ABSTRACT
A microtitre plate enzyme immunoassay (EIA) for plasma estradiol is described, involving competition between sample estradiol and an immobilized estradiol-bovine serum albumin complex for a monoclonal anti-estradiol antibody, followed by immobilized antibody quantitation using enzyme-labelled antiglobulins. The assay dose-response curve covered a range of 6-1500 fmol/well. The intra- and inter-assay coefficient of variation for the assay of three plasma pools ranged from 3.1 to 4.7% and from 4.7 to 10.6% respectively. The assay showed satisfactory correlation with a standard estradiol radioimmunoassay. Pre-coated microtitre plates were stable, dried, at 4 degrees C for up to 3 months and the anti-estradiol was stable to lyophilization and also was stable in solution at 4 degrees C for up to 1 month.
Subject(s)
Antibodies, Monoclonal , Estradiol/blood , Cross Reactions , Female , Humans , Immunoenzyme Techniques , RadioimmunoassayABSTRACT
A microtitre plate enzyme immunoassay for estradiol, using a purified monoclonal antibody covalently bound to peroxidase and a small amount of immobilized immunogen, was optimized. Decreasing the antibody concentration to 2 X 10(-10) M (Kd/5) gave optimum estradiol detectability. The enzymatic signal was, however, very low in this assay. A 14-fold enhancement could be obtained using an avidin-biotin system in which several biotin molecules are conjugated with the antibody, providing multiple sites for binding by an avidin-enzyme complex. Further reagent concentration optimization gave an assay in which a range of 2 to 140 pg estradiol/well could be assayed simply and reproducibly.
