Factors affecting enzyme-linked immunosorbent assay (ELISA) for insulin antibodies in serum.

We have evaluated the factors affecting an enzyme-linked immunosorbent assay (ELISA) for circulating insulin antibodies. Dilutions of patients' sera were incubated in polystyrene tubes coated with procine insulin. The second incubation was with alkaline phosphatase-conjugated anti-human Fab. Each of these reactions was complete after 4 h. Specificity of the reaction for insulin antibodies was demonstrated by removal of anti-insulin activity after preincubation with insulin, but not with glucagon at similar concentrations. Sensitivity of the ELISA system, assessed by performing the reaction with affinity-column purified insulin antibodies, was 10 microgram of specific antibody per liter. Using this system, we examined sera from 22 patients who had been determined by radioimmunoassay to have insulin antibodies, and sera from 23 normal individuals. The ELISA results correlated reasonably well with those of RIA (r = +0.84). Besides detecting insulin antibodies in diabetic patients who are being treated with insulin, we use this ELISA test as a screening procedure to be certain insulin antibodies are not present when we use indirect immunofluorescence methods of fixed pancreatic substrate to detect islet-cell antibodies.