title: Generation and characterization of a scFv against recombinant coat protein of the geminivirus tomato leaf curl New Delhi virus.
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.
authors.affiliation: Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, Aachen, Germany.
authors.forename: Adel M
authors.initials: AM
authors.lastname: Zakri
authors.forename: Angelika
authors.initials: A
authors.lastname: Ziegler
authors.forename: Lesley
authors.initials: L
authors.lastname: Torrance
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Ulrich
authors.initials: U
authors.lastname: Commandeur
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000235:genetics* / Q000276:immunology*; D000915:Antibody Affinity; D053839:Begomovirus / Q000276:immunology*; D015153:Blotting, Western; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018985:Epitopes, B-Lymphocyte / Q000276:immunology; D018551:Solanum lycopersicum / Q000821:virology; D051379:Mice; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology*; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D036022:Capsid Proteins; D018985:Epitopes, B-Lymphocyte; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies