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 method of detecting luciferase activity in a sample, comprising
a. incubating the sample in the presence of luciferin and ATP and a bivalent cation to allow the generation of a light signal,
b. wherein the incubation is performed in the absence of an exogenous or supplementary thiol-containing compound, and
c. wherein the total light yield of said light signal is enhanced by performing the incubation in a reaction mixture comprising ammonium ions and at least 20 mM phosphate ions, and measuring the light signal.
2. The method according to claim 1 , wherein said phosphate concentration is more than 60 mM.
3. The method according to claim 1 , wherein the phosphate ions are provided by H3PO4, H2PO4-, or HPO42-, PO43- or a combination thereof.
4. The method according to claim 1 , wherein said ammonium ion is provided by an ammonium salt, an amine or a zwitterionic compound, or a combination thereof.
5. The method according to claim 1 , wherein said ammonium ion is provided by an amine-based buffer, preferably Tris (tris(hydroxymethyl)aminomethane) or Bis-Tris (bis(2-hydroxyethyl)imino-tris(hyd roxymethyl)methane).
6. The method 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 method according to claim 1 , wherein the ammonium ion concentration is at least 20 mM and at most 60 mM.
8. The method according to claim 1 , wherein the reaction mixture pH ranges between 5 and 9.
9. The method according to claim 1 , wherein said reaction mixture furthermore comprises a reducing agent.
10. The method according to claim 9 , wherein said reducing agent is a non-thiol compound, preferably a compound selected from the group consisting oftris (2-carboxyethyl)phosphine hydrochloride (TCEP), thiosulfate, sulfite and dithionite.
11. A method according to claim 1 , wherein the ammonium ion concentration is at least 60 mM and at most 100 mM.
12. A method according to claim 1 , wherein the ammonium ion concentration is at least 100 mM and at most 250 mM.
13. A method according to claim 1 , wherein the ammonium ion concentration is at least 250 mM and at most 500 mM.