Quantitative Analysis of 17β-Estradiol in River Water by Fluorometric Enzyme Immunoassay Using Biotinylated Estradiol

Abstract

A sensitive and simple immunoassay to determine 17beta-estradiol (E2) in fresh water was developed. The method is based on a solid-phase avidin-biotin binding assay and solid phase extraction. The binding event of E2 to the antibody is detected indirectly by the competitive reaction between E2 and biotinylated estradiol (BE) as a tracer for the limited binding sites of antibodies immobilized onto the wall of a microtiter plate. Namely, E2 concentrations are determined from the strong interaction between BE and avidin conjugated with horseradish peroxidase (avidin-HRP). In order to achieve a sensitive measurement for the binding of BE to the antibody immobilized on the microtiter plate substrate, QuantaBlu fluorogenic peroxidase substrate (QFPS) was employed. The detection limit and the linear range of E2 determination were 27 pM and 27 - 7480 pM, respectively. The relative standard deviations (RSD) for the E2 assay were between 0.3 and 12.0% (n = 3). The cross-reactivities of several other estrogens in this assay system were also investigated. No serious influences from any cross-reaction caused by other estrogens tested in this experiment were observed. The determination of E2 in water samples from eight rivers and a marsh in Hokkaido was performed by the immunoassay combined with solid phase extraction. It was found that the concentration of E2 was in the range between 0.06 and 67 pM.