title: Quantification of protein-ligand dissociation kinetics in heterogeneous affinity assays.
abstract: Biochemical affinity assays inherently involve interactions of heterogeneous nature. We report a methodology to discriminate between and accurately characterize specific and nonspecific interactions in force-induced dissociation assays. Ligand-coupled superparamagnetic particles are incubated on surfaces coated with a mixture of specific receptors and nonspecifically interacting proteins. Consequently, a mixed population of surface bound particles is formed with different binding natures. Magnetic field gradients are used to apply translational forces on the bound particles. Using a multicomponent dissociation analysis, we are able to make a distinction between weak nonspecific interactions, strong nonspecific interactions, and specific interactions. We validate the model by comprehensive experiments in which the biochemical components and applied forces are varied. The low-force data yield reliable values for the spontaneous dissociation rates of single-molecule specific bonds, and at high forces, the bond barriers are modified by the applied force. The results generate a new perspective for applications of magnetic force affinity assays in studies of heterogeneous molecular biorecognition.
authors.affiliation: Department of Applied Physics, Eindhoven University of Technology, Eindhoven, The Netherlands.
authors.forename: Asha
authors.initials: A
authors.lastname: Jacob
authors.forename: Leo J
authors.initials: LJ
authors.lastname: van Ijzendoorn
authors.forename: Arthur M
authors.initials: AM
authors.lastname: de Jong
authors.forename: Menno W J
authors.initials: MW
authors.lastname: Prins
mesh_terms: D001710:Biotin / Q000378:metabolism; D007118:Immunoassay / Q000379:methods*; D007700:Kinetics; D008024:Ligands; D060328:Magnetic Phenomena; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D013499:Surface Properties
chemical_list: D008024:Ligands; D011506:Proteins; D001710:Biotin