title: A novel monoclonal antibody-based enzyme-linked immunosorbent assay to determine luteinizing hormone in bovine plasma.
abstract: 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.
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi, Milano, Italy. Electronic address: vitaliano.borromeo@unimi.it.
authors.forename: V
authors.initials: V
authors.lastname: Borromeo
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi, Milano, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Berrini
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi, Milano, Italy.
authors.forename: F
authors.initials: F
authors.lastname: De Grandi
authors.affiliation: Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare, Università degli Studi, Milano, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Cremonesi
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi, Milano, Italy.
authors.forename: N
authors.initials: N
authors.lastname: Fiandanese
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi, Milano, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Pocar
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi, Milano, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Secchi
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001681:Biological Assay; D002417:Cattle / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary*; D005260:Female; D007986:Luteinizing Hormone / Q000097:blood* / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D007986:Luteinizing Hormone
keywords: Bovine; ELISA; Immunoaffinity chromatography; Luteinizing hormone; Monoclonal antibody