A sensitive microtitre plate enzyme immunoassay of oestradiol-17 beta in the cow and mare.

Microtitre plates were coated with antiserum against oestradiol-17 beta-6-(O-carboxymethyl)-oxime bovine serum albumin raised in sheep. The plasma samples (0.2-1.0 ml) were extracted with peroxide-free diethyl ether prepared daily by treatment with Al2O3. The enzyme conjugate was prepared by coupling oestradiol-17 beta-6-(O-carboxymethyl)-oxime to horse-radish peroxidase. The conjugate was chromatographed on a Sephadex G-25 column. The standard curve ranged from 0.37 to 18.40 fmol/well of oestradiol-17 beta. The amount of oestradiol-17 beta causing a 50% reduction of maximum binding was 4.4 fmol/well. Standards and samples were incubated overnight at 4 degrees C. The conjugate solution was added followed by further incubation for 2 h at 4 degrees C. Tetramethylbenzidine was used as a chromogen, and the optical density was measured at 450 nm. The patterns of oestradiol-17 beta during a normal oestrus cycle in the cow and mare are presented.