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)