Botulism, a neuroparalytic disease caused by toxins from C. botulinum bacteria, affects more than half a million people worldwide per year. The goal of this project is to develop a rapid, sensitive and specific in vitro assay test for the diagnosis and detection of botulinum neurotoxins that can be used for effective monitoring of the food supply and diagnosis of infected patients. This assay test is meant to replace the currently used """"""""mouse lethality test"""""""". The botulinum and tetanus toxins have recently been identified as zinc metalloproteases which cleave specific proteins in the neuron. Thus, we plan to exploit the proteolytic activity of these toxins as a method of toxin detection. Specific synthetic fluorogenic and/or chromogenic substrates for these toxins will be developed for use in a commercially viable, in vitro detection kit. In Phase I, four steps will be taken toward this goal : (l) reference substrates will be developed (2) suitable reporter groups will be developed for incorporation into the substrates (3) the ability to inhibit the proteolytic activity of toxin in vitro with specific antisera will be demonstrated, and (4) potential chemistries for the immobilization of substrates on various surfaces will be examined.
In vitro detection and diagnosis of botulinum toxin has widespread application in food safety and medical diagnosis. Food manufacturers worldwide must monitor all products for the presence of C. botulinum based on the neurotoxin it elaborates. The """"""""mouse lethality test"""""""" in current use is slow, costly, and socially undesirable. Thus manufacturers are looking for an alternative, in vitro assay for botulinum toxin. For these same reasons, medical diagnosticians are also seeking alternative detection methods.
