title: Single-label time-resolved luminescence assay for estrogen receptor--ligand binding.
abstract: Homogeneous luminescence-based microplate assays are desirable in high-throughput screening of new nuclear receptor regulators. Time-resolved fluorescence resonance energy transfer (TR-FRET) assays provide high sensitivity due to low background signal. The TR-FRET concept requires labeling of both ligand and receptor, making the assay format and its development relatively expensive and complex compared with single-label methods. To overcome the limitations of the multilabel methods, we have developed a single-label method for estrogen receptor (ER)-ligand binding based on quenching resonance energy transfer (QRET), where estradiol labeled with luminescent europium(III) chelate (Eu-E(2)) is quenched using soluble quencher molecules. The luminescence signal of Eu-E(2) on binding to full-length ER is protected from quenching while increasing competitor concentrations displace Eu-E(2) from the receptor, reducing the signal. The QRET method was paralleled with a commercial fluorescence polarization (FP) assay. The measured signal-to-background (S/B) values for estradiol, estrone, fulvestrant, and tamoxifen obtained for the QRET assay (5.8-9.2) were clearly higher than the S/B values for the FP assay (1.3-1.5). A K(d) value of 30nM was calculated for binding of Eu-E(2) to ER from a saturation binding isotherm. The QRET method provides an attractive new single-label assay format for nuclear receptor ligand screening.
authors.affiliation: Laboratory of Biophysics, Department of Cell Biology and Anatomy and Medicity Research Laboratories, Institute of Biomedicine, University of Turku, 20520 Turku, Finland. roohuttu@utu.fi
authors.forename: Roope
authors.initials: R
authors.lastname: Huttunen
authors.lastname: Shweta
authors.forename: Eija
authors.initials: E
authors.lastname: Martikkala
authors.forename: Merja
authors.initials: M
authors.lastname: Lahdenranta
authors.forename: Pasi
authors.initials: P
authors.lastname: Virta
authors.forename: Pekka
authors.initials: P
authors.lastname: Hänninen
authors.forename: Harri
authors.initials: H
authors.lastname: Härmä
mesh_terms: D001681:Biological Assay; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000378:metabolism*; D005063:Europium / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D008024:Ligands; D049449:Luminescence; D011485:Protein Binding
chemical_list: C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D008024:Ligands; D005063:Europium; D004958:Estradiol