Methods for detecting botulinum toxin with applicability to screening foods against biological terrorist attacks

Seven serologically related, but antigenically different, botulinum toxins (BoNTs) have been identified including types A, B, C, D, E, F, and G. The bacterium Clostridium botulinum along with some strains of Clostridium baratti and Clostridium butyricum are known to produce botulinum toxins responsible for 4 forms of botulism poisoning including food-borne botulism, inhalation botulism, wound botulism, and infant botulism. Botulism toxins consist of a heavy chain (100 kDa), responsible for binding to target cells, and a light chain (50 kDa) responsible for catalytic protein cleaving activity. Light chain has been identified as a zinc endopeptidase that cleaves proteins forming the synaptic vesicle docking and fusion complex (Simpson 1996; Lacy and Stevens 1997). The standard for detection of BoNT toxins is the mouse bioassay, which is able to detect as little as 0.02 ng of toxin. Strengths of the mouse bioassay include conceptual simplicity and sensitivity. While the non-selectivity of the mouse bioassay enables it to detect any BoNT serotype, additional neutralization assays are necessary to determine serotype. Other limitations of the mouse bioassay include expense, expertise related to maintaining mouse-rearing facilities, and time, because as much as 4 d may be required to obtain results (Hallis and others 1996; Witcome and others 1999). Several attempts to replace the mouse bioassay have been made. Methods that have been developed and hold promise for future replacement of the mouse bioassay include mass spectroscopy, immunoassays, polymerase chain reaction (PCR) assays, and assays based upon protease activities of BoNTs. Currently, no single assay appears to be capable of replacing the broadly applicable mouse bioassay.