A rapid ELISA for measuring insulin in a large number of research samples

Abstract

An enzyme-linked immunosorbent assay (ELISA) for insulin was developed. Anti-insulin antibody was bound to the bottom of 96-well microtiter plates. Insulin conjugated to beta-galactosidase was used as a label and methyl umbilliferyl beta-D galactoside was used as an enzyme substrate. To estimate insulin, relative fluorescence was measured with a fluorescent microtiter plate reader. Unknowns from an insulin release experiment yielded results comparable to those obtained with our enzyme-immunoassay (EIA) and a conventional radioimmunoassay (RIA). The insulin ELISA is suitable for research purposes in which samples contain solutions of physiological salts and albumin, but not for samples containing serum. The insulin ELISA is as sensitive as the insulin RIA and has several advantages over the standard insulin RIA. These include (1) avoidance of hazards and inconvenience of handling radioactivity, (2) not requiring a separate test tube for each sample, (3) stability of the enzyme-labeled insulin (greater than 18 months), (4) short time period required for the assay (less than 6 hours), and (5) the possibility of long-term storage (at least 3 months) of antibody-coated microtiter plates.