would thus appear to depend on whether the pig insulin is injected as a suspension or whether it becomes precipitated at the site of injection. I t is well known in experimental immunology t h a t an antigen is more immunogenic as a suspension t h a n a s a solution, since phagoeytosis of the particles of antigen b y macrophages potentiates the antigenieity of protein (Frei et al., 1965) . I t cannot be overlooked t h a t factors other t h a n the state of the insulin m a y be of significance for potentiating the antigenieity of pig insulin. Thus, it is possible t h a t contamination of pig insulin b y small amounts of ox insulin, proinsulin (Steiner st al., 1967) or proteins alien to insulin (Brunfeldt and Deekert, 1964b) m a y be of significance for the formation of insulin antibodies in patients treated with suspensions of pig insulin. Nor can the possibility be excluded t h a t a small p a r t of the pig insulin is so modified during preparation t h a t it m a y be significant for the antigenicity of the pig insutin. For example, the splitting-off of amide groups might well be significant for the antigenic characteristics of pig insulin. I t is not clear why patients who had previously produced insulin antibodies following t r e a t m e n t with mixed pig-ox insulin preparations in acid solution could be re-immunized following t r e a t m e n t with neutral solutions of pig insulin. The result m u s t presumably m e a n t h a t neutral solutions of pig insulin represent such a weak antigenic stimulus t h a t a n t i b o d y formation does not occur unless the i m m u n e a p p a r a t u s of the organism is already sensitized. The fact t h a t the insulin antibodies formed are unable to discriminate between pig insulin and ox insulin agrees well with this. The present investigations suggest t h a t the formation of insulin antibodies in h u m a n subjects can be avoided b y using rapidly-acting neutral solutions of pig insulin crystals. However, the problem of insulin antibodies has already been reduced to a clinically insignificant phenomenon even with the use of long-acting insulin preparations. This is seen from the experience obtained in the Scandinavian countries, where insulin resistance is a practically unknown situation. During the last 15 years, several thousand diabetics with insulin requirements have undergone t r e a t m e n t at Niels Steensen's ttospitM, Gentofte. During this period, not one single ease of insulin resistance has been observed. Likewise, the insulin requirements of patients in Scandinavia appear to be lower t h a n elsewhere. At Niels Steensen's Hospitat, the 24-hour insulin dosage in diabetics was examined over 2 periods, namely 1942-1952 and 1963--1967. The patients, practically all with diabetes starting before the age of 30, had all received insulin suspensions containing mainly pig insulin for a period of more t h a n 1 year, all patients were over the age of 18 years, had normal renal function, no infectious disease, and were not overweight or pregnant. All patients had recently terminated a hospital s t a y for the control of their diabetes, were well controlled and ambulant. The 24-hour insulin dosage the d a y prior to returning home from hospital was noted. The distribution of the 24-hour insulin dosage during the 2 periods mentioned is seen in Fig. 3. This shows t h a t only 1 out of 1006 patients had an insulin requirement which was greater t h a n 100 i.u./24 h. For comparison, it m a y be mentioned t h a t out of 1089 patients examined at the university diabetes clinic in F r a n k f u r t a m Nain, 36 patients were found with an insulin requirement greater 50 ,0 30 0 / , % 20 0 <20 i 40 60 units/day 80 >100 Fig. 3. The distribution of the size of the dose of insulin in 1006 diabetic subjects treated with insulin during the periods 1942--1952 and 1963--1967. n = number of patients examined t h a n 100 i.u. per 24 hours (Ditschuneit and Federlin, 1965) . Insulin resistance is almost always due to the presence of large amounts of insulin antibodies, and as there is in addition a relationship between insulin requirement and insulin a n t i b o d y titer, the extremely rare occurrence of insulin resistance and the low insulin requirement might suggest t h a t the formation of insulin antibodies is less pronounced using the insulin preparations employed in Scandinavia. I t is not clear why there should be a shift in the distribution curve for 1963--1967 towards a lower 24-h dosage (cf. Fig. 3), b u t this is p r o b a b l y related to the fact t h a t in 1963--1967 the insulin preparations had a greater degree of purity. Acknowledgements. The author wishes to thank Physician-in-chief Jacob E. Poulsen, 3/[.D., K. Brunfeldt, ~ . Sc., and Physician-in-chief Otto Jacobsen, for their helpful criticism. 2* Berson , S.A. , Yalow , I~.S.: Antigens in insulin. Determinants of specificity of porcine insulin in man . Science 139 , 844 -- 845 ( 1963 ). Brunfeldt , K. , Deekert , T. : Antibodies in the pig against pig insulin . Acta endoer . 47 , 367 -- 370 ( 1964a ). -- - - The antigenic properties of pig insulin . Acta endoer . 47 , 353 -- 366 ( 1964b ). Deckert , T. : Insulin Antibodies. Thesis , p. 173 . Copenhagen: Munksgard, 1964 . -- Jorgcnsen , X.R. : Immunological reactivity of insulin in human serum . Aeta endocr . 53 , 673 -- 680 ( 1966 ). -- A u t o i m m u n o l o g i c a l aspects of diabetes mellitus . Acta reed. scand. Suppl . 476 , 29 -- 41 ( 1967 ). Devlin , J.G. , O ' D o n o v a n , D . K . : Preferential beef/pork insulin binding capacity . Diabetes 15 , 790 -- 797 ( 1966 ). -- D u g g a n , M. : A n t i b o d y studies following "beef", beef a n d pork a n d "pork" only insulin preparations. F o u r t h A n n u a l Meeting of the E u r o p e a n Association for the S t u d y of Diabetes, Louvain , Belgium, 1968 . Ditschuneit , H. , Federlin , K. : Pathophysiologie der Insulinresistenz. Diabetologia 1 , 2 4 8 ( 1965 ). Frankhauser , S. , Morell , B. : Antigenicity of different insulin preparations in psychiatric a n d diabetic patients. F o u r t h A n n u a l Meeting of the E u r o p e a n Association for the S t u d y of Diabetes, Louvain , B e l g i u m 1968 . Frei , P.C. , Benacerraf , B. , T h o r b e c k e G .J. : Phagocytosis of the antigen, a crucial step in the induction of the p r i m a r y response . Proc. nat. Acad. Sci . 53 , 20 -- 23 ( 1965 ). Lockwood , D . H . , P r o u t , T . E . : A n t i g e n i c i t y of heterologous a n d homologous insulin . Metabolism 14 , 530 -- 538 ( 1965 ). Skom , J . H . , Talmage , D . W . : N o n - p r e c i p i t a t i n g insulinantibodies . J. clin. I n v e s t . 37 , 783 -- 786 ( 1958 ). Steiner , D . F . , Cunningham , D. , Spigelman , L. , A t e n B.: Insulin biosynthesis: Evidence for a precursor . Science 157 , 697 -- 700 ( 1967 ).


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Torsten Deckert, Else Grundahl. The antigenicity of pig insulin, Diabetologia, 1970, 15-20, DOI: 10.1007/BF00425886