title: Generation of a recombinant full-length human antibody binding to botulinum neurotoxin A.
abstract: In order to develop a recombinant full-length human anti-botulinum neurotoxin A (BoNT/A) antibody, human peripheral blood mononuclear cells (PBMC) were collected from three healthy volunteers and induced for BoNT/A-specific immune response by in vitro immunization. The genes encoding human Fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized PBMC. Afterwards, one combinatory human antigen-binding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 10(5) Escherichia coli transformants. After screening the library by BoNT/A antigen using a plaque lifting with immunostaining approach, 55 clones were identified as positive. The Fab gene of the most reactive clone exhibiting particularly strong BoNT/A binding signal was further subcloned into a full-length human IgG1 antibody gene template in an adenoviral expression vector, in which the heavy and light chains were linked by a foot-and-mouth-disease virus-derived 2A self-cleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (K(d)) for the recombinant human antibody to bind to BoNT/A was determined by indirect enzyme-linked immunosorbent assay and results confirmed that the recombinant full-length human antibody retained BoNT/A-binding specificity with K(d) value of 10(-7) M.
authors.affiliation: Defence Research and Development Canada-Suffield, Box 4000, Station Main, Medicine Hat, Alberta, Canada T1A8K6. wei-gang.hu@drdc-rddc.gc.ca
authors.forename: Wei-Gang
authors.initials: WG
authors.lastname: Hu
authors.forename: Scott
authors.initials: S
authors.lastname: Jager
authors.forename: Damon
authors.initials: D
authors.lastname: Chau
authors.forename: Dave
authors.initials: D
authors.lastname: Mah
authors.forename: Les P
authors.initials: LP
authors.lastname: Nagata
mesh_terms: D010582:Bacteriophage lambda / Q000276:immunology; D019274:Botulinum Toxins, Type A / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology*
chemical_list: D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; C100277:immunoglobulins, Fd; D019274:Botulinum Toxins, Type A