title: Epitope characterization of sero-specific monoclonal antibody to Clostridium botulinum neurotoxin type A.
abstract: Botulinum neurotoxins (BoNTs) are extremely potent toxins that can contaminate foods and are a public health concern. Anti-BoNT antibodies have been described that are capable of detecting BoNTs; however there still exists a need for accurate and sensitive detection capabilities for BoNTs. Herein, we describe the characterization of a panel of eight monoclonal antibodies (MAbs) generated to the non-toxic receptor-binding domain of BoNT/A (H(C)50/A) developed using a high-throughput screening approach. In two independent hybridoma fusions, two groups of four IgG MAbs were developed against recombinant H(C)50/A. Of these eight, only a single MAb, F90G5-3, bound to the whole BoNT/A protein and was characterized further. The F90G5-3 MAb slightly prolonged time to death in an in vivo mouse bioassay and was mapped by pepscan to a peptide epitope in the N-terminal subdomain of H(C)50/A (H(CN)25/A) comprising amino acid residues (985)WTLQDTQEIKQRVVF(999), an epitope that is highly immunoreactive in humans. Furthermore, we demonstrate that F90G5-3 binds BoNT/A with nanomolar efficiency. Together, our results indicate that F90G5-3 is of potential value as a diagnostic immunoreagent for BoNT/A capture assay development and bio-forensic analysis.
authors.affiliation: National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Mannitoba, Canada. cindi.corbett@phac-aspc.gc.ca
authors.forename: Cindi R
authors.initials: CR
authors.lastname: Corbett
authors.forename: Erin
authors.initials: E
authors.lastname: Ballegeer
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Weedmark
authors.forename: M D
authors.initials: MD
authors.lastname: Elias
authors.forename: Fetweh H
authors.initials: FH
authors.lastname: Al-Saleem
authors.forename: Denise M
authors.initials: DM
authors.lastname: Ancharski
authors.forename: Lance L
authors.initials: LL
authors.lastname: Simpson
authors.forename: Jody D
authors.initials: JD
authors.lastname: Berry
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000097:blood / Q000276:immunology; D000915:Antibody Affinity; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D019274:Botulinum Toxins, Type A / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D047013:Clostridium botulinum type A / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D006825:Hybridomas / Q000166:cytology / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D013997:Time Factors
chemical_list: D000907:Antibodies, Bacterial; D058846:Antibodies, Monoclonal, Murine-Derived; D057134:Antibodies, Neutralizing; D000939:Epitopes; D011994:Recombinant Proteins; D019274:Botulinum Toxins, Type A