Development of a sensitive, direct luminescent enzyme immunoassay for plasma estradiol-17β

A rapid, sensitive, precise, chemiluminescent enzyme immunoassay for estradiol-17β has been developed and validated. Antibodies were produced in rabbits using estradiol-17β-6-(O-carboxymethyl)oxime coupled to bovine serum albumin, purified and immobilized on polystyrene beads (6.4 mm diameter). The same derivative was used to prepare the enzymatic tracer by coupling with horseradish peroxidase. The assay, direct on the serum sample, featured a 4-h binding step at 4°C followed by the chemiluminescent detection using luminol/H₂O₂. The detection limit was 0.15 pg/tube and the assay was carried out on 20-100 μl of sample, allowing measurement of estradiol-17β in plasma concentrations from 1.5 to 500 pg/ml. The method fulfills all the standard requisites of precision and accuracy and the results agree well with a radioimmunoassay procedure on extracted serum.