title: Enzyme inhibitor screening using a homogeneous proximity-based immunoassay for estradiol.
abstract: 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 17beta-hydroxysteroid dehydrogenase type 1. The enzyme overexpressed in MCF-7 cells catalyzed a reversible conversion of estroneto17beta-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 Zbeta 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.
authors.affiliation: Department of Biotechnology, University of Turku, Turku, Finland. leena.kokko@utu.fi
authors.forename: Leena
authors.initials: L
authors.lastname: Kokko
authors.forename: Nina
authors.initials: N
authors.lastname: Johansson
authors.forename: Timo
authors.initials: T
authors.lastname: Lövgren
authors.forename: Tero
authors.initials: T
authors.lastname: Soukka
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004958:Estradiol / Q000032:analysis*; D005240:Feasibility Studies; D006801:Humans; D007118:Immunoassay / Q000379:methods*
chemical_list: D004791:Enzyme Inhibitors; D004958:Estradiol; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase