Development of a sensitive enzyme immunoassay for LH determination in bovine plasma using the streptavidin-biotin technique.

Abstract

Using the biotin-streptavidin amplification technique, highly sensitive specific second-antibody enzyme immunoassays for determining LH in bovine plasma with long (48 h) and short (4 h) incubation periods were developed. Biotin was linked to bLH by the N-hydroxysuccinimide method and the product (biotinyl-bLH) used to bridge between streptavidin-peroxidase and the immobilised bLH antibody in competitive tests. The assays were validated and their performance compared with a radioimmunoassay currently in use. The sensitivities of the long and short incubation enzyme immunoassays (8 pg and 15 pg/well, respectively) were superior to that of 5-day incubation radioimmunoassay (100 pg/tube). Plasma interference in both assays were acceptable and volumes of 5 to 40 microliters gave parallel standard curves and comparable LH levels, 10-20 microliters plasma was sufficient to measure LH baseline levels by the long incubation enzyme immunoassay. The mean recovery of added standard bLH to plasma samples containing different endogenous LH was greater than 90% (range 91.7-112) in both assays. The intra- and inter-assay variations of both assays were less than 10 and 17%, respectively. When both enzyme immunoassay and radioimmunoassay were used to measure LH in cyclic cows, the basal levels measured by enzyme immunoassay were lower than that measured by radioimmunoassay. Enzyme immunoassay offers an attractive alternative to the lengthy radioimmunoassay in current usage, with an added advantage of employing non-isotopic label.