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RIA methodology is used widely to measure proinsulin in human serum. However, some RIAs lack the sensitivity necessary to quantify proinsulin in unextracted serum and require long incubation periods. We developed an RIA with a sensitivity of 3.5 in <48 h. This was accomplished by using a nonequilibrium binding reaction at room temperature and PEG-assisted second antibody precipitation as the method for separating bound and free proinsulin. We obtained a specific antiproinsulin antibody by adsorbing the initial goat antiserum with human C-peptide–agarose. Proinsulin produced 50% displacement of tracer at 25.6 pM, whereas both human insulin and C-peptide failed to displace tracer at concentrations as high as 1 μM. We evaluated several cleaved derivatives of proinsulin for cross-reactivity with the antibody. B-chain–C-peptide cleaved derivatives (≤50% cross-reactivity) were more potent than A-chain …