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

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Steinhauer, CorneliaLund University,Lunds universitet,Institutionen för immunteknologi,Institutioner vid LTH,Lunds Tekniska Högskola,Department of Immunotechnology,Departments at LTH,Faculty of Engineering, LTH (author)

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

Article/chapterEnglish2006

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Wiley,2006

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LIBRIS-ID:oai:lup.lub.lu.se:37cfb91f-de53-4667-87a4-e2da54506503
https://lup.lub.lu.se/record/395445URI
https://doi.org/10.1002/pmic.200600036DOI

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Language:English
Summary in:English

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SwePubSwePub

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Subject category:art swepub-publicationtype
Subject category:ref swepub-contenttype

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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 and genre

TEKNIK OCH TEKNOLOGIER Industriell bioteknik hsv//swe
ENGINEERING AND TECHNOLOGY Industrial Biotechnology hsv//eng
purification
on-chip
antibody microarray
affinity coupling
affinity tag
orientated coupling

Added entries (persons, corporate bodies, meetings, titles ...)

Wingren, ChristerLund University,Lunds universitet,Institutionen för immunteknologi,Institutioner vid LTH,Lunds Tekniska Högskola,Department of Immunotechnology,Departments at LTH,Faculty of Engineering, LTH(Swepub:lu)immt-cwi (author)
Khan, Farid (author)
He, Mingyue (author)
Taussig, Michael J. (author)
Borrebaeck, CarlLund University,Lunds universitet,Institutionen för immunteknologi,Institutioner vid LTH,Lunds Tekniska Högskola,Department of Immunotechnology,Departments at LTH,Faculty of Engineering, LTH(Swepub:lu)immt-cbo (author)
Institutionen för immunteknologiInstitutioner vid LTH (creator_code:org_t)

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In:Proteomics: Wiley6:15, s. 4227-42341615-98611615-9853

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