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Sensitive, In Vitro, Bifunctional, Potency Assay For Botulinum Neurotoxin Type A

Christian, T.; Shine, N.

Botulinum toxin progresses through a three step process leading to the interruption of synaptic transmission. The first step is binding of toxin to neuronal cells and incorporation of the toxin into an endosome; the second step is translocation of the enzymatic light chain out of the endosome and into the cytosol of the neuron; and the final step is cleavage of synaptosomal proteins to prevent vesicle docking and neurotransmitter release. In this assay, the luminal domain loop of SV2c has been attached to magnetic beads and used as a capture reagent for botulinum neurotoxin type A. After capture, the activity of the toxin is assessed using one of our FRET substrates, SNAPtide. Analysis of the cleaved peptide is via HPLC using a fluorescence detector. Optimization of reaction conditions and detection limits will be described. This assay monitors two of the three steps of toxin activity, binding and cleavage. As such, this assay has the potential to be part of an alternative method to replace the mouse bioassay.