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 2.5ng/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 invivo 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.
A novel monoclonal antibody-based enzyme-linked immunosorbent assay to determine luteinizing hormone in bovine plasma / V. Borromeo, A. Berrini, F. De Grandi, F. Cremonesi, N. Fiandanese, P. Pocar, C. Secchi. - In: DOMESTIC ANIMAL ENDOCRINOLOGY. - ISSN 0739-7240. - 48:1(2014 Jul), pp. 145-157.
|Titolo:||A novel monoclonal antibody-based enzyme-linked immunosorbent assay to determine luteinizing hormone in bovine plasma|
BORROMEO, VITALIANO (Primo)
BERRINI, ANNA (Secondo)
POCAR, PAOLA (Penultimo)
SECCHI, CAMILLO LUIGI (Ultimo)
|Parole Chiave:||Bovine; ELISA; Immunoaffinity chromatography; Luteinizing hormone; Monoclonal antibody|
|Settore Scientifico Disciplinare:||Settore BIO/12 - Biochimica Clinica e Biologia Molecolare Clinica|
Settore VET/01 - Anatomia degli Animali Domestici
Settore VET/10 - Clinica Ostetrica e Ginecologia Veterinaria
|Data di pubblicazione:||lug-2014|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1016/j.domaniend.2014.03.004|
|Appare nelle tipologie:||01 - Articolo su periodico|