title: A scFv-alkaline phosphatase fusion protein which detects potato leafroll luteovirus in plant extracts by ELISA.
abstract: A single chain Fv antibody fragment (scFv) was obtained from a synthetic phage-antibody library after four rounds of selection against purified preparations of potato leafroll luteovirus (PLRV). Nucleotide sequence analysis showed that the scFv belongs to the human V(H)3 family. DNA encoding the scFv was sub-cloned into pDAP2 such that a scFv-alkaline phosphatase fusion protein was produced by transformed bacteria following induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). The fusion protein was obtained at concentrations of 10 mg/l of Escherichia coli culture medium and these fusion protein preparations were used directly in ELISA to detect PLRV in sap extracts from infected plants. Our work is the first report of the selection of a scFv specific for a luteovirus from a synthetic phage-display library and the production of a fusion protein with alkaline phosphatase for the detection of PLRV in infected plants. The results demonstrate the potential of scFv and enzyme-scFv fusion proteins in routine testing for plant virus infection.
authors.affiliation: Scottish Crop Research Institute, Invergowrie, Dundee, UK. kharpe@scri.sari.ac.uk
authors.forename: K
authors.initials: K
authors.lastname: Harper
authors.forename: R J
authors.initials: RJ
authors.lastname: Kerschbaumer
authors.forename: A
authors.initials: A
authors.lastname: Ziegler
authors.forename: S M
authors.initials: SM
authors.lastname: Macintosh
authors.forename: G H
authors.initials: GH
authors.lastname: Cowan
authors.forename: G
authors.initials: G
authors.lastname: Himmler
authors.forename: M A
authors.initials: MA
authors.lastname: Mayo
authors.forename: L
authors.initials: L
authors.lastname: Torrance
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology*; D000956:Antigens, Viral / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D017793:Luteovirus / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010936:Plant Extracts; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D011198:Solanum tuberosum / Q000821:virology
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D004279:DNA, Viral; D007128:Immunoglobulin Fragments; D010936:Plant Extracts; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D000469:Alkaline Phosphatase