title: Isolation and characterization of recombinant single chain fragment variable anti-idiotypic antibody specific to Aspergillus fumigatus membrane protein.
abstract: Aspergillus fumigatus causes the highly lethal form of invasive aspergillosis (IA). In the present study to develop a novel anti-fungal drug for protection against invasive disease, we identified a single chain fragment variable (scFv) antibody (scFv AF1) by panning against A. fumigatus membrane fraction (AMF) or HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) as antigen. The key step was elution of bound phages with phosphate buffered saline (PBS) at pH 7.0 containing AMF. The specificity of soluble scFv AF1 antibody to antigens was verified by ELISA, which specifically binds to both AMF and nmAb-KT. After nucleotide sequencing, clone expression and purification by HisTrap HP affinity column, scFv AF1 showed in vitro anti-fungal activity against A. fumigatus. By SPR analysis it showed high binding affinity to nmAb-KT (K(d)=5.22×10(-11) M). The method used to isolate scFv AF1 was a new method and we believe that it will be applicable to isolate the specific scFv against any kind of membrane protein of yeast or fungus.
authors.affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Niigata 956-8603, Japan.
authors.forename: Senthilkumar
authors.initials: S
authors.lastname: Krishnaswamy
authors.forename: M Enamul
authors.initials: ME
authors.lastname: Kabir
authors.forename: M Mamunur
authors.initials: MM
authors.lastname: Rahman
authors.forename: Masahiko
authors.initials: M
authors.lastname: Miyamoto
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Furuichi
authors.forename: Tadazumi
authors.initials: T
authors.lastname: Komiyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D000908:Antibodies, Fungal / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D057134:Antibodies, Neutralizing / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D000915:Antibody Affinity; D000918:Antibody Specificity; D000935:Antifungal Agents / Q000302:isolation & purification / Q000494:pharmacology; D000946:Antigens, Fungal; D001232:Aspergillus fumigatus / Q000276:immunology* / Q000472:pathogenicity; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005656:Fungal Proteins / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D066298:In Vitro Techniques; D055646:Killer Factors, Yeast / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000908:Antibodies, Fungal; D057134:Antibodies, Neutralizing; D000935:Antifungal Agents; D000946:Antigens, Fungal; D017931:DNA Primers; D005656:Fungal Proteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D055646:Killer Factors, Yeast; D008565:Membrane Proteins; D019151:Peptide Library; D011994:Recombinant Proteins