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

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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.

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Acknowledgments

The authors would like to express their gratitude to Dr. Usha Barwale Zehr (Maharashtra Hybrid Seed Company, MAHYCO, India), for kindly providing the cloned of ToLCNDV DNA-A. We also thank Dr. Stephan Winter (DSMZ, Braunschweig, Germany) for providing us infected plant material and antibodies and for helpful discussion. This work was partially supported by Marie Curie Fellowship QLK3-CT-2001-60032.

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  1. Adel M. Zakri

    Present address: Center of Excellence in Biotechnology Research (CEBR), King Saud University, Riyadh, Saudi Arabia

Authors and Affiliations

  1. Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany

    Adel M. Zakri, Rainer Fischer & Ulrich Commandeur

  2. Plant Pathology Programme, Scottish Crop Research Institute (SCRI), Invergowrie, Dundee, DD2 5DA, UK

    Adel M. Zakri, Angelika Ziegler & Lesley Torrance

  3. Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074, Aachen, Germany

    Rainer Fischer

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  1. Adel M. Zakri
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Correspondence to Ulrich Commandeur.

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Zakri, A.M., Ziegler, A., Torrance, L. et al. Generation and characterization of a scFv against recombinant coat protein of the geminivirus tomato leaf curl New Delhi virus. Arch Virol 155, 335–342 (2010). https://doi.org/10.1007/s00705-010-0591-6

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  • DOI: https://doi.org/10.1007/s00705-010-0591-6

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