DETERMINATION OF LUTEINIZING HORMONE (LH) AND FOLLICLE STIMULATING HORMONE (FSH) IN BOVINE PLASMA: DEVELOPMENT AND VALIDATION OF SPECIES-SPECIFIC MONOCLONAL ANTIBODY-BASED ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISA).

Maximizing livestock reproductive potential is of primary importance in business farming, and it’s the reason for the increasing interest in the development of innovative bio-techniques addressed to the improvement of in vivo and in vitro farm animal fertility. A better knowledge of gonadotropin (i.e.: luteinizing hormone (LH) and follicle stimulating hormone (FSH)) plasma patterns can have a positive impact on animal reproductive efficiency, for example by helping detection of dysfunction of the pituitary-ovarian axis, diagnosis of reproductive disorders, monitoring of antifertility programs or superovulatory responses for embryo transfer. Gonadotropins in bovine plasma are currently measured only in a limited number of specialized laboratories. The first reason is that immunoassay for detecting bovine LH (bLH) and bovine FSH (bLH) are not easily accessible, being all in-house developed methods relying on custom reagents. Second, FSH and LH are species-specific (both amino acid and oligosaccharides differs among species), limiting the use for such assays to homologous reagents. Finally, hormone standards as well as anti-bFSH and anti-bLH antibodies are difficult to produce. The aim of the present study was therefore to develop and validate two novel species-specific immunoassay for measuring bLH or bFSH in bovine plasma, based on reagents (antibody and reference standard) produced in our laboratory. Specifically, the objectives were: (1) to produce a panel of anti-bLH and anti-bFSH mAbs by immunizing with standard hormones obtained from the USDA; (2) to select mAbs suitable for immuno-affinity chromatography purification of bLH and bFSH from pituitary glands; (3) to combine the mAbs and the purified gonadotropins to develop two enzyme-linked immunosorbent assay (ELISA) for bLH and bFSH detection. Several anti-bFSH and anti-bLH mAbs were produced and characterized. On the basis of their affinity and specificity we selected: two mAbs recognizing non-overlapping epitopes on the β-subunit of the bLH molecule; one mAbs binding to an epitope localized on the β-subunit of the bFSH molecule; and one mAb binding to the pituitary glycoprotein common α-subunit. An anti-bLH β-subunit and an anti-bFSH β-subunit mAb were used to develop immunoaffinity chromatography protocols for the one-step extraction of bLH and bFSH from pituitary glands. The methods resulted efficient, and ensure the supply of substantial amounts of highly purified biologically active bLH and bFSH. The purity of the hormones was assessed by SDS-electrophoresis, isoelectric focusing, western blotting, amino acid composition, amino acid sequence, nanoLC-ESI-MS/MS. The biological activity was measured by in vitro specific bioassays based on cell lines expressing LH and FSH receptors. The purified gonadotropins and the anti-bFSH and anti-bLH mAbs were combined to develop two ELISAs to detect plasma bLH and bFSH in cattle. These sandwich ELISAs with biotin-avidin amplification showed a good sensitivity, ranging from 0.05 to 2.5 ng/ml for bLH and from 0.25 to 10 ng/ml for bFSH. Cross-reactivity, recovery and reproducibility tests confirmed the accuracy and precision of the assays to measure gonadotropins in bovine plasma without any prior treatment of samples. The analytical specificity was finally validated in vivo by measuring plasma bLH and bFSH patterns after provocative test with gonadotropin releasing hormone (GnRH) in heifers. In conclusion, the ELISA developed satisfy all the criteria required to investigate LH and FSH secretory patterns in the bovine species. The fact that both assays are based on mAbs, together with the easiness of the one-step production of reference bLH and bFSH ensure long-term continuity in large-scale measurements of LH and FSH. These methods allow the rapid, inexpensive and quantitative measurement of the hormone and are therefore valuable tools to further our knowledge on the reproductive physiology of the bovine species.