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Sensitive and Specific Assay to Measure Endoprotease Activity of Botulinum Toxin Type A Holotoxin in Milk.

Shine, N.; Eaton, L.; Crawford, K.

An assay focused on the enzymatic property of botulinumtoxin has been developed. This assay is designed to detect specific cleavage products resulting from activity of the toxin on a synthetic peptide substrate. Measurement of the proteolyticactivity of these botulinumtoxins provides a potentially sensitive and direct means for monitoring the presence of toxin. Substantialsignal amplification can be expected as a result of catalytic turnover of the substrate. Also, while a genetically engineered toxin might evade other means of detection based on immunoassay or PCR, for example, changes in the enzyme activity which is a fundamental attribute of C. botulinumintoxication are less likely. Therefore, the endoproteaseactivity would always be available to use for detection. Also, unlike immunodetection, functional assays can distinguish active from inactive toxins. The assay described here analyzes the cleavage of a biotinylatedderivative of SNAPtideTM(US patent #6504006) a FRET substrate for botulinumneurotoxin, type A (BTA) which was designed at List Laboratories. The FRET pair has been replaced by a single dinitrophenylgroup (Dnp) for detection of the cleaved substrate. A rapid and sensitive HPLC protocol has been developed for specific detection of the substrate cleaved by BTA. In undiluted whole milk the lowest concentration of BTA holotoxin detectable is less than 1.0 ng/ml after 5 hours of digestion. The sample preparation of milk is simple and efficient, resulting in HPLC chromatograms free of interfering components. The peak for the cleaved product is well-resolved with a baseline allowing confident assignment of low intensity peaks. Still lower levels of detection could be achieved in a shorter time frame by including a BTA concentration step during the sample preparation and by using a fluorophoreinstead of the Dnp. The use of a peptidicsubstrate in serum, on the other hand, is problematic due to the presence of other proteases and the likelihood of nonspecific cleavage of the substrate. Preliminary studies with bovine serum spiked with BTA indicated nonspecific hydrolysis of the biotinylatedsubstrate. The results of the HPLC experiments will be described.