Characterization of the Epitope Region of F1-2 and F1-5, Two Monoclonal Antibodies to Botulinum Neurotoxin Type A

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 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.

Antibody Protection against Botulinum Neurotoxin Intoxication in Mice

“…In this study, the ability of MAbs to neutralize BoNT/A was modeled in both the systemic and oral mouse models of botulism. We also sought to dissect the contribution of two wellcharacterized MAbs: F1-40, a MAb against the Lc of BoNT/A that does not bind the catalytic domain, and F1-2, a MAb against the Hc of BoNT/A with a conformational epitope in the translocation domain of the toxin (22,23). Toxin neutralization studies with mice in which antibody was administered both pre-and postintoxication by i.v.…”

“…While this site is located on the Lc, it is distal from the catalytic domain. The binding site for MAb F1-2 is conformational and has been mapped to the region between R564 and K595 within the transmembrane domain of the toxin (22,23).…”

Detection of Botulinum Neurotoxin Serotype B at Sub Mouse LD50 Levels by a Sandwich Immunoassay and Its Application to Toxin Detection in Milk

“…All pGS-21a-derived plasmids were sequenced using primer pGS-F and pGS-R, to confirm the correct integration of the BoNT/A fragment into the vector. The expression and purification of all GST-fusion proteins was performed as previously described [13]. The recombinant DNA methods used in this study were approved by the Institutional Biosafety Committee.…”

Immunity to Ricin: Fundamental Insights into Toxin–Antibody Interactions

“…Pepscan analysis, which can be applied to antisera or individual mAbs, entails screening in an enzyme-linked immunosorbant assay (ELISA) format a collection of overlapping peptides that span the length of a protein of interest for antibody reactivity. Pepscan analysis has been used to localize linear B-cell epitopes on other toxins, including anthrax (Abboud et al 2009; Kelly-Cirino and Mantis 2009; Nguyen et al 2009a, b) and botulinum neurotoxin (Scotcher et al 2009a; Scotcher et al 2009b). Phage displayed peptide libraries are often used in conjunction with pepscan analysis to selectively enrich for peptide(s) that have affinity for a mAb of interest (Mullaney et al 2001; Smith and Petrenko 1997).…”