Patent ID: 7208285

Claim:
A method of determining protease activity of botulinum toxin serotype A (BoNT/A), comprising the steps of: (a) treating a BoNT/A substrate with a sample under conditions suitable for clostridial toxin protease activity, said BoNT/A substrate comprising (i) a donor fluorophore; (ii) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore; and (iii) a BoNT/A recognition sequence comprising a BoNT/A P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence, said BoNT/A P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence intervening between said donor fluorophore and said acceptor; wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor; (b) exciting said donor fluorophore; and (c) determining resonance energy transfer of said treated substrate relative to a control substrate, wherein a difference in resonance energy transfer of said treated substrate as compared to said control substrate is indicative of BoNT/A protease activity.