A radioimmunoassay for circulating human proinsulin.

Abstract

A radioimmunoassay for human proinsulin (hPl) has been developed using biosynthetic hPl prepared by recombinant DNA technology as immunogen, standard, and tracer. The antiserum was raised in a guinea pig and then adsorbed against insulin and C-peptide conjugated to Sepharose to improve its specificity. After adsorption of the antiserum, the cross-reactivities to insulin and C-peptide were each less than 0.2%. Competition studies using in vitro enzymatically split forms of proinsulin demonstrated that the major antigenic determinant recognized was the junctional region between the B-chain of insulin and the C-peptide. The range of the assay extended from 10 to 150 fmol/tube, with a 50% displacement of 45-55 fmol/tube. This sensitivity proved suitable for measurements of serum hPl concentrations during infusion of biosynthetic hPl into normal subjects and type I diabetic subjects. Eighty-five of 89 serum samples from the normal subjects and each of 20 samples from diabetic subjects diluted in parallel with the hPl standard. Since the direct assay sensitivity was not sufficient for measurement of endogenous hPl levels, a simple procedure for quantitative extraction of proinsulin-like material (PLM) from up to 40 ml of plasma on insulin antibody-Sepharose columns was developed. Logit-log slopes were calculated for dilutions of extracts of samples collected in the fasting state and 60 min after 75 g or oral glucose from eight healthy subjects. The slopes of 15 of the 16 samples did not differ significantly from the slope of the hPl standard.(ABSTRACT TRUNCATED AT 250 WORDS)