Kits for luminogenic and nonluminogenic multiplex assays
View Patent ↗Methods and kits to detect the presence or amount of at least one molecule for an enzyme-mediated reaction in a multiplex luminogenic/nonluminogenic assay are provided.
1. A kit comprising:
a fluorogenic cell-permeable substrate which is an AFC or AMC fluorophore covalently linked to a peptide; and
a luminogenic non-cell permeable substrate which is an aminoluciferin covalently linked to a peptide or carboxy protected derivative thereof.
2. The kit of claim 1 further comprising an enzyme capable of mediating a luminescence reaction with the luminogenic substrate.
3. The kit of claim 1 further comprising instructions for conducting a luminogenic reaction and a fluorogenic reaction in a single reaction vessel which comprises the fluorogenic substrate and the luminogenic substrate.
4. The kit of claim 1 , wherein the fluorogenic substrate comprises 7-amino-4-trifluoromethylcoumarin (AFC).
5. The kit of claim 1 , wherein the fluorogenic cell-permeable substrate which is an AFC or AMC fluorophore covalently linked to a peptide comprises a cleavage site for a protease selected from the group consisting of a caspase, factor Xa, tobacco etch virus (TEV) protease, tryptase and secretase.
6. The kit of claim 1 , wherein the luminogenic non-cell permeable substrate is an aminoluciferin covalently linked to a peptide comprising a cleavage site for a protease selected from the group consisting of a caspase, factor Xa, tobacco etch virus (TEV) protease, tryptase and secretase.
7. The kit of claim 1 , wherein the luminogenic non-cell permeable substrate is selected from the group consisting of DEVD-aminoluciferin (DEVD is SEQ ID NO:1), Z-LETD-aminoluciferin (LETD is SEQ ID NO:2), Z-LEHD-aminoluciferin (LEHD is SEQ ID NO:3) or a combination thereof.
8. The kit of claim 1 , wherein the peptide of the aminoluciferin covalently linked to a peptide consists of 1 to 4 amino acids which include a recognition site for a protease.
9. A kit comprising:
a fluorogenic cell-permeable substrate which is an AFC or AMC fluorophore covalently linked to a peptide; and
an enzyme capable of mediating a luminescence reaction.
10. The kit of claim 9 further comprising instructions for conducting a luminogenic reaction and a fluorogenic reaction in a single reaction vessel which comprises the fluorogenic substrate and the enzyme.
11. The kit of claim 9 , wherein the fluorogenic substrate comprises 7-amino-4-trifluoromethylcoumarin (AFC).
12. The kit of claim 11 , wherein the fluorogenic substrate comprises a peptide, the peptide comprising a cleavage site for a protease selected from the group consisting of a caspase, factor Xa, tobacco etch virus (TEV) protease, tryptase and secretase.
13. The kit of claim 9 , further comprising a luminogenic substrate for the enzyme, wherein the luminogenic substrate comprises an aminoluciferin covalently linked to a peptide.
14. The kit of claim 13 , wherein the peptide-linked aminoluciferin comprises a cleavage site for a protease selected from the group consisting of a caspase, factor Xa, tobacco etch virus (TEV) protease, tryptase and secretase.
15. The kit of claim 2 or 9 wherein the enzyme is a luciferase.