A novel monoclonal antibody-based enzyme-linked immunosorbent assay to determine luteinizing hormone in bovine plasma

• Published in: Domestic animal endocrinology Vol. 48; pp. 145 - 157
• Main Authors: Borromeo, V., Berrini, A., De Grandi, F., Cremonesi, F., Fiandanese, N., Pocar, P., Secchi, C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2014
• Subjects: Animals; Antibodies, Monoclonal - immunology; Biological Assay; Bovine; Cattle - blood; ELISA; Enzyme-Linked Immunosorbent Assay - veterinary; Female; Immunoaffinity chromatography; Luteinizing hormone; Luteinizing Hormone - blood; Luteinizing Hormone - immunology; Monoclonal antibody; Reproducibility of Results; Sensitivity and Specificity; Luteinizing hormone; Monoclonal antibody; Bovine; Immunoaffinity chromatography; ELISA
• ISSN: 0739-7240; 1879-0054
• DOI: 10.1016/j.domaniend.2014.03.004

The development of a novel enzyme-linked immunosorbent assay (ELISA) for determining luteinizing hormone (LH) in bovine plasma is described. Anti-bovine LH (bLH) monoclonal antibodies (mAbs) were produced and characterized. One mAb recognizing the bLH β subunit was used for immunoaffinity purification of substantial amounts of biologically active bLH from pituitary glands. The purified bLH in combination with 2 anti-bLH β subunit mAbs was used to develop a sandwich ELISA, which satisfied all the criteria required to investigate LH secretory patterns in the bovine species. The ELISA standard curve was linear over the range 0.05 to 2.5 ng/mL, and the assay proved suitable for measuring bLH in plasma without any prior treatment of samples. Cross-reactivity and recovery tests confirmed the specificity of the method. The intra- and inter-assay coefficients of variation ranged between 3.41% and 9.40%, and 9.29% and 15.84%, respectively. The analytical specificity of the method was validated in vivo by provocative tests for LH in heifers, using the LH releasing peptide gonadotropin-releasing hormone. In conclusion, the adoption of mAbs for this ELISA for coating the wells and labeling, combined with the easy one-step production of reference bLH, ensures long-term continuity in large-scale measurements of LH in the bovine species.