Development of an enzyme immunoassay for the determination of testosterone

Abstract

In this study a double antibody enzyme immunoassay for the direct determination of testosterone in plasma was developed. Testosterone 3-O-CMO was conjugated with horseradish peroxidase by the mixed anhydride method and the conjugate purified by column chromatography (sephadex G-25). Testosterone antibody was obtained by immunization of rabbits against Testosterone-3-O-CMO-BSA. Cross reactions of the antiserum against some steroids were found to be <0.01%. The detection limit of the assay was 2.5 pg/well and the working range of the standard curve was 0-20 ng/ml (0-200 pg/well). The recovery was found to be 98.8% after the addition of known amounts of testosterone to plasma samples. The inter-assay coefficient of variation was 12.6%. The described EIA offers a very economical alternative for the routine estimation of testosterone levels. Only minimal laboratory equipment is required and therefore the assay should be especially useful for research in animal science.