Immunoaffinity chromatography and a biotin-streptavidin amplified enzymeimmunoassay for sensitive and specific estimation of estradiol-17 beta

A sensitive test system has been developed for estimation of estradiol-17 beta (E2) in bovine plasma. Plasma extracts are first purified by a selective immunoaffinity chromatography (IAC) using an antibody raised against estradiol-6-carboxymethyloxime-bovine serum albumin and immobilized to Sepharose. The eluate was analysed by a competitive enzyme immunoassay (EIA) on microtitration plates. For the assay the wells of microtitration plates were coated with affinity purified sheep IgG (antirabbit IgG) that binds the hormone specific antibody raised in rabbits against estradiol-17-hemisuccinate-bovine serum albumin. E2 is estimated by displacement of biocytinyl-E2, that was produced by ligation of estradiol-17 beta, D-glucuronic acid and biocytin. Bound biocytinyl-E2 is detected after binding of streptavidin-peroxidase and colour production by the enzyme. A very high amplification was possible with this technique and the absolute detection limit amounted to approximately 120 fg/well at 94% relative binding. By combination of IAC and EIA the following levels of E2 were found in bovine plasma: male or female calves less than 2.7 pg/ml, cycling cow 0.5-7 pg/ml, cow during the last month of pregnancy 9-310 pg/ml, mature bull 5-30 pg/ml. However, up to 1110 pg E2/ml were found in plasma of a calf after treatment with an illicit hormone preparation used for growth promotion; after 21 days levels declined to 6 pg/ml which is hardly different from controls. In conclusion, the IAC/EIA can be used for sensitive estimation of estradiol-17 beta in plasma from all type of cattle and for control of improper use of E2 after commitment of a threshold level.