title: Ligand binding to transmembrane receptors on intact cells or membrane vesicles measured in a homogeneous 1-microliter assay format.
abstract: We have developed homogeneous miniaturized assays to measure ligand binding to either intact cells or receptor-containing membrane fragments by analysis of particle brightness. As an example, the affinities and inhibition constants of fluorescently labeled interleukin-8 (IL-8) and a low-molecular-weight antagonist toward the receptors CXCR1 and CXCR2, which belong to the superfamily of G protein-coupled receptors (GPCRs), were determined. Although the results were generally comparable between the two approaches, the cell-based measurements revealed a more complex pattern of both ligand and inhibitor titration curves, pointing to the influence of intracellular regulatory events. Both the vesicle- and cell-based membrane receptor assays were successfully miniaturized to a total volume of 1 microl without compromising their sensitivity, indicating that screening of transmembrane receptors in these formats is feasible. This is the first report of a cellular ligand-binding assay performed in such low volumes. The resulting savings in reagent could potentially enable the use of primary cells for future HTS/ultra-HTS efforts.
authors.affiliation: EVOTEC Biosystems AG, Applied Assay Development, Hamburg, Germany. martin.klumpp@evotecoai.com
authors.forename: M
authors.initials: M
authors.lastname: Klumpp
authors.forename: A
authors.initials: A
authors.lastname: Scheel
authors.forename: E
authors.initials: E
authors.lastname: Lopez-Calle
authors.forename: M
authors.initials: M
authors.lastname: Busch
authors.forename: K J
authors.initials: KJ
authors.lastname: Murray
authors.forename: A J
authors.initials: AJ
authors.lastname: Pope
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D002474:Cell-Free System; D006224:Cricetinae; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D016209:Interleukin-8 / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008081:Liposomes / Q000378:metabolism*; D008904:Miniaturization; D011956:Receptors, Cell Surface / Q000378:metabolism*; D023062:Receptors, Interleukin-8A / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D016209:Interleukin-8; D008024:Ligands; D008081:Liposomes; D011956:Receptors, Cell Surface; D023062:Receptors, Interleukin-8A; D020962:Heterotrimeric GTP-Binding Proteins