Kits and reagents for luciferase detection assay system
View Patent ↗The invention relates to methods and kits for detecting enzyme activity using bioluminescence. In particular, it relates to a novel assay system with increased light yield for a sensitive and convenient detection of luciferase activity, such as luciferase reporter enzyme activity. Provided is a method of detecting luciferase activity in a sample, comprising incubating the sample in the presence of luciferin and ATP to allow the generation of a light signal, wherein said light signal is enhanced by performing the incubation in a reaction mixture comprising phosphate and ammonium ions, and measuring the light signal. The invention also relates to kits for use in such method.
1. A kit for detecting luciferase activity, comprising an assay reagent and a luciferase substrate, wherein said assay reagent comprises at least 100 mM phosphate ions and more than 200 mM ammonium ions.
2. The kit according to claim 1 , wherein said assay reagent comprises at least 200 mM phosphate ions and/or at least 300 mM ammonium ions.
3. The kit according to claim 1 , wherein the phosphate ions in the assay reagent are provided by H 3 PO 4 , H 2 PO 4 − , or HPO 4 2− , PO 4 3− or a combination thereof.
4. The kit according to claim 1 , wherein the ammonium ions in the assay reagent are provided by an ammonium salt, an amine or a zwitterionic compound, or a combination thereof.
5. The kit according to claim 4 , wherein the ammonium ions are provided by an amine-based buffer, preferably Tris (tris(hydroxymethyl)aminomethane) or Bis-Tris (bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane).
6. The kit according to claim 4 , wherein said amine is a primary or secondary amine, preferably an amine selected from the group consisting of monoethanolamine, diethanolamine, monoethylamine and diethylamine.
7. The kit according to claim 1 , wherein ammonium ions in the assay reagent are present in excess of the phosphate ions.
8. The kit according to claim 7 , wherein ammonium ions are present in at least 1.4-fold excess of the phosphate ions.
9. The kit according to claim 1 , wherein the assay reagent comprises Tris, ethanolamine and/or ammonium phosphate neutralized with phosphoric acid to neutral pH.
10. The kit according to claim 1 , wherein the assay reagent comprises Tris-phosphate buffer pH 7, D-luciferin, ATP, Mg 2+ , EDTA, Ca 2+ , thiosulfate and a non-ionic detergent.
11. The kit according to claim 1 , wherein the assay reagent comprises no exogenous or supplementary thiol compound.
12. The kit according to claim 1 , wherein said luciferase substrate is in a lyophilised form.
13. The kit according to claim 1 , further comprising a cell lysing agent.
14. The kit according to claim 13 , wherein said cell lysing agent is present in said assay reagent.
15. The kit according to claim 1 , further comprising a luciferase standard.
16. The kit according to claim 1 , further comprising a non-thiol reducing agent.
17. The kit according to claim 16 , wherein the reducing agent is thiosulfate, sulfite, or dithionite.
18. A Luciferase detection reagent comprising at least 100 mM phosphate ions and at least 200 mM ammonium ions, luciferase substrate, ATP, divalent cation and a reducing agent.
19. The reagent according to claim 18 , comprising at least 200 mM phosphate ions, and/or at least 300 mM ammonium ions.
20. The reagent according to claim 18 , comprising no exogenous or supplementary thiol compound.
21. The reagent according to claim 18 , further comprising a stabilizer, chelating agent, reducing agent and/or a cell lysing agent.
22. The reagent according to claim 18 , comprising a Tris-phosphate buffer, D-luciferin, ATP, Mg 2+ , EDTA, Ca 2+ , thiosulfate and a non-ionic detergent.