Oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection

L Torrance, A Ziegler, H Pittman, M Paterson, R Toth, I Eggleston

Research output: Contribution to journalArticlepeer-review

77 Citations (Scopus)

Abstract

Single chain variable fragment (scFv) molecules were selected from a synthetic phage display library then cloned into a generic vector for expression of the scFv fused to the light chain constant domain of human immunoglobulin with a C-terminal cysteine residue (scFvC(L)cys). A heterobifunctional maleimide linker was synthesised and a strategy for functionalisation of gold with the scFVC(L)cys fusion proteins elaborated. Successful covalent attachment of functional scFvC(L)cys was demonstrated using a surface plasmon resonance-based sensor. The results showed that the immobilised scFvC(L)cys molecules were functional and specific binding curves (with response relative to the concentration of virus antigen) were obtained over more than 25 cycles of binding and dissociation. ScFv molecules lacking the C-terminal cysteine performed poorly in similar experiments. The work demonstrates the feasibility of using simple scFv selection and cloning procedures combined with oriented immobilisation of scFVC(L)cys fusion proteins for robust antigen sensing surfaces in immunosensor or other biotechnological applications. (c) 2005 Elsevier B.V. All rights reserved.

Original languageEnglish
Pages (from-to)164-170
Number of pages7
JournalJournal of Virological Methods
Volume134
Issue number1-2
DOIs
Publication statusPublished - Jun 2006

Keywords

  • single-chain variable fragment (scFv)
  • ScFvC(L)cys fusion protein plasmid vector
  • surface plasmon resonance (SPR)
  • phage display libraries
  • Cowpea mosaic virus
  • maleimide linker
  • PHAGE DISPLAY LIBRARIES
  • PHOSPHATASE FUSION-PROTEINS
  • RECOMBINANT ANTIBODIES
  • ANTIHAPTEN ANTIBODIES
  • VARIABLE FRAGMENTS
  • ESCHERICHIA-COLI
  • REAGENTS
  • VECTOR
  • SCFV
  • DERIVATIVES

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