A sensitive sandwich enzyme immunoassay for free or complexed Clostridium botulinum neurotoxin type A

Fourteen monoclonal antibodies (mAbs) against Clostridium botulinum type A neurotoxin were raised in mice using a carboxy terminal fragment of the toxin for immunization. A two-site immunometric assay was developed which allowed detection of 8 LD50/ml (40 pg/ml) botulinum neurotoxin A and an accurate quantification close to 25 LD50/ml (150 pg/ml). No cross-reactivity was observed with other toxinotypes. During the development of this assay, interference induced by associated protein was observed. By comparing the effect of different buffers, a buffer composed with Tris–HCl and NaCl salts was demonstrated to dissociate protein complexed with the neurotoxin A. Applied to the measurement of the toxin in different matrices, this dissociating buffer ensures the correct quantification.