Development of radioimmunoassay for estimation of C-peptide in human serum

Abstract

C-Peptide radioimmunoassay (RIA) is an invaluable aid in diagnosis of diabetes, insulinoma and hypoglycaemia. An RIA for C-peptide was developed by using a polyclonal antibody for human C-peptide raised in rabbits and validated using human serum samples. Tyrosyated C-peptide was radioiodinated to a specific activity of around 283 µCi/µg by conventional chloramine-T method. The detection limit of the assay (90% binding) was 0.43 ng/mL and the ED50 value was 8.5 ng/mL. The assay with the standard range of 0.3–18 ng/mL and low non-specific binding provide superior assay features. Separation of the bound from free complex was carried out using 22% polyethylene glycol. Intra-assay and inter-assay variations were found to be < 11.5% along with analytical recovery of 90–104%. No cross reactivity was observed with proinsulin, glucagon, somatostatin, insulin and pancreatic polypeptide.The developed assay can be used for the routine analysis of clinical samples.