title: Isolation of recombinant phage antibodies targeting the hemagglutinin cleavage site of highly pathogenic avian influenza virus.
abstract: Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS) in the hemagglutinin protein (HA). Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in Escherichia coli bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus.
authors.affiliation: Graduate School of Science and Technology, Shizuoka University, Suruga-ku, Shizuoka, Japan.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Dong
authors.forename: Akira
authors.initials: A
authors.lastname: Sakurai
authors.forename: Namiko
authors.initials: N
authors.lastname: Nomura
authors.forename: Enoch Y
authors.initials: EY
authors.lastname: Park
authors.forename: Futoshi
authors.initials: F
authors.lastname: Shibasaki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D000914:Antibodies, Viral / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D001665:Binding Sites; D004285:Dogs; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D007114:Immunization; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology*; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D019151:Peptide Library; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D012995:Solubility
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007140:Immunoglobulin Fab Fragments; D010446:Peptide Fragments; D019151:Peptide Library; D011994:Recombinant Proteins