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

Abstract

As an alternative to radioimmunoassays, a simple, highly sensitive and quick enzymeimmunoassay (EIA) for determination of cortisol in blood plasma of yaks on microtiterplates using second antibody coating technique and cortisol-horseradish peroxidase as a label has been developed. The wells of the microtiterplate were coated with affinity-purified goat IgG (antirabbit IgG) that binds the hormone specific antibody. The EIA was carried out directly in 20 microl of heat treated plasma after 1:5 dilution with PBS. The cortisol standard curve, with doses ranged from 0.4 to 100 pg/well. The sensitivity of the assay was 20 pg/ml. Cortisol standard curve in buffer showed parallelism with serially diluted yak plasma containing high endogenous cortisol. Intra- and inter-assay coefficients of variation (CV) determined using pooled plasma was found 6.58 and 11.35%, respectively. Recovery of known concentrations of added cortisol in charcoal stripped plasma was linear (r = 0.98). For biological validation of cortisol enzymeimmunoassay, the blood samples were collected from yak cows at -48 and -24h before and 0, 12, 24, 36, 48, 60, 72, 84 and 96 h after dexamethasone administration. The plasma cortisol before dexamethasone administration was significantly (P < 0.05) higher than after dexamethasone administration. The developed EIA was further validated biologically by estimating cortisol in peri-parturient cows beginning day 10 prior to calving till day 10 post-calving; the concentrations were along with the expected lines as reported in bovine. In conclusion, the EIA developed in this study is simple, highly sensitive, valid and sufficiently reliable method for estimation of cortisol directly in bovine plasma.