Measurement of human proinsulin by an indirect two-site immunoradiometric assay

Abstract

An indirect two-site immunoradiometric assay is described for the measurement of human proinsulin in plasma. Polyethylene tubes coated with purified guinea-pig antibodies to insulin were used to extract proinsulin and insulin from plasma. Rabbit antibody to human C peptide was then added to react with the C-peptide moiety of the bound proinsulin. The uptake of this antibody was measured by the subsequent binding of¹²⁵I-sheep antibody to rabbit IgG. The binding of radioactivity to the tubes was a function of the proinsulin concentration in the sample. The sensitivity of the assay was 0.006 pmol/ml. Only 200 μl of plasma was required in the assay and the¹²⁵I-labelled antibody was produced from readily available reagents. The polyethylene tubes remained stable for at least 5 months after coating. The mean fasting proinsulin level was 0.009 pmol/ml in sixteen normal subjects and 0.025 pmol/ml in twelve maturity onset diabetics. Oral glucose produced an 8 fold increase in proinsulin concentration but a decline in the plasma proinsulin/insulin molar ratio. Four patients with insulinoma had extremely elevated proinsulin levels and proinsulin/insulin ratios.