Use of Free Radical Chemistry in an Immunometric Assay for 17β-Estradiol

Background: We wished to develop an enzyme immunometric assay for 17β-estradiol (E2) in human serum using solid-phase immobilized epitope immunoassay (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-fold 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%, respectively, at concentrations of 169-2845 ng/L. No cross-reactivity was seen with 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 for the development of sensitive immunoassays.