Self-assembly of antibody fragments and polymers onto gold for immunosensing

Inger Vikholm

Research output: Contribution to journalArticleScientificpeer-review

37 Citations (Scopus)

Abstract

In order to obtain a site-specific orientation of antibodies for immunosensing and prevent non-specific binding (NSB) antibody Fab′-fragments have been immobilized directly onto gold and the remaining free space in between the antibodies have been coated by a non-ionic hydrophilic polymer of N-[tris(hydroxy-methyl)methyl]acrylamide. The polymer possesses low NSB and can be covalently attached onto the gold surface by disulfide anchors. The antibody fragments were to be embedded in the protein repellent host matrix in such a way that only the antigen binding part of the antibody sticks out from the monolayer surface. Thus, NSB binding to the host matrix and to the antibody fragments could be reduced and a high antigen binding capacity could be obtained.

Surface plasmon resonance, SPR has been used to compare the attachment of antibody F(ab)2 and Fab′-fragments of anti-human IgG onto gold. Antibody Fab′-fragments directly coupled onto gold give a three-fold response to human IgG (hIgG) to that of F(ab)2-fragments. The Fab′-fragment layer could be regenerated with a glycine–HCl solution. The F(ab)2-fragment layer, on the other hand, could not be regenerated. This indicates a difference in immobilisation of the fragments. The F(ab)2-fragments were adsorbed on the surface and thus randomly oriented. The much higher binding capacity and the ability to regenerate the Fab′-fragment layer indicates that the Fab′-fragments were site-directly immobilised on the gold surface. When antibody Fab′-fragments were allowed to interact with the surface and the remaining free space in between the antibodies were filled with the protein repellent polymer the NSB was considerably reduced.

Original languageEnglish
Pages (from-to)311-316
JournalSensors and Actuators B: Chemical
Volume106
Issue number1
DOIs
Publication statusPublished - 2005
MoE publication typeA1 Journal article-refereed

Fingerprint

Immunoglobulin Fragments
Immunoglobulin Fab Fragments
antibodies
Antibodies
Gold
Self assembly
self assembly
Polymers
fragments
gold
polymers
Antigens
Immunoglobulin G
antigens
Acrylamide
Surface plasmon resonance
Proteins
Anchors
Disulfides
Monolayers

Keywords

  • antibody fragments
  • non-specific binding
  • repellent
  • polymers
  • surface plasmon resonance
  • antigen
  • antibodies

Cite this

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Self-assembly of antibody fragments and polymers onto gold for immunosensing. / Vikholm, Inger.

In: Sensors and Actuators B: Chemical, Vol. 106, No. 1, 2005, p. 311-316.

Research output: Contribution to journalArticleScientificpeer-review

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AB - In order to obtain a site-specific orientation of antibodies for immunosensing and prevent non-specific binding (NSB) antibody Fab′-fragments have been immobilized directly onto gold and the remaining free space in between the antibodies have been coated by a non-ionic hydrophilic polymer of N-[tris(hydroxy-methyl)methyl]acrylamide. The polymer possesses low NSB and can be covalently attached onto the gold surface by disulfide anchors. The antibody fragments were to be embedded in the protein repellent host matrix in such a way that only the antigen binding part of the antibody sticks out from the monolayer surface. Thus, NSB binding to the host matrix and to the antibody fragments could be reduced and a high antigen binding capacity could be obtained.Surface plasmon resonance, SPR has been used to compare the attachment of antibody F(ab)2 and Fab′-fragments of anti-human IgG onto gold. Antibody Fab′-fragments directly coupled onto gold give a three-fold response to human IgG (hIgG) to that of F(ab)2-fragments. The Fab′-fragment layer could be regenerated with a glycine–HCl solution. The F(ab)2-fragment layer, on the other hand, could not be regenerated. This indicates a difference in immobilisation of the fragments. The F(ab)2-fragments were adsorbed on the surface and thus randomly oriented. The much higher binding capacity and the ability to regenerate the Fab′-fragment layer indicates that the Fab′-fragments were site-directly immobilised on the gold surface. When antibody Fab′-fragments were allowed to interact with the surface and the remaining free space in between the antibodies were filled with the protein repellent polymer the NSB was considerably reduced.

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