A radioimmunoassay for human proinsulin (hPI) has been developed using biosynthetic hPI 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 <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 hPI concentrations during infusion of biosynthetic hPI into normal subjects and type I diabetic subjects. Eightyfive of 89 serum samples from the normal subjects and each of 20 samples from diabetic subjects diluted in parallel with the hPI standard.
Since the direct assay sensitivity was not sufficient for measurement of endogenous hPI 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. Log it-log slopes were calculated for dilutions of extracts of samples collected in the fasting state and 60 min after 75 g of oral glucose from eight healthy subjects. The slopes of 15 of the 16 samples did not differ significantly from the slope of the hPI standard. The fasting PLM concentration was 5.8 ± 3.0 fmol/ml (mean ± S.D, N = 8) and rose to 22.5 ± 6.7 fmol/ml (N = 6) 60 min after a 75-g oral glucose load. This assay will be useful in characterizing the metabolism and metabolic effects of biosynthetic human proinsulin and its intermediate forms and determining their therapeutic potential, and for measuring endogenous PLM in extracted plasma samples.