Characterization of the Monoclonal Antibody Response to Botulinum Neurotoxin Type A in the Complexed and Uncomplexed Forms

Abstract

Clostridium botulinum produces large complex toxins, which include botulinum neurotoxin (BoNT) and auxiliary non-toxic proteins. We prepared monoclonal antibodies (mAbs) from mice that were immunized several times with BoNT/A after basal immunization with toxoid. We then examined the reactivities of these mAbs to BoNT and toxoid and showed that some mAbs reacted to only BoNT. This result indicates that the antigenicity of BoNT/A partially disappeared with formalin treatment. Some mAbs that specifically recognized either BoNT/A1 or BoNT/A2 were considered useful as detection antibodies specific for the BoNT/A subtype. Results of a neutralizing test with mAbs against either BoNT/A1 or BoNT/A2 showed that neutralizing antibody recognition sites were present in the light chain, heavy chain (N-terminal half), and heavy chain (C-terminal half) domains. Investigation of the different binding capabilities of the mAbs to BoNT and the complex toxin by immunoprecipitation suggested that the light chain of BoNT is exposed at the molecular surface of the complex toxin since there was no difference in the binding of light chain-specific mAb to BoNT and the complex toxin. The heavy chain is related to BoNT binding to non-toxic components, because the reactivity of the heavy chain to some mAbs was influenced by non-toxic components.