Oriented antibody immobilization by site-specific UV photocrosslinking of biotin at the conserved nucleotide binding site for enhanced antigen detection

Nathan Alves, Nur Mustafaoglu, Basar Bilgicer

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

The nucleotide binding site (NBS) is an under-utilized, highly conserved binding site found within the variable region of nearly all antibody Fab arms. Here, we describe an NBS specific UV photocrosslinking biotinylation method (UV-NBSBiotin) for the oriented immobilization of antibodies to streptavidin-coated surfaces, such that the antigen binding activity remains unaffected. An optimal UV exposure of 1J/cm2 yielded an average conjugation efficiency of ~1 biotin per antibody resulting in significant immobilization efficiency while maintaining maximal antigen binding activity. With the continued push for miniaturization of medical diagnostics to reduce cost and increase patient accessibility the ever shrinking on chip detection areas necessitate the highest level of immobilized antibody activity to maximize assay detection capabilities. The UV-NBSBiotin method yielded surfaces with significantly enhanced antigen detection capabilities, improved antigen detection sensitivity and the highest amount of active surface immobilized antibody when compared to other common immobilization methods including: ε-NH3 + surface conjugation, NHS-Biotin, and direct physical adsorption. Taken together, the UV-NBSBiotin method provides a universal, site-specific immobilization method that is amenable to any available assay detection modality with potential significant implications in the development of miniaturized medical diagnostics and lab on a chip technologies.

Original languageEnglish (US)
Pages (from-to)387-393
Number of pages7
JournalBiosensors and Bioelectronics
Volume49
DOIs
StatePublished - Nov 5 2013
Externally publishedYes

Fingerprint

Binding sites
Antigens
Biotin
Nucleotides
Antibodies
Immobilization
Immobilized Antibodies
Binding Sites
Assays
Lab-on-a-chip
Streptavidin
Surface Antigens
Biotinylation
Miniaturization
Adsorption
Costs
Technology
Costs and Cost Analysis

Keywords

  • Biosensor
  • ELISA
  • Immunoglobulin
  • Oriented immobilization
  • Site-specific crosslinking

ASJC Scopus subject areas

  • Biophysics
  • Biomedical Engineering
  • Biotechnology
  • Electrochemistry

Cite this

Oriented antibody immobilization by site-specific UV photocrosslinking of biotin at the conserved nucleotide binding site for enhanced antigen detection. / Alves, Nathan; Mustafaoglu, Nur; Bilgicer, Basar.

In: Biosensors and Bioelectronics, Vol. 49, 05.11.2013, p. 387-393.

Research output: Contribution to journalArticle

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