Development of a simple enzyme immunoassay for the determination of ovine luteinizing hormone.
The present study describes the development and validation of a simple sensitive and specific sandwich enzyme immunoassay (EIA) for the quantification of ovine luteinizing hormone (LH) in plasma. Microtitre plates were coated with the capture antibody 518b7 anti-bovine LH. A second peroxidase-labelled anti-ovine LH antibody was used as tracer. A simple 3-step procedure was used for the sample analysis; (1) incubation of standards and samples with the pre-coated antibody plates for 2 h at 37 degrees C; (2) incubation with the peroxidase-labelled antibody for 1 h at room temperature; and (3) colour development with TMB substrate. A linear dose-response curve was obtained in the range 0-10 ng/ml (r2 > 0.99). The detection limit was 0.05 ng/ml, and the intra-assay and inter-assay coefficients of variation were 7% and 11.7%, respectively. The theoretical stability of microplates and reagents was calculated, this being greater than one year. Low or undetectable cross-reactivities were recorded for follicle-stimulating hormone, bovine thyroid-stimulating hormone, equine chorionic gonadotrophin and a gonadotrophin-releasing hormone (GnRH) analogue. The EIA was biologically validated by the determination of plasma LH concentrations of nine Rasa Aragonesa ovariectomized and estradiol-implanted ewes after a double GnRH challenge. In conclusion, this enzyme immunoassay provides an efficient, simple and sensitive method for the routine analysis of ovine LH.
Valares
J A
JA
Departamento de Producción Animal y Ciencia de los Alimentos, Facultad de Veterinaria de Zaragoza, Zaragoza, Spain.
Abecia
J A
JA
Forcada
F
F
Palacín
I
I
Mata
L
L
Razquin
P
P
D000818:Animals; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D007124:Immunoenzyme Techniques / Q000662:veterinary*; D007986:Luteinizing Hormone / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012756:Sheep / Q000097:blood*; D013997:Time Factors
D007987:Gonadotropin-Releasing Hormone; D007986:Luteinizing Hormone