Analytical Sciences

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

Yuko MATSUMOTO,* Hideki KURAMITZ,** Shinji ITOH,*** and Shunitz TANAKA*

*Division of Material Science, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan
**Department of Environmental Biology and Chemistry, Faculty of Science, Toyama University, Gofuku, Toyama 930-8555, Japan
***Hokkaido College of Pharmacy, Otaru, Hokkaido 047-0264, Japan

A sensitive and simple immunoassay to determine 17 β-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.

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