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. Microtiter
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 l plasma was
similar to that observed in buffer standards, all assays were conducted using 80 l of the
unknown plasma samples. A standard curve was obtained in the range of 25–12,800 pg
LH/well in 80 l 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 sensitivemethod for routine
analysis of goat plasma LH.
