title: Kinetics of ligand binding to membrane receptors from equilibrium fluctuation analysis of single binding events.
abstract: Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies. The single-molecule sensitivity made the analysis possible without overexpressed membrane proteins, which makes the assay attractive in early drug-screening applications.
authors.affiliation: Department of Applied Physics, Chalmers University of Technology, Göteborg, Sweden. anders.gunnarsson@chalmers.se
authors.forename: Anders
authors.initials: A
authors.lastname: Gunnarsson
authors.forename: Linda
authors.initials: L
authors.lastname: Dexlin
authors.forename: Patric
authors.initials: P
authors.lastname: Wallin
authors.forename: Sofia
authors.initials: S
authors.lastname: Svedhem
authors.forename: Peter
authors.initials: P
authors.lastname: Jönsson
authors.forename: Christer
authors.initials: C
authors.lastname: Wingren
authors.forename: Fredrik
authors.initials: F
authors.lastname: Höök
mesh_terms: D001402:B-Lymphocytes / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013499:Surface Properties; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D008024:Ligands; D008081:Liposomes; D011956:Receptors, Cell Surface; D057127:Single-Chain Antibodies