A Double-antibody Enzyme Immunoassay for Determination of Gonadotropin-releasing Hormone in Bovine Plasma

Abstract

A double-antibody enzyme immunoassay (EIA) for the determining GnRH in bovine plasma was studied. Antiserum raised against [Glu¹]-GnRH-rabbit serum albumin conjugate in a male horse was used. Enzyme-labelled antigens were prepared by coupling synthetic GnRH with horseradish peroxidase using bis-diazotized benzidine. GnRH analogues and fragment of GnRH were tested for cross-reactivity in the EIA system. These results suggested that the C-terminus of GnRH molecule plays more important role in antigen-antibody binding than the N-terminus of it.
Plasma specimens were extracted with acid-methanol. Process standard curve was suitable for plasma assay. The sensitivity of this EIA system was as little as 50 pg/tube of GnRH. After intravenous injection of 200 μg of synthetic GnRH in 2 steers, plasma concentrations of GnRH were determined in this EIA system. The levels abruptly increased just after injection and decreased thereafter.