Oriented surface immobilization of antibodies at the conserved nucleotide binding site for enhanced antigen detection

Nathan J. Alves, Tanyel Kiziltepe, Basar Bilgicer

Research output: Contribution to journalArticle

35 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)9640-9648
Number of pages9
JournalLangmuir
Volume28
Issue number25
DOIs
StatePublished - Jun 26 2012
Externally publishedYes

ASJC Scopus subject areas

  • Materials Science(all)
  • Condensed Matter Physics
  • Surfaces and Interfaces
  • Spectroscopy
  • Electrochemistry

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