Estimation of progesterone in buffalo milk by radioimmunoassay

The standardization of a radioimmunoassay (RIA) for the direct estimation of progesterone in buffalo milk using I-125-progesterone tracer prepared by radiolabeling the tyrosine methyl ester (TME) conjugated to progesterone and a specific antibody is described. Progesterone standards were prepared in progesterone free milk. Milk collected from buffaloes during their heat period was used as progesterone free milk. Various reaction conditions were optimized to arrive at a suitable assay system. The optimized assay system had a working range of 0.25 - 50 ng/ml and 50 mu l sample. The assay involved an incubation of progesterone antibody with standards or sample and the radiolabeled tracer for 2 hours at room temperature. The separation of bound and free fractions was achieved using a precipitating reagent consisting of second antibody and polyethylene glycol ( PEG). The sensitivity of the assay was 0.05 ng/ml. The intra-assay and inter-assay variations were < 10% and < 5%, respectively. The analytical recovery was 91 - 115% and the dilution test showed a recovery of 96 - 104%. Assay drift was observed to be < 12%. Levels of progesterone in milk samples during heat period and at various months of pregnancy as well as levels after artificial inseminations estimated by the developed assay correlated well with clinical diagnosis.