Use of free radical chemistry in an immunometric assay for 17 beta-estradiol

Background: We wished to develop an enzyme immunometric assay for 17 beta - estradiol (E2) in human serum using solid-phase immobilized epitope immunoa ssay (SPIE-IA) technology and free radical chemistry. Methods: We used an anti-estradiol monoclonal antibody as capture antibody and Fenton-like reagents to cross-link it to E2. The same antibody, labeled with acetylcholinesterase, was used for detection. Serum was diluted 10-fo ld before assay. Results: After correction by the dilution factor, the detection limit was 5 ng/L for human serum and intra-and interassay CVs were <7% and 15%, respec tively, at concentrations of 169-2845 ng/L. No cross-reactivity was seen wi th other natural steroids. In comparison with a competitive commercial RIA performed on 88 undiluted human sera, the slope (SD) of the regression line was 1.05 (+/- 0.02) and the intercept was 47 (+/-27) ng/L (S-y/x 186 ng/L) at concentrations of 20-5000 ng/L (r(2) = 0.97). Conclusions: The use of Fenton-like chemistry in SPIE-IA technology allows a sensitive measurement of E2 in human serum and could be a new approach fo r the development of sensitive immunoassays. (C) 2001 American Association for Clinical Chemistry.