|Johnson, Eric -|
Submitted to: Hybridoma
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 13, 2009
Publication Date: May 5, 2009
Citation: Scotcher, M.C., Johnson, E.A., Stanker, L.H. 2009. Characterization of the epitope region of F1-2 and F1-5, two monoclonal antibodies to Botulinum Neurotoxin Type A. Hybridoma. 28(5):315-325 Interpretive Summary: Botulism is a serious, often fatal foodborne disease caused by a toxin produced by the bacterium Clostridium botulinum. The toxin, Botulinum toxin, is considered the most toxic biological agent known and it is classified as a class ‘A’ bioterrorism agent. The ‘gold standard’ for detection of botulinum toxin in food is a time consuming test using live mice. A faster, non animal-based test is needed. In a previous report we described the development of such a test that is even more sensitive than the mouse bioassay. This faster test uses monoclonal antibodies for toxin detection. Our antibodies are highly specific and only bind Botulinum toxin. The precise nature of antibody binding, in other words the location on the toxin molecule bound by the antibody, has been determined in this new study. This information not only extends our knowledge of the performance of this new test, but also it improves our ability to design even better tests.
Technical Abstract: 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 speci'c 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.