First direct assay for intact human proinsulin

¹ University of Liège, Division of Nuclear Pediatrics, Sart Tilman, 4000 Liège, Belgium.

² Steno Diabetes Center, Niels Steensens Vej 2, 2820 Gentofte, Denmark.

³ Lilly Research Laboratories, 307 East Mccarty Street, Indianapolis, IN 46285.

⁴ Fakulteit Geneeskunde in Farmacie, Laarbeeklaan 103, Vrije Universiteit, 1000 Brussels, Belgium.
^a Author for correspondence. Fax 32-4-366-82-55.

We describe a sensitive two-site sandwich enzyme-linked immunosorbent assay for the measurement of intact human proinsulin in 100 µL of serum or plasma. The assay is based on the use of two monoclonal antibodies specific for epitopes at the C-peptide/insulin A chain junction and at the insulin B chain/C-peptide junction, respectively. Cross-reactivities with insulin, C-peptide, and the four proinsulin conversion intermediates were negligible. The detection limit in buffer was 0.2 pmol/L (3 standard deviations from zero). The working range was 0.2–100 pmol/L. The mean intra- and interassay coefficients of variation were 2.4% and 8.9%, respectively. The mean recovery of added proinsulin was 103%. Dilution curves of 40 serum samples are parallel to the proinsulin calibration curve. Proinsulin concentrations in 20 fasting healthy subjects were all above the limit of detection: median (range), 2.7 pmol/L (1.1–6.9 pmol/L). Six fasting non-insulin-dependent diabetes mellitus and five insulinoma patients had proinsulin concentrations significantly higher than healthy subjects: median (range), 7.7 pmol/L (3.2–18 pmol/L) and 153 pmol/L (98–320 pmol/L), respectively.