title: Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.
abstract: Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine.
authors.affiliation: Molecular Plant Pathology Lab, USDA-ARS, Beltsville, MD 20705, United States.
authors.forename: Qing
authors.initials: Q
authors.lastname: Yuan
authors.affiliation: Floral and Nursery Plants Research Unit, U.S. National Arboretum, USDA-ARS, Beltsville, MD 20705, United States.
authors.forename: Ramon
authors.initials: R
authors.lastname: Jordan
authors.affiliation: Citrus Research and Education Center, University of Florida, Lake Alfred, FL 33850, United States.
authors.forename: Ronald H
authors.initials: RH
authors.lastname: Brlansky
authors.affiliation: Sigma-Tau Pharmaceuticals, Rome, Italy.
authors.forename: Olga
authors.initials: O
authors.lastname: Istomina
authors.affiliation: Molecular Plant Pathology Lab, USDA-ARS, Beltsville, MD 20705, United States. Electronic address: john.hartung@ars.usda.gov.
authors.forename: John
authors.initials: J
authors.lastname: Hartung
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D061505:Cell Surface Display Techniques / Q000379:methods*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D044167:Xylella / Q000276:immunology*
chemical_list: D057127:Single-Chain Antibodies
keywords: Phage display; Single chain variable fragment; Xylella fastidiosa