Fluorescence polarization assays for determining clostridial toxin activity

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

• (a) measuring fluorescence polarization of a clostridial toxin substrate by exciting said clostridial toxin substrate with plane polarized light, said clostridial toxin substrate comprising
• (i) a donor fluorophore;
• (ii) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;
• and
• (iii) a clostridial toxin recognition sequence comprising a cleavage site, wherein said cleavage site intervenes between said donor fluorophore and said acceptor;
• (b) treating said clostridial toxin substrate with a sample, under conditions suitable for clostridial toxin protease activity;
• and
• (c) measuring fluorescence polarization of said treated clostridial toxin substrate by exciting said treated clostridial toxin substrate with plane polarized light;
• wherein an increase in said fluorescence polarization measured in step (c) relative to said fluorescence polarization measured in step (a) is indicative of the activity of said clostridial toxin in said sample due to cleavage of said treated clostridial toxin substrate by said Clostridial toxin;
• wherein a decrease in said fluorescence polarization measured in step (c) relative to said fluorescence polarization measured in step (a) is indicative of the presence of said clostridial toxin in said sample due to complex formation of said clostridial toxin with said treated clostridial toxin substrate;
• and
• wherein no substantial change in said fluorescence polarization measured in step (c) relative to said fluorescence polarization measured in step (a) is indicative of the lack of presence or activity of said clostridial toxin in said sample.