Validation of a simple, sensitive enzyme immunoassay (EIA) for the determination of caprine plasma LH

Abstract

The study describes the development and validation of a simple and highly sensitive enzyme immunoassay (EIA) for the determination of goat plasma LH, utilizing the biotin–streptavidin peroxidase amplification system in a competitive-binding assay. Micro-titer plates were coated with goat anti-rabbit globulin as the second antibody and biotin was coupled to oLH and used as a bridge between streptavidin peroxidase and immobilized oLH beta antisera. A simple 4-step procedure was used at room temperature for the sample analysis: (1) overnight incubation of LH standards and plasma samples with the LH antibody in a 96-well micro-titer plate, pre-coated with a second antibody; (2) incubation in a biotinylated–LH conjugate for 30 min; (3) incubation with streptavidin peroxidase for a further 30 min and (4) lastly incubation in tetramethyl benzidine substrate for 40 min to develop colour. A two-dimensional titer determination test proved the antibody titer of 1:100,00,000 and the biotinylated–LH conjugate titer of 1:4000 to be the most suitable. As the absolute binding sensitivity of different concentrations of oLH in 80 μl plasma was similar to that observed in buffer standards, all assays were conducted using 80 μl of the unknown plasma samples. A standard curve was obtained in the range of 25–12,800 pg LH/well in 80 μl hormone-free plasma. The sensitivity of the EIA procedure was 25 pg/well LH, which corresponds to 0.31 ng/ml plasma. In a parallelism test, the relative percentage binding curve for serially diluted goat plasma samples containing high levels of endogenous LH and for the oLH standard ran parallel to each other, thereby confirming the actual LH estimation in goat plasma. The classical patterns of plasma LH during the estrous cycle and the gradual increases in LH concentrations after gonadotrophin releasing hormone (GnRH) administration provided biological validation of the assay for the determination of plasma LH in caprine species. This EIA technique provides a simple and sensitive method for routine analysis of goat plasma LH.