Single-label time-resolved luminescence assay for estrogen receptor--ligand binding

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.