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

Nathan Alves, Tanyel Kiziltepe, Basar Bilgicer

Research output: Contribution to journalArticle

32 Citations (Scopus)

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

Fingerprint

nucleotides
antigens
Binding sites
Antigens
Nucleotides
antibodies
immobilization
Antibodies
Binding Sites
Butyric acid
butyric acid
indoles
Immobilized Antibodies
Surface Antigens
conjugation
Ultraviolet radiation
affinity
platforms
damage
Adsorption

ASJC Scopus subject areas

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

Cite this

Oriented surface immobilization of antibodies at the conserved nucleotide binding site for enhanced antigen detection. / Alves, Nathan; Kiziltepe, Tanyel; Bilgicer, Basar.

In: Langmuir, Vol. 28, No. 25, 26.06.2012, p. 9640-9648.

Research output: Contribution to journalArticle

@article{c4890cb3eabc45c4a5d4f2e4f5386cb3,
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.",
author = "Nathan Alves and Tanyel Kiziltepe and Basar Bilgicer",
year = "2012",
month = "6",
day = "26",
doi = "10.1021/la301887s",
language = "English (US)",
volume = "28",
pages = "9640--9648",
journal = "Langmuir",
issn = "0743-7463",
publisher = "American Chemical Society",
number = "25",

}

TY - JOUR

T1 - Oriented surface immobilization of antibodies at the conserved nucleotide binding site for enhanced antigen detection

AU - Alves, Nathan

AU - Kiziltepe, Tanyel

AU - Bilgicer, Basar

PY - 2012/6/26

Y1 - 2012/6/26

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84862889117&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84862889117&partnerID=8YFLogxK

U2 - 10.1021/la301887s

DO - 10.1021/la301887s

M3 - Article

VL - 28

SP - 9640

EP - 9648

JO - Langmuir

JF - Langmuir

SN - 0743-7463

IS - 25

ER -