Assays for detecting enzymatic activity
The present invention relates to method of detecting activity of a sample. Some methods involve detecting a neo-binding-site created by the activity such as detecting an activity of a protease by detecting a neo-binding-site created by cleavage of a substrate by the protease.
1. A method of detecting protease activity in a sample, comprising:
combining in a solution at least the sample and a substrate under conditions compatible for the protease activity,
wherein the substrate comprises an amino acid sequence selected from the group consisting of IEGRDYKDDDDKGSHHHHHH (SEQ ID NO:8), IDGRDYKDDDDK (SEQ ID NO:41), IDGRDYKDDDDKGS (SEQ ID NO:42), IDGRDYKDDDDKGSHHHHHH (SEQ ID NO:43), AEGRDYKDDDDK (SEQ ID NO:44), AEGRDYKDDDDKGS (SEQ ID NO:45), and AEGRDYKDDDDKGSHHHHHH (SEQ ID NO:46),
wherein the amino acid sequence comprises a cleavage site for the protease,
wherein cleavage at the cleavage site generates a neo-binding-site, and
wherein the cleavage site is heterologous with respect to the neo-binding-site;
previously, subsequently or concurrently adding to the solution a first binding molecule, wherein the first binding molecule preferentially binds the neo-binding-site after cleavage of the substrate by the protease activity as compared to binding to the neo-binding-site in the uncleaved substrate; and
detecting the binding of the first binding molecule to a fragment of the cleaved substrate.
2. The method of claim 1 , wherein the protease activity is factor Xa (FXa) activity.
3. The method of claim 2 , wherein the cleavage site comprises an amino acid sequence selected from the group consisting of IEGR (SEQ ID NO:1), IDGR (SEQ ID NO:39), and AEGR (SEQ ID NO:40).
4. The method of claim 1 , wherein the neo-binding-site is located or bound to the uncleaved substrate at a site carboxy-terminal to the cleavage site.
5. The method of claim 1 , method of claim 1 , wherein the cleavage site comprises an amino acid sequence selected from the group consisting of IEGR (SEQ ID NO:1), IDGR (SEQ ID NO:39), AEGR (SEQ ID NO:40), DEVD (SEQ ID NO:13), IETD (SEQ ID NO:14), VDVAD (SEQ ID NO:15), VEID (SEQ ID NO:16), YVAD (SEQ ID NO:17), DALD (SEQ ID NO:18), VEVD (SEQ ID NO:19), VEMD (SEQ ID NO:20), SSTD (SEQ ID NO:21) and WEND (SEQ ID NO:22).
6. The method of claim 1 , wherein the solution comprises GPRP-NH2 (SEQ ID NO:32).
7. The method of claim 1 , wherein the solution comprises calcium.
8. The method of claim 1 , wherein the solution comprises a blocker of fibrin network formation.
9. The method of claim 2 , wherein the method is used to quantify the amount of the factor Xa activity in the sample.
10. The method of claim 1 , wherein the substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4.
11. The method of claim 10 , wherein amino acid sequence SEQ ID NO:2 or SEQ ID NO:4 is immediately carboxy-terminal to the FXa cleavage site.
12. The method of claim 1 , wherein the substrate is bound to a surface and the surface binds the substrate at a site carboxy-terminal of the cleavage site.
13. The method of claim 1 , wherein the neo-binding-site comprises an amino acid sequence.
14. The method of claim 13 , wherein the neo-binding-site comprises an amino acid sequence selected from the group consisting of DYKDDDDK (SEQ ID NO:2), DIP EN (SEQ ID NO:33), ARG (SEQ ID NO:34), ARGSV (SEQ ID NO:35), ARGSVIL (SEQ ID NO:36) and FFGV (SEQ ID NO:37).
15. The method of claim 1 , wherein the first binding molecule is selected from the group consisting of an antibody, an aptamer, a ligand and a receptor.
16. The method of claim 1 , wherein the first binding molecule comprises at least one detection label.
17. The method of claim 16 , wherein the at least one detection label is selected from an electrochemiluminescence label, an enzyme label, a fluorophore, a latex particle, a magnetic particle, a radioactive element, a phosphorescent dye, a dye crystalite, a gold particle, a silver colloidal particle, a selenium colloidal particle, a metal chelate, a coenzyme, an electro active group, an oligonucleotide and a stable radical.
18. The method of claim 17 , wherein the metal chelate is a ruthenium or an osmium metal chelate.
19. A method of detecting protease activity in a sample, comprising:
combining in a solution at least the sample and a substrate under conditions compatible for the protease activity,
wherein the substrate comprises an amino acid sequence consisting of the sequence set forth in IEGRDYKDDDDK (SEQ ID NO:3) or IEGRDYKDDDDKGS (SEQ ID NO:5),
wherein the amino acid sequence comprises a cleavage site for the protease,
wherein cleavage at the cleavage site generates a neo-binding-site, and
wherein the cleavage site is heterologous with respect to the neo-binding-site;
previously, subsequently or concurrently adding to the solution a first binding molecule, wherein the first binding molecule preferentially binds the neo-binding-site after cleavage of the substrate by the protease activity as compared to binding to the neo-binding-site in the uncleaved substrate; and
detecting the binding of the first binding molecule to a fragment of the cleaved substrate.
20. The method of claim 19 , wherein the protease activity is factor Xa (FXa) activity.
21. The method of claim 20 , wherein the cleavage site comprises an amino acid sequence selected from the group consisting of IEGR (SEQ ID NO:1), IDGR (SEQ ID NO:39), and AEGR (SEQ ID NO:40).
22. The method of claim 19 , wherein the solution comprises a blocker of fibrin network formation.
23. The method of claim 22 , wherein the method is used to quantify the amount of the factor Xa activity in the sample.
24. The method of claim 19 , wherein the substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4.
25. The method of claim 24 , wherein amino acid sequence SEQ ID NO:2 or SEQ ID NO:4 is immediately carboxy-terminal to the FXa cleavage site.
26. The method of claim 19 , wherein the substrate is bound to a surface and the surface binds the substrate at a site carboxy-terminal of the cleavage site.
27. The method of claim 19 , wherein the neo-binding-site comprises an amino acid sequence.
28. The method of claim 27 , wherein the neo-binding-site comprises an amino acid sequence selected from the group consisting of DYKDDDDK (SEQ ID NO:2), DIP EN (SEQ ID NO:33), ARG (SEQ ID NO:34), ARGSV (SEQ ID NO:35), ARGSVIL (SEQ ID NO:36) and FFGV (SEQ ID NO:37).
29. The method of claim 19 , wherein the first binding molecule is selected from the group consisting of an antibody, an aptamer, a ligand and a receptor.
30. The method of claim 19 , wherein the first binding molecule comprises at least one detection label.
31. The method of claim 30 , wherein the at least one detection label is selected from an electrochemiluminescence label, an enzyme label, a fluorophore, a latex particle, a magnetic particle, a radioactive element, a phosphorescent dye, a dye crystalite, a gold particle, a silver colloidal particle, a selenium colloidal particle, a metal chelate, a coenzyme, an electro active group, an oligonucleotide and a stable radical.
32. The method of claim 31 , wherein the metal chelate is a ruthenium or an osmium metal chelate.