Carl Borrebaeck
Professor
Improved affinity coupling for antibody microarrays: Engineering of double-(His)(6)-tagged single framework recombinant antibody fragments
Author
Summary, in English
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.
Department/s
- Department of Immunotechnology
Publishing year
2006
Language
English
Pages
4227-4234
Publication/Series
Proteomics
Volume
6
Issue
15
Document type
Journal article
Publisher
John Wiley & Sons Inc.
Topic
- Industrial Biotechnology
Keywords
- purification
- on-chip
- antibody microarray
- affinity coupling
- affinity tag
- orientated coupling
Status
Published
ISBN/ISSN/Other
- ISSN: 1615-9861