A sensitive radioimmunoassay of insulin autoantibody: reduction of non-specific binding of [125I]insulin

There has been a lack of consensus among results of assays for insulin autoantibody (IAA) carried out by different laboratories, despite a reduction in the non-specific effect using a cold insulin competitor in radioimmunoassays (RIAs) for IAA in type I diabetes. We speculated that the discrepancies are partly a result of the non-specific binding (NSB) of [125I]insulin to unidentified molecules in serum on polyethylene glycol separation, and tried to improve IAA RIAs. The molecular weight of a candidate for the factor causing NSB was estimated to be about 700 kDa by gel filtration analysis, resembling that of alpha 2-macroglobulin (a2M). Further, the addition of purified a2M to the assay resulted in an increase in NSB. Screening revealed that heterocyclic compounds, such as isothiazolinone derivatives (ProClin300), were greatly effective at reducing NSB in control subjects from 2.904+/-0.909% to 1.347+/-0.254% (n=283, mean+/-SD, p<0.0001). Using our newly developed IAA RIA with ProClin300, the sensitivity for newly diagnosed type I diabetes patients (n=55) was 32.7% and 30.9% with or without insulin competition, respectively, whereas that of the former assay without ProClin300 was only 20.0%.