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We describe a two-site "sandwich"-type time-resolved immunofluorometric assay for human insulin, based on use of two monoclonal antibodies with different specificities. The first antibody is immobilized on the surface of microtiter plate strip wells, the other is labeled with Eu3+. Serum samples can be assayed with one incubation step; two incubation steps are required when plasma samples are assayed. After the immunoreactions are complete, the bound fraction of Eu3+-label is quantified by dissociating it in a fluorescence-enhancement solution and measuring its fluorescence with a fluorometer with time-resolution. The sensitivity of the assay is 0.24 micro-int. units/mL. The standard curve is linear from 0.24 to 2400 micro-int. units/mL.

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