title: A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses.
abstract: Potyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins of Dasheen mosaic potyvirus (DsMV), Konjak mosaic potyvirus (KoMV), and Zantedeschia mild mosaic potyvirus (ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression in Escherichia coli. Moreover, the pectate lyase E (PelE) signal peptide of Erwinia chrysanthemi S3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs in E. coli.
authors.affiliation: Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan.
authors.forename: Han-Lin
authors.initials: HL
authors.lastname: Liu
authors.affiliation: Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan.
authors.forename: Wei-Fang
authors.initials: WF
authors.lastname: Lin
authors.affiliation: Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan.
authors.forename: Wen-Chi
authors.initials: WC
authors.lastname: Hu
authors.affiliation: Department of Life Science, Fu Jen Catholic University, New Taipei City, Taiwan.
authors.forename: Yung-An
authors.initials: YA
authors.lastname: Lee
authors.affiliation: Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan ycchang@ntu.edu.tw.
authors.forename: Ya-Chun
authors.initials: YC
authors.lastname: Chang
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D017800:Potyvirus / Q000187:drug effects* / Q000502:physiology; D016415:Sequence Alignment; D057127:Single-Chain Antibodies / Q000032:analysis / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D057127:Single-Chain Antibodies; D014764:Viral Proteins