Covert cross reactants in a two-site immunoassay studied with monoclonal antibodies

A one-step two-site immunoradiometric assay for the measurement of free beta subunit of human chorionic gonadotrophin (beta-hCG) was developed using monoclonal antibodies. The immobilized antibody was specific for free beta subunit and the radiolabeled antibody recognized both intact human chorionic gonadotrophin (hCG) and free beta subunit. Although the level of hCG "cross-reaction" was low when studied using conventional techniques, the apparent beta-hCG content of samples was found to be inversely proportional to the hCG level. From both experimental evidence and computer simulation studies this was found to be due to the binding of hCG to the limited amount of 125I-labeled antibody present. The term covert cross reactants has been introduced to describe substances which bind to only one of the antibodies in a two-site immunoassay. When establishing such an assay the effect of covert cross reactants on the response of an analyte should be investigated.