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 release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses 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 immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic 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 specific humoral response against BTx-A. These results suggest that phage-displayed 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 identification of the BTx-A receptor and the design of a BTx-A vaccine.
Wu
H C
HC
Institute of Preventive Medicine, National Defense Medical Center, San-Hsia, Taiwan. hancw@pchome.com.tw
Yeh
C T
CT
Huang
Y L
YL
Tarn
L J
LJ
Lung
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001906:Botulism / Q000382:microbiology / Q000517:prevention & control; D003014:Clostridium botulinum / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007114:Immunization; D015151:Immunoblotting; D051379:Mice; D008813:Mice, Inbred ICR; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology / Q000378:metabolism
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D018985:Epitopes, B-Lymphocyte; D019151:Peptide Library; D010455:Peptides; D019274:Botulinum Toxins, Type A