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    Kinetics of Ligand Binding to Membrane Receptors from Equilibrium Fluctuation Analysis of Single Binding Events

    Year: 2011

    Journal: J. Am. Chem. Soc., 2011, 133 (38), pp 14852–14855, 20111207

    Authors: Gunnarsson A.*†, Dexlin L.‡, Wallin P.†, Svedhem S.†, Jönsson P. †, Wingren C.‡, Höök F.*†

    Last authors: Fredrik Höök

    Organizations: Department of Applied Physics, Chalmers University of Technology, SE-41133 Go teborg, Sweden Department of Immunotechnology and CREATE Health, Lund University, SE-221 84 Lund, Sweden

    Country: Sweden, Sverige

    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.