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Improved affinity c...
Improved affinity coupling for antibody microarrays: Engineering of double-(His)(6)-tagged single framework recombinant antibody fragments
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- Steinhauer, Cornelia (author)
- Lund University,Lunds universitet,Institutionen för immunteknologi,Institutioner vid LTH,Lunds Tekniska Högskola,Department of Immunotechnology,Departments at LTH,Faculty of Engineering, LTH
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- Wingren, Christer (author)
- Lund University,Lunds universitet,Institutionen för immunteknologi,Institutioner vid LTH,Lunds Tekniska Högskola,Department of Immunotechnology,Departments at LTH,Faculty of Engineering, LTH
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Khan, Farid (author)
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He, Mingyue (author)
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Taussig, Michael J. (author)
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- Borrebaeck, Carl (author)
- Lund University,Lunds universitet,Institutionen för immunteknologi,Institutioner vid LTH,Lunds Tekniska Högskola,Department of Immunotechnology,Departments at LTH,Faculty of Engineering, LTH
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(creator_code:org_t)
- Wiley, 2006
- 2006
- English.
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In: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 6:15, s. 4227-4234
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http://dx.doi.org/10...
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https://doi.org/10.1...
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Abstract
Subject headings
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- 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.
Subject headings
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology (hsv//eng)
Keyword
- purification
- on-chip
- antibody microarray
- affinity coupling
- affinity tag
- orientated coupling
Publication and Content Type
- art (subject category)
- ref (subject category)
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