A sensitive and useful radioimmunoassay for neurotoxin and its haemagglutinin complex from Clostridium botulinum

A sensitive radioimmunoassay for the detection of botulinum toxin, produced by Clostridium botulinum, was developed. This employs homogeneous botulinum neurotoxin type A and its 125I-labelled derivative of high specific radioactivity, rather than its complex with haemagglutinin as used hitherto. The sensitivity of the assay is 1 ng of neurotoxin per ml, which is equivalent to 80 LD50 units (half-lethal doses) in mice. Neurotoxin and its complex with haemagglutinin were measurable with equal sensitivity when using antibodies against botulinum neurotoxin type A. Specificity of the assay was demonstrated by the lack of response to type B and E botulinum toxins and to heat-inactivated botulinum toxin or extracts of Clostridium sporogenes strain BL46, which contains many surface antigenic determinants common to Clostridium botulinum. Using appropriate conditions, neurotoxin added to fish extract could be quantified accurately, proportionality being observed between the amounts of standard toxin added. In addition, the amounts of toxin species produced by culturing Clostridium botulinum in canned fish was measurable; the values obtained were comparable to those observed by the mouse bioassay. Moreover, the fish samples gave a dose-response curve in the competition radioimmunoassay which was paralleled by the response of botulinum neurotoxin standards. This assay offers the most sensitive, reliable immunological method available for the quantitation of molecular forms of botulinum toxin. As the technique can be used with unpurified fish extracts, it should be widely applicable to different types of samples contaminated with botulinum toxin; furthermore, the clinical diagnosis of human botulism could be substantiated with this method.