An improved radioimmunoassay of C-peptide and its application in a multiyear study

A commercial radioimmunoassay (RIA) for human proinsulin C-peptide was modified to improve its ruggedness and specificity, to decrease the influence of specimen matrix, and to shorten "hands-on" time. In the new protocol, we prepare calibrators in a C-peptide-free serum pool, prepared by treatment with activated charcoal (biological matrix), instead of in a defined matrix. This yielded essentially 100% analytical recoveries for C-peptide concentrations up to 300 pmol/L, a broader analytical range. We also corrected calibrators and unknown samples for nonspecific binding (NSB). Decreasing the concentration of ethanol (from 950 to 880 mL/L) for differential precipitation of the antigen-antibody complex resulted in an NSB of less than 10%, while maintaining high bound/total count percentages for samples and calibrators. C-peptide is thermally unstable without aprotinin at -20 degrees C and with or without aprotinin at 4 degrees C or above, but multiple freeze-thaw cycles do not affect C-peptide in serum. The modified C-peptide assay was applied to plasma from a multiyear study (fasting and post-carbohydrate-challenge subjects). During the four years of the study CVs ranged from 1.9% to 8.6% for replicate analyses of C-peptide in samples with concentrations less than or equal to 500 pmol/L. Between-run CVs were 3.8% to 8.2%, total CVs 3.8% to 10.7%.