title: Lanthanide-based time-resolved fluorescence of in cyto ligand-receptor interactions.
abstract: A lanthanide-based assay for ligand-receptor interactions provides an attractive alternative to the traditional radiolabeled determinations in terms of sensitivity, throughput, and biohazards. We designed and tested peptide ligands modified with an Eu-DTPA chelate. These labeled ligands were used in competitive binding assays with results comparable to those obtained using the traditional radiolabeled binding assays. The sensitivity of time-resolved fluorescence is sufficient to detect attomoles of europium, allowing assays in 96-well plates, compared with 30-mm dishes for (125)I binding assays to whole cells. We verified binding of Eu-DTPA-NDP-alpha-MSH to cells overexpressing the human melanocortin-4 receptor. The Eu-labeled ligand bound to these cells with an affinity similar to that of unlabeled NDP-alpha-MSH and was used to optimize a competitive binding assay. The lanthanide-based assays provided superior results with higher throughput and eliminated the need for radioactive waste disposal. This assay is appropriate for high-throughput screening of ligand libraries.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, University of Arizona, and Arizona Cancer Center, Tucson, AZ 85724, USA.
authors.forename: Heather L
authors.initials: HL
authors.lastname: Handl
authors.forename: Josef
authors.initials: J
authors.lastname: Vagner
authors.forename: Henry I
authors.initials: HI
authors.lastname: Yamamura
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Hruby
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Gillies
mesh_terms: D001667:Binding, Competitive; D003584:Cytological Techniques; D005453:Fluorescence; D006801:Humans; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008024:Ligands; D010455:Peptides / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D044105:Receptor, Melanocortin, Type 4 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013995:Time
chemical_list: D028581:Lanthanoid Series Elements; D008024:Ligands; D010455:Peptides; D044105:Receptor, Melanocortin, Type 4; D011956:Receptors, Cell Surface