Characterization and Use in Immunoradiometric Assay of Monoclonal Antibodies Directed Against Human Proinsulin

Abstract

Proinsulin in human serum is heterogenous. Existing assay methods do not distinguish between the various forms intermediate on the pathway of processing from proinsulin to insulin. We report the production of mouse monoclonal antibodies against human proinsulin with biosynthetic human proinsulin (produced by recombinant DNA technology) as immunogen. Four monoclonal antibody-producing cell lines were obtained from two separate fusions. Two of the antibodies had affinity constants for intact proinsulin of 3.1 X 10(10) and 5.6 X 10(10) L/mol and bound all forms of proinsulin and insulin. Cross-reactivity with intact proinsulin was greater than with 65-66 and 32-33 split proinsulins and des 64-65 and des 31-32 proinsulins. The third antibody had an affinity constant for intact proinsulin of 6.3 X 10(10) L/mol and reacted only with intact proinsulin, 65-66 split proinsulin, and des 64-65 proinsulin; there was no reaction with 32-33 split proinsulin, des 31-32 proinsulin, or C-peptide. The fourth antibody reacted only with intact proinsulin and had an affinity constant of 4.1 X 10(8) L/mol. We report the use of two antibodies in a sensitive two-site immunoradiometric assay for intact proinsulin. Insulin, C-peptide, and 32-33 split proinsulin did not react in the assay up to a concentration of 100 pM. The 65-66 split proinsulin and des 64-65 proinsulin reacted with a potency of approximately 55% relative to intact proinsulin. Serum proinsulin concentrations measured with this assay were compared with those determined by an alternative method that detects only split proinsulins.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of IgG Autoantibodies Against Human Proinsulin in Patients With IDDM Before Insulin Treatment

“…Support for our observation of IgG specific for proinsulin comes from the demonstration that human proinsulin induces specific antibodies in mice (10). Furthermore, Fineberg et al (11) recently found antibodies to proinsulin in patients with IDDM after treatment with insulin or proinsulin.…”

“…The presence of autoantibodies to proinsulin in early IDDM indicates that precursors of insulin are involved in the immune process of IDDM. Immunization with proinsulin also leads to the formation of insulin-proinsulin-specific antibodies (10). IAA and PAA may thus be induced by the same immunogen, which may be proinsulin and/or its precursor molecules.…”

Sensitive and specific two-site immunoradiometric assays for human insulin, proinsulin, 65-66 split and 32-33 split proinsulins

“…The mouse serum and cell supernatants were assayed as below. The monoclonal antibody 3B1 was prepared as previously described (Gray et al, 1987).…”

“…We have, therefore, attempted to produce a panel of monoclonal antibodies with different binding specificities for these immunologically insulin-like molecules, such that their use in different combinations would allow us to determine specifically the individual concentrations of these molecules. We have reported our early attempts to assay intact proinsulin by this means (Gray et al, 1987). We now report the successful outcome of the overall objective, together with the concentrations of insulin, intact, and partially processed proinsulins in plasma from normal male subjects.…”

Biosynthesis of betagranin in pancreatic β-cells. Identification of a chromogranin A-like precursor and its parallel processing with proinsulin

“…Insulin. A mouse monoclonal antibody (3B7) raised to human proinsulin, which recognizes epitopes within the insulin structure (Gray et al, 1987), was partially purified by (NH4)2SO4 precipitation and coupled to diazotized cellulose by established procedures (Hales & Woodhead, 1980). Extracted samples (1 x 106 d.p.m.…”