Characterization of the epitope region of F1-2 and F1-5, two monoclonal antibodies to Botulinum neurotoxin type A.
F1-2 and F1-5 are mouse IgG1 monoclonal antibodies that bind the heavy chain of Botulinum neurotoxin serotype A (BoNT/A). To characterize the epitopes of F1-2 and F1-5, three complementary experimental approaches were selected. First, recombinant peptide fragments of BoNT/A heavy-chain were used in Western blots to identify the epitope regions. Second, a peptide phage display library was used to identify specific amino acids bound by F1-2 and F1-5, and these amino acids were mapped onto the three-dimensional structure of BoNT/A. Third, selected amino acids were mutated to alanine and the effects of the mutations on F1-2 and F1-5 binding were evaluated. Data from recombinant peptide fragment binding experiments suggested that the epitopes for antibodies F1-2 and F1-5 are located between amino acids R564 and S793 on the toxin heavy chain. Furthermore, elimination of amino acids from the amino terminus (R564-K595), or from the carboxyl terminus (N759-S793) of this fragment abolished binding of both F1-2 and F1-5, suggesting a conformational epitope for these antibodies. Peptide sequences deduced from antibody binding to the peptide phage display library suggested that tyrosine residues located at positions 748, 750, and 753 might form a significant part of the F1-2 and F1-5 epitope motif. Mutation of Y750 or Y753 to alanine significantly reduced binding of either antibody, while mutation of Y748 to alanine had no effect on antibody binding. The nucleotide and deduced amino acid sequences of the variable regions of the heavy chains of F1-2 and F1-5 are reported. The complementarity determining regions (CDRs) of the heavy chains were found to be 78% identical. It is possible that F1-2 and F1-5 bind the same epitope via the common amino acids within their CDRs.
U.S. Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, California 94710, USA.
Miles C
MC
Scotcher
Eric A
EA
Johnson
Larry H
LH
Stanker
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000276:immunology*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D000939:Epitopes; D019274:Botulinum Toxins, Type A