Development and validation of a highly sensitive economic enzymeimmunoassay for prolactin determination in blood plasma of mithun (Bos frontalis) and its application during milk let down and cyclicity

The objective of the present study was to develop and validate highly sensitive and economic enzymeimmunoassay (EIA) for prolactin determination in mithun blood plasma on microtitreplates using the biotin–streptavidin amplification system and second antibody coating technique and to apply this procedure during milk let down and cyclicity in mithuns (Bos frontalis), a semi-wild ruminant. Biotin was coupled to prolactin and used to bridge between streptavidin peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 50 μl mithun plasma. The sensitivity of the EIA procedure was 0.1 ng/ml plasma. Plasma volumes viz., 12.5, 25 and 50 μl did not influence much the shape of standard curve though a slight drop in the OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous mithun plasma prolactin with bovine prolactin standards used in the assay. It showed good parallelism with the bovine standard curve. Plasma prolactin was estimated in six cyclic mithun cows during an estrous cycle. Mean plasma prolactin concentrations around the day of estrus were recorded to be higher than any other day of the cycle. Prolactin profiles were also obtained in three mithuns before, during and after milking. A sharp release of prolactin shortly after udder stimulation was observed. High levels of prolactin were maintained during milking, falling sharply thereafter. In conclusion, the EIA developed for prolactin determination in mithun blood plasma is sufficiently reliable, economic and sensitive enough to estimate prolactin in all physiological variation in mithun.