Summary
The insulin-binding potency of guinea pig anti-insulin serum (GPAIS) is determined following reaction for 30 minutes at room temperature between solutions containing GPAIS and mixtures of unlabeled and131I-labeled insulin. Insulin which has not reacted with the antibodies is extracted from solution by a suspension of finely divided cellulose. From the radioactive contents of supernatant solutions from reaction mixtures containing the sample, an excess of non-precipitating (reference) GPAIS, and the buffered diluent, the potency of the sample can be determined. Factors affecting the assay procedure are considered and it is shown that, within defined limits, results are reproduceable and the method sensitive and rapid. The technique has potential uses for the study of insulin-antibody reactions and has practical applications in the production and rational use of GPAIS.
Résumé
Les auteurs présentent une méthode pour l'estimation du pouvoir de liaison avec l'insuline, du sérum anti-insulinique, obtenu par immunisation de cobayes. Les solutions contenant l'immun-sérum sont incubées durant 30 minutes entre 20 et 25° C, en présence d'un mélange d'insuline ordinaire et de radioinsuline-131I. L'insuline libre, non liée aux anticorps, est extraite de la solution par addition d'une suspension de cellulose en fines particules. Le titre en anticorps d'un échantillon est calculé à partir des radioactivités mesurées dans les surnageants de trois différents milieux, contenant respectivement les anticorps à doser, un excès d'immun-sérum non-précipitant (sérum de référence) et le milieu tampon. Différents facteurs susceptibles d'interférer avec le dosage sont envisagés et la méthode est prouvée sensible, rapide et, endéans des limites définies, reproductible. Cette méthode peut être utilisée pour l'étude des réactions entre l'insuline et ses anticorps et a également trouvé un champ d'application dans la production et l'emploi rationnels du sérum anti-insulinique.
Zusammenfassung
Es wird berichtet über eine Methode, die insulinbindende Kraft des Anti-Insulin-Serum beim Meerschweinchen (GPAIS) zu bestimmen. Die Lösungen, die das Immunserum enthalten, werden während 30 min bei einer Temperatur zwischen 20° und 25° C inkubiert in Gegenwart einer Mischung von gewöhnlichem Insulin und mit131I markiertem Radioinsulin. Das freie Insulin, das nicht an Antikörper gebunden ist, wird aus der Lösung durch Hinzufügung einer Aufschwemmung von feinverteilter Zellulose extrahiert. Aus dem radioaktiven Gehalt der überstehenden Lösungen der Reaktionsgemische, die die Probe, einen Überschuß von nicht präzipitierendem GPAIS (Bezugserum) und die gepufferte Verdünnungsflüssigkeit enthalten, läßt sich die insulinbindende Kraft bestimmen. Faktoren, die die Bestimmungsmethode beeinflussen können, werden diskutiert. Es läßt sich zeigen, daß die Ergebnisse innerhalb definierter Grenzen reproduzierbar sind, während die Methode empfindlich und schnell durchzuführen ist. Die Technik läßt sich mit Vorteil zum Studium der Reaktionen zwischen Insulin und seinen Antikörpern verwenden. Ihr praktischer Anwendungsbereich liegt in der Produktion und dem zweckmäßigen Gebrauch des Anti-Insulin-Serum.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Antoniades, H.N., J.A. Bougas, R. Camerini-Davalos andH.M. Pyle: Insulin regulating mechanism and diabetes mellitus. Diabetes13, 230–240 (1964).
Arquilla, E.R., andA.B. Stavitsky: The production and identification of antibodies to insulin and their use in assaying insulin. J. clin. Invest.35, 458–466 (1956).
—, andJ. Finn: Insulin antibody variations in rabbits and guinea pigs, and multiple antigenic determinants on insulin. J. exp. Med.118, 55–71 (1963).
Berson, S.A., andR.S. Yalow: Immunoassay of protein hormones. In “The Hormones”. (Pincus, G., K.V. Thimann andE.B. Astwood, Eds.) Vol. IV, New York, Academic Press, pp. 557–630, 1964.
— —: Quantitative aspect of the reaction between insulin and insulin-binding antibody. J. clin. Invest.38, 1996–2016 (1959).
— —: Recent studies on insulin binding antibodies. Ann. N.Y. Acad. Sci.82, 338–344 (1959).
Bray, G. A.: Liquid scintillator for counting aqueous solution in a liquid scintillation counter. Analyt. Biochem.1, 279–285 (1960).
Corcos, J.M., andZ. Ovary: Biological properties of guinea pig anti-insulin antibodies. Proc. Soc. exp. Biol.119, 142–148 (1965).
Franckson, J.R.M., Y. Arnould, W. Malaisse andV. Conard: Glucose metabolism in the normal anesthetised dog injected successively with anti-insulin serum and insulin. Diabetes13, 532–541 (1964).
Froesch, E.R., H. Burgi, E.B. Ramseier, P. Bally andA. Labhart: Antibody suppressible and nonsuppressible insulin-like activities in human serum and their physiological significance. An insulin assay with adipose tissue of increased precision and specificity. J. clin. Invest.42, 1816–1834 (1963).
Gordis, E.: Detection of insulin-binding antibodies and separation of free and antibody bound insulin by rapid chemical procedure. Proc. Soc. exp. Biol.103, 542–544 (1960).
Grodsky, G.M., andP.H. Forsham: Immunochemical assay of total extractable insulin in man. J. clin. Invest.39, 1070–1079 (1960).
Herbert, V., K.S. Lau, C.W. Gottlieb andS.J. Bleicher: Coated charcoal immunoassay of insulin. J. clin. Endocr.25, 1375–1384 (1965).
Hirata, Y., andH.T. Blumenthal: Demonstration of precipitating insulin-binding antibody in the serum of insulin treated guinea pigs and rabbits. J. Lab. clin. Med.62, 683–691 (1963).
Jones, V.E., andA.C. Cunliffe: A precipitating antibody to insulin. Nature.192, 136–138 (1961).
Kologlu, Y., L.L. Wiesel, V. Positano andG.E. Anderson: An immune Chromatographie method for assay in serum of antibodies to glucagon and insulin. Proc. Soc. exp. Biol.112, 518–523 (1963)
Malaisse, W.J., F.Malaisse and P.H.Wright: A new method for the measurementin vitro of pancreatic insulin secretion. Endocrinology. in press.
Mitchell, M.L., andA.H. Bradford: The measurement of insulin binding by resin paperin vitro. Diabetes.12, 257–261 (1963).
Morgan, C.R., andA. Lazarow: Immunoassay of insulin: two antibody system. Plasma insulin levels in normal, sub-diabetic and diabetic rats. Diabetes12, 115–126 (1963).
Morse, J.H.: Heterogeneity of insulin-antibody complexes in rabbits and guinea pigs. Proc. Soc. exp. Biol.103, 494–496 (1960).
Pope, C.G.: The immunology of insulin. In “Advances in Immunology”. (Dixon, F.J., andJ.H. Humphrey, Eds.), Vol. 5, New York, Academic Press, pp. 209–244, 1966.
Robinson, B.H.B., andP.H. Wright: Guinea pig anti-insulin serum. J. Physiol.155, 302–310 (1961).
Samaan, N., R. Frazer andW.J. Dempster: The “typical” and “atypical” forms of serum insulin. Diabetes12, 339–348 (1963).
Skom, J.H., andD.W. Talmage: Non-precipitating insulin antibodies. J. clin. Invest.37, 783–786 (1958).
Wright, P.H.: Experimental diabetes induced by insulin antibodies. In “On the nature and treatment of diabetes”, (Wrenshall, G.A., and B.S.Leibel, Eds.) New York, Excerpta Medica Foundation, pp. 354–360, 1965.
—: The effect of insulin antibodies on glucose uptake by the isolated rat diaphragm. Biochem. J.71, 633–638 (1959).
-, and L.Norman: Some factors affecting insulin antibody production in guinea pigs. Diabetes, in press.
—, andL.R. Calimlim: Assay of insulin antibodies produced by the guinea pig. Nature207, 995–996 (1965).
- - and W.J.Malaisse: Endogenous insulin secretion in the rat following injection of anti-insulin serum. Amer. J. Physiol. in press.
Yagi, Y., P. Maier andD. Pressman: Two different anti-insulin antibodies in guinea pig antisera. J. Immunol.89, 442–451 (1962).
Yalow, R.S., andS.A. Berson: Immunoassay of endogenous plasma insulin in man. J. clin. Invest.39, 1157 -1175 (1960).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Wright, P.H., Malaisse, W.J. A simple method for the assay of guinea pig anti-insulin serum. Diabetologia 2, 178–188 (1966). https://doi.org/10.1007/BF01222068
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF01222068