Development and Validation of a Sensitive Enzymeimmunoassay for Determination of Plasma Metastin in Mithun (Bos frontalis)

Abstract

Metastin, also known as kisspeptin-10, is a potent stimulator of gonadotropin-releasing hormone (GnRH) neurons in the central nervous system. Recently, it has been emerged as a key player in the regulation of reproduction in mammals. Blood concentrations of metastin during different physiological stages in bovine species in general and mithun (Bos frontalis) in particular are not available. Lacking of such information may probably be due to non-availability of simple assay procedure to measure the peptide. Therefore, the objective of this study was to develop and validate a simple and sufficiently sensitive enzyme immunoassay (EIA) for metastin determination in mithun plasma using the biotin-streptavidin amplification system and second antibody coating technique. Biotin was coupled to metastin and used to bridge between streptavidin-peroxidase and the immobilized metastin antiserum in the competitive assay. The EIA was conducted directly in 150 μL of unknown mithun plasma. Metastin standards ranging from 0.01–51.2 ng/150 μL/well were prepared in hormone-free plasma. The lowest detection limit was 0.07 ng/mL plasma. Plasma volumes for the EIA, viz., 75, 150, and 200 μL did not influence the shape of standard curve even though a drop in OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous mithun metastin with metastin standard used. It showed good parallelism with the metastin standard curve. For the biological validation of the assay, metastin was measured in (a) blood samples collected from 12 pregnant mithun cows during different stages of pregnancy, (b) in blood from seven early pregnant and 12 non-pregnant mithuns, and (c) in follicular fluid obtained from different types of follicle. It was found that the plasma metastin concentrations increased (P < 0.001) from first through last trimester of pregnancy. Plasma metastin levels were much higher (P < 0.001) in early pregnant than non-pregnant cows. Follicular fluid metastin concentrations were found to increase (P < 0.001) as the follicles grow and the highest levels were recorded in preovulatory follicles. In conclusion, a simple, sufficiently sensitive and direct EIA procedure has been developed for the first time to determine metastin levels in mithun. A wide range of metastin concentrations can be detected during different physiological stages in mithun using this metastin-EIA procedure.