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FP5

ACE Résumé de rapport

Project ID: EVK1-CT-2001-00100
Financé au titre de: FP5-EESD
Pays: Netherlands

A rapid microplate-based assay for measuring estrogen receptor binding of individual chemicals and mixtures of chemicals

A fast assay for binding of compounds to the estrogen receptor alpha in 96-well plate format was developed, based on the competition between fluorescent coumestrol and estrogenic compounds. Displacement of coumestrol was measured as a decrease in fluorescence intensity using a microplate reader. Competitive binding curves for the well-known estrogenic compounds 17a-estradiol (E2), ethinylestradiol, 4-nonylphenol, 4-octylphenol, genistein, bisphenol A, tamoxifen and diethylstilbestrol and 9 other estrogenic compounds were constructed by using 7-10 different concentrations of the compounds and a fixed concentration of ER-a-LBD (14nmol) and coumestrol (100nmol). IC50 values and relative potencies (compared to E2) of the estrogenic compounds were determined. The assay was validated by comparing the relative potencies to those from standard radioligand binding assays in the literature. Low CV values were obtained from both negative and positive controls analyzed both in an experiment day (3% and 12% resp.) and in different experiment days (5% and 7% resp.). Z' values of 0.73 and 0.66 obtained from within day and between day experiments, respectively, indicate the high quality of the present assay. The present fluorescent binding assay has proven to be fast and easy, and allows accurately quantifying the binding affinity of estrogenic ligands. The method is also suitable as a high-throughput screening assay for ER ligands in pharmaceutical as well as environmental research.

Informations connexes

Reported by

University of Leiden, Pharmacogenomics Unit, LACDR
P.O. Box 9502
2300RA Leiden
Netherlands
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