期刊
JOURNAL OF BIOMOLECULAR SCREENING
卷 10, 期 4, 页码 348-354出版社
SAGE PUBLICATIONS INC
DOI: 10.1177/1087057104272191
关键词
homogeneous; high-throughput screening; europium(III) chelate nanoparticle; time-resolved fluorometry
The authors have previously reported a homogeneous time-resolved fluorescence proximity immunoassay for estradiol. The assay was based on luminescence resonance energy transfer between a long lifetime fluorescent europium(III) chelate-dyed nanoparticle donor and a short lifetime, near- infrared fluorescent acceptor. The energy transfer prolonged the lifetime of the sensitized acceptor emission, and the fluorescence of the acceptor was measured using a time-resolved detection. The developed immunoassay was employed to screen inhibitors for enzyme 17 beta-hydroxysteroid dehydrogenase type 1. The enzyme overexpressed in MCF-7 cells catalyzed a reversible conversion of estrone to 17 beta-estradiol. The inhibition efficiency of the tested molecule was obtained by comparing the final concentration of converted estradiol after 60 min of conversion reaction in a sample and in a conversion control not containing an inhibitor. The Z beta factor calculated using the E2 concentrations of the homogeneous assay was 0.64, demonstrating a relatively good performance of the assay. The results from the homogeneous assay were comparable with the results obtained using radioactively labeled estrone as a substrate and high-performance liquid chromatography (HPLC) separation of estrone and converted estradiol after the enzyme reaction. Thus, this homogeneous assay can simplify the primary screening of potential new drug molecules by replacing a tedious radiometric HPLC method.
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