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Journal of Biomolecular Screening, Vol. 10, No. 4, 348-354 (2005)
DOI: 10.1177/1087057104272191

Enzyme Inhibitor Screening Using a Homogeneous Proximity-Based Immunoassay for Estradiol

Leena Kokko

Department of Biotechnology, University of Turku, Turku, Finland

Nina Johansson

Hormos Medical Corp., Turku, Finland

Timo Lövgren

Tero Soukka

Department of Biotechnology, University of Turku, Turku, Finland

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 17ß-hydroxysteroid dehydrogenase type 1. The enzyme overexpressed in MCF-7 cells catalyzed a reversible conversion of estroneto17ß-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 Zß 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.

Key Words: homogeneous • high-throughput screening • europium(III) chelate nanoparticle • time-resolved fluorometry


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