**8. Enzymatic assays to detect toxins**

Rapidly distinguishing between the presence of active versus inactive toxin is critical for effective medical intervention in toxicoses. As BoNTs are zinc metalloproteases, knowledge of the human targets for these enzymes has enabled development of enzyme-substrate assays. Activity assays have been developed using a wide variety of detection systems. Toxin samples can be treated with recombinant versions of host–target substrates (such as SNAP-25), and the cleavage products can be detected using immunoblotting. Alternatively, fluorogenic peptide substrates emit a signal when cleaved. One such system uses a peptide ("SNAPtide") with reverse-phase HPLC and a fluorescence detector to detect as low as 5 pg/mL of BoNT/A in skim milk [45]. Other peptide substrates (VAMPtide and SYNTAXtide) have been used for detection of their cognate BoNTs [46]. The levels of substrate cleavage correlate well with toxin activity.
