title: Development of single chain variable fragment (scFv) antibodies against surface proteins of 'Ca. Liberibacter asiaticus'.
abstract: 'Candidatus Liberibacter asiaticus' is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19. The antibody population is enriched for antibodies that bind antigens of 'Ca. Liberibacter asiaticus'. The primary library has more than 10(7) unique antibodies and the genes that encode them. We have screened this library for antibodies that bind to specifically-chosen proteins that are present on the surface of 'Ca. Liberibacter asiaticus'. These proteins were used as targets for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule protein KpsF; a protein component of the type IV pilus (CapF); and, two flagellar proteins FlhA and FlgI. These scFvs have been used in ELISA and dot blot assays against purified protein antigens and 'Ca. Liberibacter asiaticus' infected plant extracts. We have also recloned many of these scFvs into a plasmid expression vector designed for the production of scFvs. Screening of these scFvs was more efficient when phage-bound, rather than soluble scFvs, were used. We have demonstrated a technology to produce antibodies at will and against any protein target encoded by 'Ca. Liberibacter asiaticus'. Applications could include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications.
authors.affiliation: Molecular Plant Pathology Lab, USDA-ARS, Beltsville, MD 20705, United States.
authors.forename: Qing
authors.initials: Q
authors.lastname: Yuan
authors.affiliation: United States 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: Minenkova
authors.affiliation: Molecular Plant Pathology Lab, USDA-ARS, Beltsville, MD 20705, United States.
authors.forename: John
authors.initials: J
authors.lastname: Hartung
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics / Q000276:immunology; D002957:Citrus / Q000382:microbiology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D015723:Gene Library; D006430:Hemiptera / Q000382:microbiology; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008828:Microbiological Techniques / Q000379:methods; D010935:Plant Diseases / Q000276:immunology / Q000382:microbiology* / Q000517:prevention & control; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D012230:Rhizobiaceae / Q000235:genetics / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000494:pharmacology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C080688:FlhA protein, Bacteria; D008565:Membrane Proteins; D057127:Single-Chain Antibodies; C074899:flgI protein, Bacteria; C078167:OMPA outer membrane proteins
keywords: Citrus greening; HLB; Huanglongbing; Phage display; Phagemid; scFv