Development and validation of a simple, sensitive, second antibody format enzyme immunoassay for LH determination in...

The objective of this study was to develop and validate a direct simple and highly sensitive enzyme immunoassay (EIA) for luteinizing hormone (LH) determination in buffalo plasma on microtiter plates using the biotin-streptavidin amplification system and the second antibody coating. Biotin was coupled to LH and used to bridge between streptavidin-peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 20 microl buffalo plasma. The LH standards ranging from 6.25 to 200 pg/well/20 microl were prepared in hormone free plasma collected from a buffalo on day 4 post-calving. The sensitivity of EIA procedure was 6.25 pg/well LH, which corresponds to 0.31 ng/ml plasma; the 50% relative binding sensitivity was seen at 50 pg/well/20 microl. Plasma volumes for the EIA, viz. 10 and 20 microl, did not influence the shape of standard curve even though a slight drop in the OD450 was seen with higher plasma volumes. A parallelism was carried out to compare the endogenous buffalo plasma LH with bovine LH standards. For the biological validation of assay, 10 Murrah buffaloes were used. These were administered (10 microg im) with a synthetic analogue of gonadotropin-releasing hormone (GnRH) and blood samples were collected at 15-min interval using indwelling jugular catheter beginning just prior to GnRH injection till 6 h and thereafter 2-h interval for another 18 h. In all animals, sharp increases in LH concentrations were recorded post-GnRH administration, which confirms the biological validation of the EIA. To record the LH peak during periestrus in a cycling buffalo, the blood samples were collected at 2-h intervals from onset of behavioral estrus signs till ovulation. The LH peak was observed after the initial behavioural estrus signs followed by the gradual decline in the levels towards the ovulation.