Cell-based fluorescence resonance energy transfer (FRET) assays for clostridial toxins

A method of determining clostridial toxin activity, comprising the steps of:

• (a) contacting a cell with a sample, said cell comprising
• (i) at least one receptor that binds a clostridial toxin, and
• (ii) a genetically encoded clostridial toxin substrate comprising
• (1) a donor fluorophore;
• (2) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;
• and
• (3) a clostridial toxin recognition sequence comprising a clostridial toxin P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ cleavage site sequence, said clostridial toxin P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ 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 contacted cell relative to a control cell, wherein a difference in resonance energy transfer of said contacted cell as compared to said control cell is indicative of clostridial toxin activity, said clostridial toxin activity comprising cellular uptake of said toxin, membrane translocation and protease activity.