Recombinant anti-botulinum neurotoxin A single-chain variable fragment antibody generated using a phage display system.
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.
Mah
David C W
DC
Canada West Biosciences, Calgary, Alberta, Canada.
Hu
Wei-Gang
WG
Pon
J Kelly
JK
Masri
Saad A
SA
Fulton
R Elaine
RE
Monette
Paul L
PL
Nagata
Les P
LP
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
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D019274:Botulinum Toxins, Type A