A Sensitive Microtitre Plate Enzyme Immunoassay of Oestradiol-17β in the Cow and Mare

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Abstract
Microtitre plates were coated with antiserum against oest-radiol-17β-6-(0-carboxymethyl)-oxime bovine serum albumin raised in sheep. The plasma samples (0.2–1.0 ml) were extracted with peroxide-free dlethyl ether prepared daily by treatment with Al2O3. The enzyme conjugate was prepared by coupling oestradiol-17β-6-(0-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-17β. The amount of oestradiol-17β causing a 50% reduction of maximum binding was 4.4 fmol/well. Standards and samples were Incubated overnight at 4°C. The conjugate solution was added followed by further incubation for 2 h at 4°C. Tetramethylbenzidine was used as a chromogen, and the optical density was measured at 450 nm. The patterns of oestradiol-17β during a normal oestrus cycle in the cow and mare are presented.