METHODS FOR DETECTION OF BOTULINUM NEUROTOXIN

摘要

Provided herein is a large immuno-sorbent surface area assay (ALISSA) for the rapid and sensitive detection of botulinum neurotoxins (BoNTs) and anthrax toxin. This assay is designed to capture a low number of toxin molecules and to measure their intrinsic protease activity via conversion of a fluorogenic or luminescent substrate. Also provided herein are novel peptides that can be specifically cleaved by BoNT and novel peptides that are resistant to cleavage by BoNT. The combination of these cleavable and control peptides can be used for implementation of an exemplary ALISSA used to specifically detect BoNT enzymatic activity. Furthermore, the ALISSA as described herein may also be used in a column based format for use in a high-throughput system for testing large quantities of samples.

主权项

1. A method for detecting the presence of a BoNT toxin in a sample comprising:
a) exposing the sample putatively containing a BoNT toxin to an enrichment matrix comprising a substrate fusion protein capable of eliciting a detectable luminogenic signal following modification by the BoNT toxin, the exposure occurring under conditions permitting the modification of the substrate fusion protein by the BoNT toxin, the substrate fusion protein comprising a BoNT cleavage site wherein:
(i) if the BoNT toxin is BoNT/A, the substrate fusion protein comprises a fragment of the synaptosome-associated protein of 25 kDa (SNAP25) containing the BoNT/A cleavage site;(ii) if the BoNT toxin is BoNT/B, the substrate fusion protein comprises a fragment of vesicle-associated membrane protein (VAMP) containing the BoNT/B cleavage site;(iii) if the BoNT toxin is BoNT/C, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/C cleavage site or a fragment of SNAP25 containing the BoNT/C cleavage site;(iv) if the BoNT toxin is BoNT/D, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/D cleavage site;(v) if the BoNT toxin is BoNT/E, the substrate fusion protein comprises a fragment of SNAP25 containing the BoNT/E cleavage site;(vi) if the BoNT toxin is BoNT/F, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/F cleavage site; and(vii) if the BoNT toxin is BoNT/G, the substrate fusion protein comprises a fragment of VAMP containing the BoNT/G cleavage site; and b) detecting the presence of the BoNT toxin by measuring a change in light emission in the sample.