Development and Validation of a Simple, Sensitive, Second Antibody Format Enzyme Immunoassay (EIA) for LH Determination in Mithun (Bos Frontalis) Plasma

Abstract

The objective of this study was to develop and validate a simple and highly sensitive enzyme immunoassay (EIA) for LH determination in mithun plasma on microtitreplates using the biotin‐streptavidin amplification system and the second antibody coating technique. 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 µL mithun plasma. The LH standards ranging from 6.25 pg/well/20 µL to 400 pg/well/20 µL were prepared in hormone free plasma collected from a mithun on day 3 post calving. The sensitivity of EIA procedure was 6.25 pg/well LH, which corresponds to 0.31 ng/mL plasma; the 50 percent relative binding sensitivity was seen at 100 pg/well/20 µL. Plasma volumes for the EIA viz. 10 and 20 µL did not influence the shape of standard curve even 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 LH with bovine LH standards. It showed good parallelism with the bovine standard curve. For the biological validation of the assay, 3 mithuns were used. These were administered 10 µg i.v., with a synthetic analogue of GnRH (Buserelin‐Acetate, Intervet, India) and blood samples were collected at 15 min intervals using indwelling jugular catheter beginning 1 h prior to GnRH injection till 8 h post injection. In all animals, sharp increases in LH concentrations were recorded post GnRH administration, which confirms the biological validation of the EIA. In conclusion, the EIA developed for LH determination in mithun blood plasma is sufficiently reliable, economical, and sensitive enough to estimate LH in all physiological variations in mithun.

Keywords:

• Mithun endocrinology
• LH
• Plasma
• EIA
• Semi‐wild
• Validation

Acknowledgments

The authors wish to thank USDA Animal Hormone Program, Beltsville, MD, USA, for supplying a highly purified reference preparation of bovine LH and bovine LH antiserum. The authors also wish to thank the Director, National Dairy Research Institute, Karnal‐132 001 (Haryana), India for providing part of facilities for the present work.