Clostridium botulinum type B toxin was purified by column chromatography steps with DEAE-Sephadex and Sephacryl S-300. Heavy chain and light chain of type B-toxin were prepared by elution from SDS-polyacrylamide gels. The toxin and subunits were labeled with [$^{125}I$]iodine.
To clarify the role of each subunit in binding of botulinum toxin to target cells, binding of labeled toxin and subunits to rat brain synaptosomes was studied. When labeled toxin or subunit was incubated with various amounts of rat brain synaptosomes, light chain did not bind to synaptosomes. In contrast, specific binding of toxin and heavy chain was observed and there was no significant difference in the amounts of toxin and heavy chain bound. When competitive effects of unlabeled toxin and heavy chain on the binding of labeled toxin and heavy chain were examined, binding of labeled toxin and heavy chain to synaptosomes was inhibited to a similar degree by unlabeled toxin. Unlabeled heavy chain showed similar inhibitory effects as unlabeled toxin. The half-inhibition concentrations of unlabeled heavy chain for the bindings of labeled toxin and heavy chain were similar to those of unlabeled toxin. These results indicate that the heavy chain has similar binding properties to synaptosomes as the intact toxin and may also suggest that the heavy chain alone is involved in the binding of botulinum type B toxin to synaptosomes.
BALB/c mice were immunized with type B toxoid or heavy chain, and spleen cells from immunized mice were fused with Sp2/O-Ag14 myeloma cells. Antibody-secreting hybridomas were screened by enzyme-linked immunosorbent assay, and cloned by limiting dilution. Four cell lines were established. The four monoclonal antibodies produced by them were characterized (Sub) Classes were determined by double immunodiffusion tests. ELISA and Immunoblotting tests with heavy and light chain showed that the all recognize the heavy chain of type B toxin. In the neutraliation tests with mice, antibody B3 had much higher neutralizing activity than the other three. When antibodies were examined for the effects on binding of labeled toxin and heavy chain to synaptosomes antibody B3 inhibited the binding effectively and the others had little inhibitory effects. This result indicates that antibody B3 blocks the 'binding site' in the heavy chain and thus prevents binding of the toxin to target cell which results in neutralizing toxic activity of the toxin whereas antibodies B1, B2, and B4 recognize some other sites in the heavy chain rather than the 'binding site.'
