Luciferase Detection Assay System

Abstract

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 incubating the sample in the presence of luciferin and ATP and a bivalent cation to allow the generation of a light signal, 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 . Method according to  claim 1 , wherein said phosphate concentration is more than 60 mM. 
   
   
       3 . Method according to  claim 1 , wherein the phosphate ions are provided by H 3 PO 4 , H 2 PO 4   − , or HPO 4   2− , PO 4   3−  or a combination thereof. 
   
   
       4 . 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 . 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(hydroxymethyl)methane). 
   
   
       6 . 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 . Method according to  claim 1 , wherein the ammonium ion concentration is at least 20 mM, preferably at least 60 mM, more preferably more than 100 mM. 
   
   
       8 . Method according to  claim 1 , wherein the reaction mixture pH ranges between 5 and 9. 
   
   
       9 . Method according to  claim 1 , wherein said incubation is performed in the absence of exogenously added or supplementary thiol-containing compound, such as DTT, glutathione or CoA, preferably wherein the incubation is performed in the absence of DTT. 
   
   
       10 . Method according to  claim 1 , wherein said reaction mixture furthermore comprises a reducing agent. 
   
   
       11 . Method according to  claim 10 , wherein said reducing agent is a non-thiol compound, preferably a compound selected from the group consisting of tris (2-carboxyethyl)phosphine hydrochloride (TCEP), thiosulfate, sulfite and dithionite. 
   
   
       12 - 17 . (canceled)