Application of a Rapid Enzyme-linked Immunosorbent Microassay (ELISA) to Study Human Anti-insulin Antibody

A rapid, reproducible enzyme-linked immunosorbent assay (ELISA) for antibody to insulin is characterized and used to study antibodies from insulin-treated diabetic subjects. No radioactivity is involved in the ELISA; rather, peroxidase-conjugated anti-human immunoglobulin is used to detect binding of antibodies to insulin-coated microplates. Color is produced by action of peroxidase on a substrate and an automated reader then measures binding spectrophotometrically. This ELISA was optimized to be at least as sensitive as measurement of antibody by direct ¹²⁵I-insulin binding. Although detection of IgG directly binding to insulincoated plates in the ELISA does not reveal speciesspecific differences, avidity differences are shown in competitive inhibition with insulins from several species. Insulin-binding IgG was purified by affinity chromatography and used to construct a standard curve of the optical density (OD) in the ELISA relative to the concentration of antibody. This assay has been used to quantify and to characterize insulin antibody of sera from a large number of diabetic subjects, ranging from insulin resistant to those who had received only highly purified and human insulins. The assay is shown to be most useful to screen for insulin antibodies in resistant patients.