title: A comparison of binding surfaces for SPR biosensing using an antibody-antigen system and affinity distribution analysis.
abstract: The application of optical biosensors in the study of macromolecular interactions requires immobilization of one binding partner to the surface. It is often highly desirable that the immobilization is uniform and does not affect the thermodynamic and kinetic binding parameters to soluble ligands. To achieve this goal, a variety of sensor surfaces, coupling strategies and surface chemistries are available. Previously, we have introduced a technique for determining the distribution of affinities and kinetic rate constants from families of binding and dissociation traces acquired at different concentrations of soluble ligand. In the present work, we explore how this affinity distribution analysis can be useful in the assessment and optimization of surface immobilization. With this goal, using an antibody-antigen interaction as a model system, we study the activity, thermodynamic and kinetic binding parameters, and heterogeneity of surface sites produced with different commonly used sensor surfaces, at different total surface densities and with direct immobilization or affinity capture.
authors.affiliation: Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA. zhaoh3@mail.nih.gov
authors.forename: Huaying
authors.initials: H
authors.lastname: Zhao
authors.forename: Inna I
authors.initials: II
authors.lastname: Gorshkova
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Fu
authors.forename: Peter
authors.initials: P
authors.lastname: Schuck
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D001666:Binding Sites, Antibody; D006801:Humans; D007700:Kinetics; D051379:Mice; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D001613:beta 2-Microglobulin