Immunoreactive proinsulin detected by enzyme-linked immunosorbent assay

A sensitive enzyme-linked immunosorbent assay for human proinsulin was developed by the modification of the method reported using monoclonal antibodies. In the present method, two monoclonal antibodies, an anti-C-peptide antibody bound to microtiter plate, and a biotin-labeled anti-insulin antibody were used. This assay was specific for proinsulin and failed to detect both insulin and C-peptide. The minimal detection limit of this assay was approximately 0.1 pmol/l. Immunoreactive proinsulin levels in serum of normal subjects, ranged from 1.7 to 8.7 pmol/l with the mean of 4.6 pmol/l. The ranges for the intra- and inter-assay coefficients of variance were 3.1-3.7% and 5.0-14.9%, respectively. Reverse phase HPLC analysis of serum of normal subject, as measured with this assay system, revealed two immunoreactive (IR-) forms. One form eluted at the same position as that of authentic proinsulin and the other was detected in a more hydrophilic part of the chromatogram (shorter retention time). Elution profiles of IR-insulin and IR-C-peptide in human serum were also examined by the present reverse phase HPLC and compared to those of IR-proinsulins.