title: Oriented surface immobilization of antibodies at the conserved nucleotide binding site for enhanced antigen detection.
abstract: The conserved nucleotide binding site (NBS), found on the Fab variable domain of all antibody isotypes, remains a not-so-widely known and unutilized site. Here, we describe a UV photo-cross-linking method (UV-NBS) that utilizes the NBS for oriented immobilization of antibodies onto surfaces, such that the antigen binding activity remains unaffected. Indole-3-butyric acid (IBA) has an affinity for the NBS with a K(d) ranging from 1 to 8 μM for different antibody isotypes and can be covalently photo-cross-linked to the antibody at the NBS upon exposure to UV light. Using the UV-NBS method, antibody was successfully immobilized on synthetic surfaces displaying IBA via UV photo-cross-linking at the NBS. An optimal UV exposure of 2 J/cm(2) yielded significant antibody immobilization on the surface with maximal relative antibody activity per immobilized antibody without any detectable damage to antigen binding activity. Comparison of the UV-NBS method with two other commonly used methods, ε-NH(3)(+) conjugation and physical adsorption, demonstrated that the UV-NBS method yields surfaces with significantly enhanced antigen detection efficiency, higher relative antibody activity, and improved antigen detection sensitivity. Taken together, the UV-NBS method provides a practical, site-specific surface immobilization method, with significant implications in the development of a large array of platforms with diverse sensor and diagnostic applications.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana 46556, United States.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Alves
authors.forename: Tanyel
authors.initials: T
authors.lastname: Kiziltepe
authors.forename: Basar
authors.initials: B
authors.lastname: Bilgicer
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D000941:Antigens / Q000276:immunology*; D001665:Binding Sites; D017124:Conserved Sequence; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism*; D055668:Photochemical Processes; D011487:Protein Conformation; D013499:Surface Properties
chemical_list: D055910:Antibodies, Immobilized; D000941:Antigens; D007074:Immunoglobulin G; D009711:Nucleotides