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.