Highly Specific Radioimmunoassay for Human Insulin Based on Immune Exclusion of All Insulin Precursors

[en] We describe a rapid and simple insulin RIA in which proinsulin and conversion intermediates do not interfere. Three monoclonal antibodies (S1, S2, and S53) were selected for their specificity (directed, respectively, against the B10 region, the junction between A chain and C-peptide, and the junction between B chain and C-peptide), their affinity constant (approximately 10(10) L/mol), and their interactive properties in mixture. S2 and S53 were able to bind simultaneously to the same proinsulin molecule, whereas neither could bind simultaneously with S1. Preincubation of serum samples with an excess of S2 resulted in capture of proinsulin and conversion intermediates modified at the junction between B chain and C-peptide into immune complexes that no longer reacted with S1. Similarly, preincubation with S53 prevented proinsulin and conversion intermediates modified at the junction between A chain and C-peptide from reacting with S1. Preincubation with an excess of both S2 and S53 left insulin as the sole reactant with S1. Thus, separation of insulin precursors from insulin by mutually exclusive antibodies is feasible, and on the basis of this new principle, a highly specific RIA for insulin was designed. The detection limit was 11 pmol/L, and the inter- and intraassay coefficients of variation were 11% and 5%, respectively. The potential of the assay for use in clinical studies was verified by application to serum samples from control subjects and patients with diabetes or insulinoma.

Disciplines :

Endocrinology, metabolism & nutrition

Author, co-author :

Deberg, Michelle ; Université de Liège - ULiège > Unité de recherche sur l'os et le cartillage (U.R.O.C.)

Houssa, P.

Frank, B. H.

Sodoyez-Goffaux, F.

Sodoyez, J. C.

Language :

English

Title :

Highly Specific Radioimmunoassay for Human Insulin Based on Immune Exclusion of All Insulin Precursors

Publication date :

July 1998

Journal title :

Clinical Chemistry

ISSN :

0009-9147

eISSN :

1530-8561

Publisher :

American Association for Clinical Chemistry, Washington, United States - District of Columbia

Volume :

Issue :

Pages :

1504-13

Peer reviewed :

Peer Reviewed verified by ORBi

Available on ORBi :

since 26 November 2010

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Bibliography

Steiner DF, Kemmler W, Tager HS, Peterson JD. Proteolytic processing in the biosynthesis of insulin and other proteins. Fed Proc 1974;33:2105-15.
Docherty K, Hutton JC. Carboxypeptidase activity in the insulin secretory granule. FEBS Lett 1983;162:137-41.
Davidson HW, Hutton JC. The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing. Biochem J 1987;245:575-82.
Davidson HW, Rhodes CJ, Hutton JC. Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic β cell via two distinct site specific endopeptidases. Nature 1988;333:93-6.
Rhodes CJ, Lincoln B, Shoelson SE. Preferential cleavage of des-31,32-proinsulin over intact proinsulin by the insulin secretory granule type II endopeptidase. Implications of a favored route for prohormone processing. J Biol Chem 1992;267:22719-25.
Temple RC, Carrington CA, Luzio SD, Owens DR, Schneider AE, Sobey WJ, Hales CN. Insulin deficiency in non-insulin-dependent diabetes. Lancet 1989;1:293-5.
Sobey WJ, Beer SF, Carrington CA, Clark PMS, Frank BH, Gray IP. et al. Sensitive and specific two-site immunoradiometric assays for human insulin, proinsulin, 65-66 split and 32-33 split proinsulins. Biochem J 1989;260:535-41.
Sizonenko S, Irminger JC, Buhler L, Deng S, Morel P, Halban PA. Kinetics of proinsulin conversion in human islets. Diabetes 1993; 42:933-6.
Halban PA. Proinsulin conversion intermediates: a possible source of confusion [Letter]. Diabetologia 1991;34:202-3.
Ludvigsson J, Heding L. Abnormal proinsulin/C-peptide ratio in juvenile diabetes. Acta Diabetol Lat 1982;19:351-8.
Mako ME, Starr JI, Rubenstein AH. Circulating proinsulin in patients with maturity onset diabetes. Am J Med 1977;63:865-9.
Yoshioka N, Kuzuya T, Matsuda A, Taniguchi M. Iwamoto U. Serum proinsulin levels at fasting and after oral glucose load in patients with type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia 1988;31:355-60.
Reaven GM, Chen YDI, Hollenbeck CB, Sheu WHH, Ostrega D. Polonsky KS. Plasma insulin, C-peptide and proinsulin concentrations in obese and non obese individuals with varying degrees of glucose tolerance. J Clin Endocrinol Metab 1993;76:44-8.
Gabbay KH, DeLuca K, Fisher JN, Mako ME, Rubenstein AH. Familial hyperproinsulinemia. An autosomal dominant defect. N Engl J Med 1976;294:911-5.
Barbetti F, Raben N, Kadowaki T, Cama A, Accili D, Gabbay KH, et al. Two unrelated patients with familial hyperproinsulinemia due to a mutation substituting histidine for arginine at position 65 in the proinsulin molecule: identification of the mutation by direct sequencing of genomic deoxyribonucleic acid amplified by polymerase chain reaction. J Clin Endocrinol Metab 1990;71:164-9.
Cohen RM, Given BD, Lucinio-Paixao J, Provow SA, Rue PA, Frank BH, et al. Proinsulin radioimmunoassay in the evaluation of insulinomas and familial hyperproinsulinemia. Metabolism 1986;35:1137-46.
Sodoyez JC, Koch M, Lemaire I, Sodoyez-Goffaux F, Rapaille A, François-Gerard C, Sondag D. Influence of affinity of antibodies upon their detection by liquid phase radiobinding assay and solid phase enzyme linked immunosorbent assay. Demonstration using monoclonal antibodies raised against rDNA human proinsulin. Diabetologia 1991;34:463-8.
Scatchard G. The attraction of proteins for small molecules and ions. Ann N Y Acad Sci 1949;51:660-72.
Brodai BP. The influence of haemolysis on the radioimmunoassay of insulin. Scand J Clin Lab Invest 1971;28:287-90.
Yalow RS, Berson SA. Assay of plasma insulin in human subjects by immunological methods. Nature 1959;184:1648-9.
Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495-7.
Alpha B, Cox L, Crowther N, Clark PMS, Hales CN. Sensitive amplified immunoenzymometric assay (IEMA) for human insulin and intact proinsulin. Eur J Clin Chem Clin Biochem 1992;30:27-32.
Andersen L, Dinesen B, Jorgensen PN, Poulsen F, Roder ME. Enzyme immunoassay for intact human insulin in serum or plasma. Clin Chem 1993;39:578-82.
Monti LD, Sandoli EP, Phan VC, Piatti PM, Costa S, Secchi A, Pozza G. A sensitive and reliable method for assaying true human insulin without interaction with human proinsulin-like molecules. Acta Diabetol 1995;32:57-63.
Siohan E, Allauzen S, Mani JC, Demuynck A, Rousset M, Laprade M, et al. A highly sensitive and specific immunoradiometric assay for human insulin using monoclonal antibodies [Abstract]. Diabetologia 1996;39(Suppl 1):A86.
Chevenne D, Letailleur A, Trivin F, Porquet D. Dosage des proinsulines, insuline et peptide-C plasmatiques [Review]. Ann Biol Clin 1995;53:3-9.
Petersen K-G, Storch M-J, Rother K, Licht T, Kerp L. Insulin and anti-insulin antibody interaction. Evidence for the formation of 7S and 10S structures. Diabetes 1985;34:799-802.
Hales CN, Randle PJ. Immunoassay of insulin with insulin-antibody precipitate. Biochem J 1963;88:137-46.
Hale PJ, Djurup R, Baddeley RM, Nattrass M. Insulin and proinsulin concentrations in patients with insulinoma before and after surgical treatment. Diabetes Nutr Metab 1991;4:113-6.
Creutzfeldt W, Arnold R, Creutzfeldt C, Deuticke U, Frerichs H, Track NS. Biochemical and morphological investigations of 30 human insulinomas. Correlation between the tumor content of insulin and proinsulin-like components and the histological and ultrastructural appearance. Diabetologia 1973;9:217-31.
Berger M, Bordi C, Cüppers H-J, Berchtold P, Gries A, Münterfering H, et al. Functional and morphologic characterization of human insulinomas. Diabetes 1983;32:921-31.
Smeekens SP, Montag AG, Thomas G, Albige-Rizo C, Carroll R, Bening M, et al. Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3. Proc Natl Acad Sci USA 1992;89:8822-6.
Neerman-Arbez M, Sizonenko SV, Halban PA. Slow cleavage at the proinsulin B-chain/connecting peptide junction associated with low levels of endoprotease PCI/3 in transformed β cells. J Biol Chem 1993;268:16098-100.