Two-Site Time-Resolved Immunofluorometric Assay For Insulin In Human Serum

We describe a two-site "sandwich"-type time-resolved immunofluorometric assay for human insulin, based on use of two monoclonal antibodies that bind to two different epitopes on the insulin molecule. The first antibody is immobilized on the surface of microtiter plate, the other is labeled with Eu³⁺. Standard, samples containing insulin are reacted simultaneously with immobilized monoclonal antibodies and with europium-labeled monoclonal antibodies. The complete assay requires only one incubation step. Enhancement Solution dissociates europium ions from the labeled antibody into solution, where they form highly fluorescence chelates with components of the enhancement solution. The fluorescence is directly proportional to the concentration of insulin.The work range of the assay is 5-160 mlU/L. The sensitivity is better than 0.35mIU/L. The within-run coefficient variations are all less than 5%. The analytical recoveries are within 97.7-102.9%. A correlation study was carried out with 20 samples measured by DSL Insulin ELISA and with 60 samples detected by Insulin ELISA afforded by our laboratory, the correlation coefficient was 0.9810 and 0.9412, respectively.