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Oriented surface immobilization of antibodies at the conserved nucleotide binding site for enhanced antigen detection.
- Source :
-
Langmuir : the ACS journal of surfaces and colloids [Langmuir] 2012 Jun 26; Vol. 28 (25), pp. 9640-8. Date of Electronic Publication: 2012 Jun 04. - Publication Year :
- 2012
-
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.
- Subjects :
- Antibodies, Immobilized metabolism
Binding Sites
Immunoglobulin G chemistry
Immunoglobulin G immunology
Immunoglobulin G metabolism
Models, Molecular
Photochemical Processes
Protein Conformation
Surface Properties
Antibodies, Immobilized chemistry
Antibodies, Immobilized immunology
Antigens immunology
Conserved Sequence
Nucleotides metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1520-5827
- Volume :
- 28
- Issue :
- 25
- Database :
- MEDLINE
- Journal :
- Langmuir : the ACS journal of surfaces and colloids
- Publication Type :
- Academic Journal
- Accession number :
- 22612330
- Full Text :
- https://doi.org/10.1021/la301887s