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

Cornelia Steinhauer; Christer Wingren; Farid Khan; Mingyue He; Michael J Taussig; Carl A K Borrebaeck

doi:10.1002/pmic.200600036

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

Cornelia Steinhauer, Christer Wingren, Farid Khan, Mingyue He, Michael J Taussig, Carl A K Borrebaeck

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

51 Citationer (Scopus)

Abstract

Udgivelsesdato: 2006-Aug

Originalsprog	Engelsk
Tidsskrift	Proteomics
Vol/bind	6
Udgave nummer	15
Sider (fra-til)	4227-34
Antal sider	7
ISSN	1615-9853
DOI	https://doi.org/10.1002/pmic.200600036
Status	Udgivet - 2006
Udgivet eksternt	Ja

Bibliografisk note

Keywords: Antibodies; Binding, Competitive; Histidine; Humans; Immunoglobulin Fragments; Protein Array Analysis; Protein Engineering; Proteomics; Recombinant Proteins; Surface Plasmon Resonance

Adgang til dokumentet

10.1002/pmic.200600036

Citationsformater

Improved affinity coupling for antibody microarrays: engineering of double-(His)6-tagged single framework recombinant antibody fragments. / Steinhauer, Cornelia; Wingren, Christer; Khan, Farid; He, Mingyue; Taussig, Michael J; Borrebaeck, Carl A K.

I: Proteomics, Bind 6, Nr. 15, 2006, s. 4227-34.

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

@article{2ddb5ba057c411dd8d9f000ea68e967b,

title = "Improved affinity coupling for antibody microarrays: engineering of double-(His)6-tagged single framework recombinant antibody fragments.",

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 Ni(2+)-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 = "Cornelia Steinhauer and Christer Wingren and Farid Khan and Mingyue He and Taussig, {Michael J} and Borrebaeck, {Carl A K}",

note = "Keywords: Antibodies; Binding, Competitive; Histidine; Humans; Immunoglobulin Fragments; Protein Array Analysis; Protein Engineering; Proteomics; Recombinant Proteins; Surface Plasmon Resonance",

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AU - Steinhauer, Cornelia

AU - Wingren, Christer

AU - Khan, Farid

AU - He, Mingyue

AU - Taussig, Michael J

AU - Borrebaeck, Carl A K

N1 - Keywords: Antibodies; Binding, Competitive; Histidine; Humans; Immunoglobulin Fragments; Protein Array Analysis; Protein Engineering; Proteomics; Recombinant Proteins; Surface Plasmon Resonance

PY - 2006

Y1 - 2006

N2 - 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 Ni(2+)-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.

AB - 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 Ni(2+)-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.

U2 - 10.1002/pmic.200600036

DO - 10.1002/pmic.200600036

M3 - Journal article

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