A time-resolved fluoroimmunoassay for human insulin based on two monoclonal antibodies

The insulin time-resolved fluoroimmunoassay (TRFIA) described here is a 'sandwich type' immunoassay using two monoclonal antibodies recognizing different epitopes of insulin. One of the antibodies is fixed to a solid phase and the other is biotinylated. After sandwich formation the biotinylated antibody is detected by time-resolved fluorometry with the fluorophore europium which is coupled to streptavidin. Serum samples of 100 microliters can be analyzed within a concentration range of 4 microIU/ml up to 500 microIU/ml. The detection limit of this assay, 3.9 microIU/ml, is comparable to the conventional radioimmunoassay and the results overall correlate well with those of the radioimmunoassay (r = 0.99). There is no detectable crossreaction with human proinsulin even using a proinsulin concentration of 2000 microIU/ml. The assay is performed with the DELFIA system (Pharmacia).