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

Författare:
Publiceringsår: 2006
Språk: Engelska
Sidor: 4227-4234
Publikation/Tidskrift/Serie: Proteomics
Volym: 6
Nummer: 15
Dokumenttyp: Artikel
Förlag: WILEY-VCH Verlag GmbH & Co

Sammanfattning

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.

Disputation

Nyckelord

  • Medicine and Health Sciences
  • purification
  • on-chip
  • antibody microarray
  • affinity coupling
  • affinity tag
  • orientated coupling

Övriga

Published
Yes
  • ISSN: 1615-9853

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