Differences in immunological responses of polyclonal botulinum A1 and A2 antitoxin against A1 and A2 toxin

Botulinum type A antitoxin in standard and therapeutic preparation is a polyclonal antibody purified from immunized sera with subtype A1 toxin. To investigate the difference between immunological responses of antitoxin against toxin among different subtypes, we examined the response of polyclonal A1 and A2 antitoxins with A1 and A2 toxins. In the mouse neutralization test, the A1 antitoxin had equivalent potency against both the A1 and A2 toxins. However, the neutralization titer of the A2 antitoxin was 4-9 fold higher against the A2 toxin than against the A1 toxin. Since the titers of the antitoxins were calculated relative to the standard antitoxin, we assumed that the difference between the antibody titers against the test toxins was due the standard antitoxin having different reactivities with the toxins. The binding volume of the A2 toxin against the standard and A1 antitoxins was 3-4% of the binding volume of the A1 toxin. The neutralization curve of the standard antitoxin was parallel against both the A1 and A2 toxins. However, the curve of the A2 antitoxin was not parallel against the A1 and A2 toxins. Furthermore, binding analysis comparing these antitoxins and toxins showed that the A1 antitoxin had a higher binding affinity and slower dissociation speed with the A1 toxin than with the A2 toxin. In contrast, the A2 antitoxin showed a higher binding affinity with the A2 toxin than with the A1 toxin. These findings indicated that antitoxin reacted strongly against the same subtype, and showed a weak response against the toxin of different subtypes. We propose that the existing standard type A antitoxin should be used for reaction with A1 toxins, and that it is necessary to establish a new standard antitoxin for each subtype of toxin.