Application of sensitive enzymeimmunoassay for determination of testosterone in blood plasma of yaks (Poephagus grunniens L.)

As an alternative to radioimmunoassays, a simple, highly sensitive and quick enzymeimmunoassay (EIA) for determination of testosterone in blood plasma of yaks on microtitreplates using second antibody coating technique and testosterone–horseradish peroxidase as a label has been developed for the first time. The wells of the microtitreplate were coated with affinity-purified goat immunoglobulin (antirabbit IgG) that binds the hormone specific antibody. The EIA was carried out directly in 20 μl of plasma after 1:10 dilution with assay buffer. The testosterone standard curve ranged from 0.2 to 200 pg/well. The sensitivity of the assay was 0.20 pg/well. Testosterone standard curve in buffer showed parallelism with serially diluted yak plasma containing high endogenous testosterone. Intra- and inter-assay coefficients of variation (CV) determined using pooled plasma was found 5.24 and 8.54%, respectively.Recovery of known concentrations of added testosterone in charcoal stripped plasma was linear (r = 0.98). For biological validation of testosterone enzymeimmunoassay, the blood samples were collected from yak cows at −2 h before and thereafter at 2 h interval until 24 h. after gonadotropin releasing hormone (GnRH) administration. There was a rapid increase (p < 0.01) of luteinizing hormone (LH) and testosterone 2 and 6 h after GnRH administration. Plasma testosterone concentration in normal adult yak bulls was found to be 0.52 ± 0.09 ng/ml. In conclusion, the EIA developed in this study is simple, highly sensitive, valid and sufficiently reliable method for estimation of testosterone directly in yak plasma.