Development of a multipurpose time-resolved fluorescence immunoassay for rat insulin

We present a time-resolved fluorescence immunoassay (TR-FIA) for the measurement of rat insulin in cell extracts and culture media. This assay is based on the binding of two monoclonal antibodies to different parts of the insulin molecule in a 96-well microtiter plate. For the detection, europium-labeled streptavidin that interacts with the second biotinylated antibody is used. Samples of 25 microl could be analyzed in less than 2 days with a measuring range between 5 and 1250 pg (0.2-50 microg/L or 34.4-8600 pM). The inter- and intraassay percentage coefficients of variation were less than 8.3 and 5.1, respectively. Recoveries of 0.48 to 40 microg/L rat insulin, added to culture medium, ranged between 94 and 107%. Results were significantly correlated with those of an in-house radioimmunoassay (RIA) for rodent insulin (P<0.0001, r(2)=0.99). The TR-FIA method had a similar detection limit (0.16 microg/L), but its working range was at least 5-fold larger. Additional advantages include the lower cost, the applicability to measurements in tissue and serum, and the quantification of insulin from other species.