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Improved affinity coupling for antibody microarrays: Engineering of double-(His)(6)-tagged single framework recombinant antibody fragments

Steinhauer, Cornelia LU ; Wingren, Christer LU ; Khan, Farid ; He, Mingyue ; Taussig, Michael J. and Borrebaeck, Carl LU (2006) In Proteomics 6(15). p.4227-4234
Abstract
Antibody-based microarray is a novel technology with great promise in biomedicine that will provide unique means to perform global proteome analysis. In the process of designing the high-density antibody microarrays required, several critical key issues have been identified that remain to be resolved. In particular, there is a great need for specific and selective approaches enabling non-purified probes to be directly purified, orientated and coupled in a generic one-step procedure directly on the chip. In this study, we report on the successful design of affinity-tagged human recombinant single-chain fragment variable antibody fragments for improved affinity coupling in array applications. By replacing the standard single-histidine... (More)
Antibody-based microarray is a novel technology with great promise in biomedicine that will provide unique means to perform global proteome analysis. In the process of designing the high-density antibody microarrays required, several critical key issues have been identified that remain to be resolved. In particular, there is a great need for specific and selective approaches enabling non-purified probes to be directly purified, orientated and coupled in a generic one-step procedure directly on the chip. In this study, we report on the successful design of affinity-tagged human recombinant single-chain fragment variable antibody fragments for improved affinity coupling in array applications. By replacing the standard single-histidine (His)(6)-tag with a consecutive double-(His)(6)-tag, the binding to Ni2+-nitrilotriacetic acid-coated substrates was significantly improved. Surface plasmon resonance analysis showed a significantly tighter binding with at least a threefold slower dissociation. The improved binding characteristics thus enabled non-purified probes even in the format of crude expression supernatants to be directly applied thereby eliminating the need for any time-consuming pre-purification step(s) prior to the immobilization. While the double-(HiS)(6)-tag probes were found to be expressed equally well as compared to the single-(His)(6)-tag probes, they displayed better long-term functional on-chip stability. Taken together, the results demonstrate the generic potential of double-(HiS)(6)-tag recombinant antibodies for the facile fabrication of high-density antibody microarrays. (Less)
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author
; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
purification, on-chip, antibody microarray, affinity coupling, affinity tag, orientated coupling
in
Proteomics
volume
6
issue
15
pages
4227 - 4234
publisher
John Wiley and Sons
external identifiers
  • wos:000239867600003
  • pmid:16826567
  • scopus:33747782971
ISSN
1615-9861
DOI
10.1002/pmic.200600036
language
English
LU publication?
yes
id
37cfb91f-de53-4667-87a4-e2da54506503 (old id 395445)
date added to LUP
2016-04-01 12:21:30
date last changed
2021-02-17 05:56:52
@article{37cfb91f-de53-4667-87a4-e2da54506503,
  abstract     = {Antibody-based microarray is a novel technology with great promise in biomedicine that will provide unique means to perform global proteome analysis. In the process of designing the high-density antibody microarrays required, several critical key issues have been identified that remain to be resolved. In particular, there is a great need for specific and selective approaches enabling non-purified probes to be directly purified, orientated and coupled in a generic one-step procedure directly on the chip. In this study, we report on the successful design of affinity-tagged human recombinant single-chain fragment variable antibody fragments for improved affinity coupling in array applications. By replacing the standard single-histidine (His)(6)-tag with a consecutive double-(His)(6)-tag, the binding to Ni2+-nitrilotriacetic acid-coated substrates was significantly improved. Surface plasmon resonance analysis showed a significantly tighter binding with at least a threefold slower dissociation. The improved binding characteristics thus enabled non-purified probes even in the format of crude expression supernatants to be directly applied thereby eliminating the need for any time-consuming pre-purification step(s) prior to the immobilization. While the double-(HiS)(6)-tag probes were found to be expressed equally well as compared to the single-(His)(6)-tag probes, they displayed better long-term functional on-chip stability. Taken together, the results demonstrate the generic potential of double-(HiS)(6)-tag recombinant antibodies for the facile fabrication of high-density antibody microarrays.},
  author       = {Steinhauer, Cornelia and Wingren, Christer and Khan, Farid and He, Mingyue and Taussig, Michael J. and Borrebaeck, Carl},
  issn         = {1615-9861},
  language     = {eng},
  number       = {15},
  pages        = {4227--4234},
  publisher    = {John Wiley and Sons},
  series       = {Proteomics},
  title        = {Improved affinity coupling for antibody microarrays: Engineering of double-(His)(6)-tagged single framework recombinant antibody fragments},
  url          = {http://dx.doi.org/10.1002/pmic.200600036},
  doi          = {10.1002/pmic.200600036},
  volume       = {6},
  year         = {2006},
}