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Title: Epitope characterization and variable region sequencing of F1-40, a high-affinity monoclonal antibody to botulinum neurotoxin

Author
item Scotcher, Miles
item McGarvey, Jeffery - Jeff
item JOHNSON, ERIC - UNIV OF WISCONSIN
item Stanker, Larry

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/2/2009
Publication Date: 3/17/2009
Citation: Scotcher, M.C., Mcgarvey, J.A., Johnson, E.A., Stanker, L.H. 2009. Epitope characterization and variable region sequencing of F1-40, a high-affinity monoclonal antibody to botulinum neurotoxin Type A (Hall Strain). PLoS ONE 4(3). Available:http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2654115

Interpretive Summary: Botulism is a serious, often fatal neuroparalytic disease in humans and animals caused by a protein toxin (botulinum toxin, BoNT) produced by the bacterium Clostridium botulinum. BoNT is considered the most toxic biological toxin known. Because of its high toxicity, the need for a long recovery period requiring extensive treatment, and the ease of producing BoNT, it is considered a class A bioterrorism agent. The ‘gold standard’ for detection of botulinum toxin is the mouse bioassay. In a previous report we described the development of a simple, non-rodent-based rapid detection based on newly developed monoclonal antibodies. This simple assay will detect BoNT at levels below the mouse bioassay. In this report we describe the location on the BoNT that are bound by one of the antibodies used in the above assay. This information extends our knowledge of the parameters controlling the immunoassay and improves our ability to design even better tests for toxin and predict assay performance.

Technical Abstract: F1-40 is a mouse-derived, IgG1 monoclonal antibody that binds the light chain of Botulinum Neurotoxin serotype A (BoNT/A). The nucleotide and deduced amino acid sequence of the variable regions of the heavy and light chains of F1-40 is reported. To characterize the epitope of F1-40, three complementary experimental approaches were selected. Firstly, recombinant fragments of BoNT/A light-chain were used in Western blots to identify the epitope regions. Secondly, peptide phage-display libraries were used to identify the specific amino acid sequence of the epitope. Thirdly, the three-dimensional structure of BoNT/A was examined in silico, and the amino acid sequences determined from the phage-display studies were mapped onto the three-dimensional structure in order to visualize the epitope. F1-40 was found to bind a peptide fragment of BoNT/A, designated L1-3, which spans from T125 to L200. The motif QPDRS was identified by phage-display, and was mapped to a region within L1-3. When the three amino acids Q-138, P-139 and D-140 were all mutated to glycine, binding of F1-40 to the recombinant BoNT/A light chain peptide was abolished. Q-138, P-139 and D-140 form a loop on the external surface of BoNT/A, exposed to solvent and accessible to F1-40 binding.