Characterization of neutralizing antibodies and identification of neutralizing epitope mimics on the Clostridium botulinum neurotoxin type A

Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the rel ease of acetylcholine at the neuromuscular junctions and synapses and to ca use neuroparalysis and death. In this study, we have identified two monoclo nal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal d oses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 10(3) and 10(4) times the 50% lethal dose, respectively. Using immuno blotting analysis, BT57-1 was recognized as a light chain and BT150-3 was r ecognized as a heavy chain of BTx-A. Also, applying the phage display metho d, we investigated the antibodies' neutralizing B-cell epitopes, These immu nopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-li-Asp-Cys for BT150, The synthetic peptide P4M (KGTFDPLQEPRT) corr esponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically, This peptide was also shown by competitiv e inhibition assay to be able to inhibit phage clone binding to BT57-1, Asp artic acid (D-5) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine ( K-5). Finally, immunizing mice with the selected phage clones elicited a sp ecific humoral response against BTx-A, These results suggest that phage-dis played random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies, In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the i dentification of the BTx-A receptor and the design of a BTx-A vaccine.