Luminescence resonance energy transfer (LRET) assays for clostridial toxin activity

1. A Clostridial toxin substrate comprising:

a. a lanthanide donor complex comprising a lanthanide binding site and a lanthanide ion;

b. an acceptor; and

c. a Clostridial toxin recognition sequence including a P 1 -P 1 ′ cleavage site that intervenes between the lanthanide donor complex and the acceptor;

wherein the acceptor has an absorbance spectrum overlapping the emission spectrum of the lanthanide donor complex; and

wherein, under the appropriate conditions, resonance energy transfer is exhibited between the lanthanide donor complex and the acceptor.

2. The substrate of claim 1 , wherein the lanthanide binding site has a Kd for a lanthanide ion of less than 1 μM.

3. The substrate of claim 1 , wherein the lanthanide binding site comprises a chelate, a cryptate, or an EF-hand motif.

4. The substrate of claim 1 , wherein the acceptor is an acceptor fluorophore.

5. The substrate of claim 4 , wherein the acceptor fluorophore comprises a fluorescent protein, a fluorophore binding protein, or a fluorescent dye.

6. The substrate of claim 1 , wherein the acceptor is a non fluorescent acceptor.

7. The substrate of claim 1 , wherein the lanthanide donor complex further comprises an antenna.

8. The substrate of claim 7 , wherein the antenna is distinct from the lanthanide-binding site.

9. The substrate of claim 7 , wherein the antenna is incorporated with lanthanide biding site.

10. The substrate of claim 1 , wherein the substrate is a peptide having a length of at most 20 residues, at most 50 residues, at most 100 residues, or at most 150 residues.

11. The substrate of claim 1 , wherein the substrate is a peptide having a length of at least 100 residues, at least 300 residues, at least 500 residues, or at least 700 residues.

12. The substrate of claim 1 , wherein the Clostridial toxin recognition sequence is a peptide having a length of at most 20 residues, at most 30 residues, at most 40 residues or at most 50 residues.

13. The substrate of claim 1 , wherein the Clostridial toxin recognition sequence is a peptide having a length of at least 20 residues, at least 50 residues, at least 100 residues, or at least 200 residues.

14. The substrate of claim 1 , wherein the substrate can be cleaved with an activity of at least 1 nanomole/minute/milligram toxin, at least 20 nanomoles/minute/milligram toxin or at least 100 nanomoles/minute/milligram toxin.

15. A method of detecting activity of a Clostridial toxin, the method comprising the steps of:

a. treating with a test sample, under conditions suitable for Clostridial toxin protease activity, a Clostridial toxin substrate according to claim 1 ;

b. exciting the lanthanide donor complex;

c. detecting resonance energy transfer of the treated substrate from the test sample; and

d. comparing the resonance energy transfer detected from the test sample with the resonance energy transfer detected from a control sample subjected to steps (a)-(c);

wherein a difference in fluorescence resonance energy transfer of the test sample as compared to a control sample is indicative of activity from a Clostridial toxin.

16. The method of claim 15 , wherein the sample is selected from the group consisting of a crude cell lysate, a bulk Clostridial toxin, a partially purified Clostridial toxin, a purified Clostridial toxin, an isolated Clostridial toxin light chain, and a formulated Clostridial toxin product.

17. The method of claim 16 , wherein the sample comprises a formulated Clostridial toxin product.

18. The method of claim 15 , wherein the sample is selected from the group consisting of a raw food, a partially cooked or processed food, a cooked or processed food, a beverage, an animal feed, a soil sample, a water sample, and a pond sediments.

19. The substrate of claim 1 , wherein the Clostridial toxin recognition sequence comprises SEQ ID NO: 96.

20. The substrate of claim 1 , wherein the Clostridial toxin recognition sequence comprises SEQ ID NO: 97.

21. The substrate of claim 1 , wherein the Clostridial toxin recognition sequence comprises SEQ ID NO: 98.

22. The substrate of claim 1 , wherein the Clostridial toxin recognition sequence comprises SEQ ID NO: 99.

23. The substrate of claim 1 , wherein the Clostridial toxin recognition sequence comprises SEQ ID NO: 100.

24. The substrate of claim 1 , wherein the Clostridial toxin recognition sequence comprises SEQ ID NO: 101.

25. The substrate of claim 1 , wherein the Clostridial toxin recognition sequence comprises SEQ ID NO: 102.

26. The substrate of claim 1 , wherein the Clostridial toxin recognition sequence comprises SEQ ID NO: 103.

27. The substrate of claim 1 , wherein the Clostridial toxin recognition sequence comprises SEQ ID NO: 104.