title: Recombinant anti-botulinum neurotoxin A single-chain variable fragment antibody generated using a phage display system.
abstract: A recombinant single-chain fragment variable antibody (scFv) to botulinum A neurotoxin (BoNT/A) was developed. BALB/C mice were immunized with BoNT/A. Splenomic RNA was isolated from the hyperimmune mice and used to prepare a cDNA library, from which the variable regions of the heavy and light chain antibody genes were generated and connected by a DNA linker. The resulting scFv genes were cloned into the phagemid vector pCANTAB5 in order to construct phage display scFv libraries. Individual anti-BoNT/A phage clones were isolated from the phage display libraries by immunoaffinity selection using immobilized BoNT/A and further evaluated by enzyme-linked immunosorbant assay, immunoprecipitation and Western blotting. Forty-eight clones were found to be BoNT/A-reactive. The most reactive clone, designated D12, was selected for further study. The scFv gene of D12 was subcloned into a Pichia pastoris vector, and expression in yeast was evaluated.
authors.affiliation: Canada West Biosciences, Calgary, Alberta, Canada.
authors.forename: David C W
authors.initials: DC
authors.lastname: Mah
authors.forename: Wei-Gang
authors.initials: WG
authors.lastname: Hu
authors.forename: J Kelly
authors.initials: JK
authors.lastname: Pon
authors.forename: Saad A
authors.initials: SA
authors.lastname: Masri
authors.forename: R Elaine
authors.initials: RE
authors.lastname: Fulton
authors.forename: Paul L
authors.initials: PL
authors.lastname: Monette
authors.forename: Les P
authors.initials: LP
authors.lastname: Nagata
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D019274:Botulinum Toxins, Type A / Q000235:genetics*; D003001:Cloning, Molecular; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019151:Peptide Library; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D019274:Botulinum Toxins, Type A