Improved affinity coupling for antibody microarrays: Engineering of double-(His)(6)-tagged single framework recombinant antibody fragments
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/395445
- author
- Steinhauer, Cornelia LU ; Wingren, Christer LU ; Khan, Farid ; He, Mingyue ; Taussig, Michael J. and Borrebaeck, Carl LU
- organization
- publishing date
- 2006
- 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 & Sons Inc.
- 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
- 2022-03-28 23:51:37
@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}},
keywords = {{purification; on-chip; antibody microarray; affinity coupling; affinity tag; orientated coupling}},
language = {{eng}},
number = {{15}},
pages = {{4227--4234}},
publisher = {{John Wiley & Sons Inc.}},
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}},
}