Development of a Simple Enzyme Immunoassay for the Determination of Ovine Luteinizing Hormone

Abstract

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.

Desenvolvimento de imunoensaios e biossensores para determinação de LDL eletronegativa

“…Considerando que a LDL(-) está presente na circulação sanguínea e que alguns estudos comprovaram sua aterogenicidade, a quantificação de LDL(-) e de seus anticorpos no plasma pode fornecer informações adicionais no estudo do processo aterosclerótico e doenças cardiovasculares. Há alguns imunoensaios descritos na literatura e disponíveis comercialmente para uso em pesquisa para determinação de LDL oxidada e/ou LDL-MDA (Holvoet et al, 2006), fosfatidilcolina oxidada (Itabe et al, 1996;Fraley et al, 2006) (Magliulo et al, 2005;Eidem et al, 2006;Sanchez et al, 2007;Valares et al, 2007). É importante esclarecer que guias são documentos que sugerem uma linha a ser seguida e são, portanto, abertos para interpretação.…”