Generation and characterization of a scFv against recombinant coat protein of the geminivirus tomato leaf curl New Delhi virus

Adel M. Zakri, Angelika Ziegler, Lesley Torrance, Rainer Fischer, Ulrich Commandeur

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

Abstract

We report the establishment of a hybridoma cell line secreting the monoclonal antibody (mAb) HAV, which recognizes the coat (AV1) protein of tomato leaf curl New Delhi virus (ToLCNDV), a begomovirus. The cell line was obtained following immunization of mice with purified recombinant AV1 fused to glutathione S-transferase (GST). A single-chain variable fragment (scFv-SAV) was assembled from hybridoma cDNA, but sequence analysis revealed a single nucleotide deletion causing a frame shift that resulted in a 21-residue N-terminal truncation. The missing nucleotide was restored by in vitro site-directed mutagenesis to create scFv-RWAV. The binding properties of mAb HAV and the corresponding scFvs were characterized by western blot, ELISA and surface plasmon resonance spectroscopy. MAb HAV bound to AV1 with nanomolar affinity but reacted neither with the N-terminal region of the protein nor with the GST fusion partner. This suggested that the antibody recognized a linear epitope in a region of the coat protein that is conserved among begomoviruses. Both scFvs retained the antigen specificity of mAb HAV, although the dissociation rate constant of scFv-RWAV was tenfold greater than that of scFv-SAV, showing the importance of restoring the 21 N-terminal amino acids.

Original languageEnglish
Pages (from-to)335-342
Number of pages8
JournalArchives of Virology
Volume155
Issue number3
DOIs
Publication statusPublished - Mar 2010

Keywords

  • SINGLE-CHAIN ANTIBODIES
  • WHITEFLY TRANSMISSION
  • MOSAIC GEMINIVIRUS
  • FRAMEWORK REGION
  • VIRAL MOVEMENT
  • DNA
  • PLANT
  • RESISTANCE
  • RESIDUE
  • BINDING

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