Oriented Surface Immobilization of Antibodies at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection

• Published in: Langmuir 2012-06, Vol.28 (25), p.9640-9648
• Main Authors: Alves, Nathan J, Kiziltepe, Tanyel, Bilgicer, Basar
• Format: Article
• Language: English
• Subjects: Antibodies, Immobilized - chemistry; Antibodies, Immobilized - immunology; Antibodies, Immobilized - metabolism; Antigens - immunology; Binding Sites; Chemistry; Conserved Sequence; Exact sciences and technology; General and physical chemistry; Immunoglobulin G - chemistry; Immunoglobulin G - immunology; Immunoglobulin G - metabolism; Models, Molecular; Nucleotides - metabolism; Photochemical Processes; Protein Conformation; Surface physical chemistry; Surface Properties
• ISSN: 0743-7463; 1520-5827
• DOI: 10.1021/la301887s

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/cm2 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-NH3 + 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.