title: High throughput fluorescence polarization: a homogeneous alternative to radioligand binding for cell surface receptors.
abstract: High throughput fluorescence polarization (FP) assays are described that offer a nonradioactive, homogeneous, and low-cost alternative to radioligand binding assays for cell surface receptors (G protein-coupled receptors and ligand-gated ion channels). FP assays were shown to work across a range of both peptide (vasopressin V1a and delta-opioid) and nonpeptide (beta1-adrenoceptor, 5-hydroxytryptamine3) receptors. Structure-activity relationships were investigated at beta1-receptors and were found to be consistent with radioligand binding assays. FP was shown to tolerate up to 5% DMSO with no loss in sensitivity or signal window. From a random set of 1,280 compounds, 1.9% were found to significantly interfere with FP measurement. If fluorescent or quenching compounds were eliminated (3% of all compounds), less than 0.4% of compounds were found to interfere with FP measurement. Assays could be run in 384-well plates with little loss of signal window or sensitivity compared to 96-well plate assays. New advances in FP measurement have therefore enabled FP to offer a high throughput alternative to radioligand binding for cell surface receptors.
authors.affiliation: Receptor & Enzyme Screening Technologies, Glaxo Wellcome Medicines Research Centre, Stevenage, Herts, UK. ma16490@GlaxoWellcome.co.uk
authors.forename: M
authors.initials: M
authors.lastname: Allen
authors.forename: J
authors.initials: J
authors.lastname: Reeves
authors.forename: G
authors.initials: G
authors.lastname: Mellor
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D006224:Cricetinae; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D007473:Ion Channels / Q000032:analysis*; D011869:Radioligand Assay / Q000379:methods*; D018342:Receptors, Adrenergic, beta-1 / Q000032:analysis; D011956:Receptors, Cell Surface / Q000032:analysis*; D017465:Receptors, Opioid, delta / Q000032:analysis; D011985:Receptors, Serotonin / Q000032:analysis; D044406:Receptors, Serotonin, 5-HT3; D017483:Receptors, Vasopressin / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis; D014162:Transfection
chemical_list: D007473:Ion Channels; D018342:Receptors, Adrenergic, beta-1; D011956:Receptors, Cell Surface; D017465:Receptors, Opioid, delta; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D017483:Receptors, Vasopressin; D011994:Recombinant Proteins