Screening for testosterone, methyltestosterone, 19-nortestosterone residues and their metabolites in bovine urine with enzyme-linked immunosorbent assay (ELISA)

This work reports on the development of rapid enzyme-linked immunosorbent assay for testosterone, methyltestosterone, 19-nortestosterone and their metabolites in real samples of bovine urine. The assays were based on the competition between an immobilised testosterone-BSA conjugate and the analyte for corresponding antibodies, followed by the use of secondary anti-species (α-IgG-HRP) to determine the degree of competition. Urine samples were analysed after 10-fold dilution with the buffer, omitting extraction and hydrolysis. The limits of detection calculated for the original sample were ca. 74, 266 and 131 pg ml⁻¹ (ppt) for testosterone, methyltestosterone and 19-nortestosterone, respectively. In particular, testosterone and methyltestosterone assays offer the advantage to pick up both parent compounds and their major metabolites due to the high cross-reactivity pattern with corresponding antibody used in each assay. The concentration in bovine urine detected by developed method does indicate 19-nortestosterone administration to heifers. The developed assays were applied to urine samples of heifers treated with above androgens.