Luciferase signal enhancing compositions

Abstract

Reagents and compositions for use in reactions catalysed by luciferase enzymes, and in particular for use in luciferase-based gene reporter assays are described. The invention also provides methods and compositions for, inter alia, increasing the sensitivity and/or improving the kinetics of luciferase-catalysed reactions.

The invention claimed is: 
     
       1. A method for conducting a luciferase-based assay comprising,
 expressing within a eukaryotic cell a luciferase that is secreted in its native form, wherein the luciferase lacks a functional signal peptide such that it is expressed intracellularly in the eukaryotic cell; 
 recovering the activity of the luciferase by contacting the luciferase with a reagent composition that provides an environment suitable for conversion of the luciferase into its active folded form, wherein the reagent composition comprises an agent selected from the group consisting of bromide anion, chelator, redox buffer, and buffer with a pH greater than 8.0; 
 contacting the luciferase with a substrate of the luciferase enzyme; and 
 detecting or measuring the bioluminescence. 
 
     
     
       2. The method of  claim 1 , wherein the luciferase in its active form includes disulphide bonds between cysteine residues. 
     
     
       3. The method of  claim 1 , wherein the recovering activity comprises a cell lysis step. 
     
     
       4. The method of  claim 1 , wherein the method is employed in a reporter gene assay. 
     
     
       5. A method for conducting a luciferase-based assay comprising,
 expressing within a eukaryotic cell a luciferase that is secreted in its native form, wherein the luciferase lacks a functional signal peptide such that it is expressed intracellularly in the eukaryotic cell; 
 recovering the activity of the luciferase by contacting the luciferase with a reagent composition that provides an environment suitable for conversion of the luciferase into its an active folded form state, wherein the reagent composition comprises one or more of bromide anion, chelator, redox buffer, an oxidising agent, and buffer with a pH greater than 8.0; 
 contacting the luciferase with a substrate of the luciferase enzyme; and 
 detecting or measuring the bioluminescence. 
 
     
     
       6. The method of  claim 5 , wherein the luciferase in its active form state includes disulphide bonds between cysteine residues. 
     
     
       7. The method of  claim 5 , wherein the recovering activity comprises further comprising a cell lysis step. 
     
     
       8. The method of  claim 5 , wherein the method is employed in a reporter gene assay. 
     
     
       9. The method of claim 5, wherein conversion of the luciferase into an active state means either the conversion from an inactive state to an active state, or the conversion from a partially active or less active state to a more active state.  
     
     
       10. A method for detecting or quantifying the amount of a recombinant luciferase in a sample, the method comprising:
 (a) contacting the sample with a reagent composition comprising one or more of bromide anion, a chelator, an oxidising agent, redox buffer, and a buffer with a pH greater than 8.0;   (b) contacting the sample with a substrate of the luciferase enzyme; and   (c) detecting or quantifying bioluminescence in the sample;   wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.    
     
     
       11. A method for detecting or quantifying the amount of a recombinant luciferase in a sample, the method comprising:
 (a) contacting the sample with a reagent composition that provides an environment that enables conversion of the luciferase into an active state, the reagent composition comprising one or more of bromide anion, a chelator, an oxidising agent, redox buffer, and a buffer with a pH greater than 8.0;   (b) contacting the sample with a substrate of the luciferase enzyme; and   (c) detecting or quantifying bioluminescence in the sample;   wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.    
     
     
       12. The method of claim 11, wherein conversion of the luciferase into an active state means either the conversion from an inactive state to an active state, or the conversion from a partially active or less active state to a more active state.  
     
     
       13. The method of claim 11, wherein step (b) occurs simultaneously with, or following step (a).  
     
     
       14. The method of claim 11, wherein the luciferase utilizes coelenterazine or an analogue or derivative thereof as a substrate.  
     
     
       15. The method of claim 11, wherein the luciferase is of marine origin.  
     
     
       16. The method of claim 11, wherein the luciferase in its active state includes disulphide bonds between cysteine residues.  
     
     
       17. The method of claim 11, wherein the luciferase lacks a functional signal peptide thereby providing intracellular expression.  
     
     
       18. The method of claim 11, wherein the method is part of a reporter gene assay.  
     
     
       19. The method of claim 11, further comprising contacting the sample with a non-ionic detergent.  
     
     
       20. The method of claim 11, further comprising contacting the sample with a zwitterionic detergent.  
     
     
       21. The method of claim 11, wherein the reagent composition comprises one or more of a chelator and a redox buffer.  
     
     
       22. A method for determining the amount and/or activity of a recombinant luciferase in a cell or sample of cells, the method comprising:
 a) lysing the cell or sample of cells to prepare a cell lysate;   b) contacting the cell lysate with a reagent composition comprising one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a pH greater than 8.0;   c) adding a substrate of the luciferase; and   d) detecting bioluminescence in the sample;   wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.    
     
     
       23. A method for determining the amount and/or activity of a recombinant luciferase in a cell or sample of cells, the method comprising:
 a) lysing the cell or sample of cells to prepare a cell lysate;   b) contacting the cell lysate with a reagent composition that provides an environment that enables conversion of the luciferase into an active state, comprising one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a pH greater than 8.0;   c) adding a substrate of the luciferase; and   d) detecting bioluminescence in the sample;   wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.    
     
     
       24. The method of claim 23, wherein conversion of the luciferase into an active state means either the conversion from an inactive state to an active state, or the conversion from a partially active or less active state to a more active state.  
     
     
       25. The method of claim 23, wherein step (c) occurs simultaneously with, or following step (b).  
     
     
       26. The method of claim 23, wherein the luciferase lacks a functional signal peptide.  
     
     
       27. The method of claim 23, wherein the reagent composition comprises one or more of a chelator and a redox buffer.  
     
     
       28. A reagent composition for the detection or quantification of a recombinant luciferase in a sample, wherein the reagent composition provides an environment that enables conversion of the luciferase into an active state and comprises an optional detergent, bromide anion and a luciferase substrate selected from coelenterazine or an analogue or derivative thereof or luciferin or an analog thereof, wherein the optional detergent, if present, is a non-ionic or zwitterionic detergent, and wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.  
     
     
       29. The reagent composition of claim 28, further comprising a non-ionic detergent.  
     
     
       30. The reagent composition of claim 28, further comprising a zwitterionic detergent.  
     
     
       31. The reagent composition of claim 28, further comprising one or more of a chelator, an oxidising agent, redox buffer, and a buffer with a pH greater than 8.0.  
     
     
       32. The reagent composition of claim 28, wherein the bromide anion is present at a concentration of at least 1 mM.  
     
     
       33. The reagent composition of claim 28, wherein the bromide anion is present at a concentration of between 5 mM and 500 mM.  
     
     
       34. The reagent composition of claim 28, wherein the luciferase lacks a functional signal peptide.  
     
     
       35. A reagent composition for the detection or quantification of a recombinant luciferase in a sample, wherein the reagent composition comprises an optional detergent, a bromide anion and a luciferase substrate selected from coelenterazine or an analogue or derivative thereof, wherein the optional detergent, if present, is a non-ionic or zwitterionic detergent, and wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.  
     
     
       36. A reagent composition for the detection or quantification of a recombinant luciferase in a sample, wherein the reagent composition comprises an optional detergent, a bromide anion and a luciferase substrate; wherein the composition does not comprise a luciferase, wherein the optional detergent, if present, is a non-ionic or zwitterionic detergent, and wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.  
     
     
       37. A reagent composition for the detection or quantification of a recombinant luciferase in a sample, wherein the reagent composition comprises one or more of bromide anion, a chelator, an oxidising agent, redox buffer, and a buffer with a pH greater than 8.0, an optional detergent, and a luciferase substrate selected from coelenterazine or an analogue or derivative thereof, wherein the optional detergent, if present, is a non-ionic or zwitterionic detergent, and wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.  
     
     
       38. The reagent composition of claim 37, wherein the detergent is present and is a non-ionic detergent.  
     
     
       39. The reagent composition of claim 37, wherein the detergent is present and is a zwitterionic detergent.  
     
     
       40. The reagent composition of claim 37, comprising one or more of a chelator and a redox buffer.  
     
     
       41. The reagent composition of claim 37, wherein the recombinant luciferase lacks a functional signal peptide.  
     
     
       42. A kit for the detection or quantification of a recombinant luciferase in a sample, the kit comprising bromide anion, a luciferase substrate, and an optional detergent, wherein the bromide anion provides an environment that enables conversion of the luciferase into an active state, the luciferase substrate is selected from coelenterazine or an analogue or derivative thereof or luciferin or an analog thereof, the optional detergent is a non-ionic or zwitterionic detergent, and the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.  
     
     
       43. The kit of claim 42, further comprising a non-ionic detergent.  
     
     
       44. The kit of claim 42, further comprising a zwitterionic detergent.  
     
     
       45. The kit of claim 42, further comprising one or more of a chelator, an oxidising agent, redox buffer, and a buffer with a pH greater than 8.0.  
     
     
       46. The kit of claim 42, wherein the bromide anion is present at a concentration of at least 1 mM.  
     
     
       47. The kit of claim 42, wherein the bromide anion is present at a concentration of between 5 mM to 500 mM.  
     
     
       48. The kit of claim 42, wherein the luciferase lacks a functional signal peptide.  
     
     
       49. A kit for the detection or quantification of a recombinant luciferase in a sample, the kit comprising an optional detergent, a bromide anion, and a luciferase substrate selected from coelenterazine or an analogue or derivative thereof, wherein the optional detergent is a non-ionic or zwitterionic detergent, and wherein the recombinant luciferase is a non-secreted variant or derivative thereof of a luciferase that is secreted in its native form.  
     
     
       50. A kit for the detection or quantification of a recombinant luciferase in a sample, the kit comprising an optional detergent, a bromide anion, and a luciferase substrate; wherein the kit component comprising bromide anion does not comprise a luciferase, wherein the optional detergent is a non-ionic or zwitterionic detergent, and wherein the recombinant luciferase is a non-secreted variant or derivative thereof of a luciferase that is secreted in its native form.  
     
     
       51. A kit for the detection or quantification of a recombinant luciferase in a sample, the kit comprising one or more of bromide anion, a chelator, an oxidising agent, redox buffer, and a buffer with a pH greater than 8.0, an optional detergent, and a luciferase substrate selected from coelenterazine or an analogue or derivative thereof, wherein the optional detergent is a non-ionic or zwitterionic detergent, and wherein the recombinant luciferase is a non-secreted variant or derivative thereof of a luciferase that is secreted in its native form.  
     
     
       52. The kit of claim 51, wherein the detergent is present and is a non-ionic detergent.  
     
     
       53. The kit of claim 51, wherein the detergent is present and is a zwitterionic detergent.  
     
     
       54. The kit of claim 51, comprising one or more of a chelator and a redox buffer.  
     
     
       55. The kit of claim 51, wherein the recombinant luciferase lacks a functional signal peptide.  
     
     
       56. A system for conducting a luciferase-based assay comprising,
 a eukaryotic cell that expresses a luciferase that is a non-secreted variant or derivative of a luciferase that is secreted in its native form;   a reagent composition that provides an environment suitable for conversion of the luciferase into an active state, wherein the reagent composition comprises one or more of bromide anion, chelator, redox buffer, an oxidizing agent, and buffer with a pH greater than 8.0;   a substrate of the luciferase enzyme; and   a detector for measuring bioluminescence.    
     
     
       57. The system of claim 56, wherein the luciferase in its active state includes disulphide bonds between cysteine residues.  
     
     
       58. The system of claim 56, further comprising a cell lysis buffer.  
     
     
       59. The system of claim 56, further comprising a reporter gene assay.  
     
     
       60. The system of claim 56, wherein conversion of the luciferase into an active state means either the conversion from an inactive state to an active state, or the conversion from a partially active or less active state to a more active state.  
     
     
       61. The system of claim 56, wherein the detector is selected from the group consisting of a luminometer, a scintillation counter, a photometer, a photomultiplier photometer, a photoemulsion film, and a charge-coupled device.  
     
     
       62. The system of claim 56, further comprising a 96-well plate.  
     
     
       63. The system of claim 56, wherein the recombinant luciferase lacks a functional signal peptide.  
     
     
       64. The system of claim 56, wherein the reagent composition comprises one or more of a chelator and a redox buffer.