Direct Enzyme Immunoassay of Estradiol in Serum of Women Enrolled in an in Vitro Fertilization and Embryo Transfer Program

We present a fast and simple direct enzyme immunoassay for the sexual steroid hormone estradiol-17 beta in serum of women enrolled in an in vitro fertilization and embryo transfer program. We added 25 microL of standards or samples and 125 microL of a mixture of monoclonal antibody and displacing agents to the wells of a microtiter plate previously coated with a second antibody. After 30 min 50 microL estradiol-horseradish peroxidase conjugate is added. Total incubation time is 75 min. The immunoassay is stopped by washing the microtiter plate. The amount of bound conjugate is then measured at 450 nm with hydrogen peroxide as a substrate and 3,3',5,5'-tetramethylbenzidine as chromogen. The detection limit is 0.24 nmol/L. The assay is thus limited in its application. Analytical recovery is 90.8% (range 80.4-102.9%); within- and between-assay CVs range between 1.3% and 15.4%. Total assay time, including preparation of reagents and data reduction, is only 2.5 h. Results of this enzyme immunoassay compare well with those obtained with four different commercial radioimmunoassays (r = 0.88-0.93).