Bioluminescence measurement system

• US 5,618,682 A
• Filed: 02/08/1994
• Issued: 04/08/1997
• Est. Priority Date: 02/10/1993
• Status: Expired due to Term

• Current Assignee: Packard Instrument Co., Inc. (Revvity, Inc.)
• Original Assignee: Packard Instrument Co., Inc. (Revvity, Inc.)
• Inventors: Scheirer, Winfried
• Primary Examiner(s): Weimar, Elizabeth C.
• Assistant Examiner(s): Mohamed, Abdel A.
• Application Number:
  US08/193,679
• Time in Patent Office: 1,155 Days
• Field of Search: 435/8, 435/4, 435/6, 435/184, 435/968, 436/35, 436/164, 436/166, 436/172
• US Class Current: 435/8
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 530/802 Chromogenic or luminescent peptides

Abstract

The present invention provides a method of increasing the duration of detectable photon emission of a luciferase-luciferin reaction. The method provides a luciferase-luciferin reaction in which photon emission can be detected for up to and including eight hours. A method of the present invention can also be used to detect the presence of luciferase in biological samples. The present invention also provides a composition used in detecting the presence of luciferase in biological samples.

Claims

1. A method for detecting the presence of luciferase in a biological sample by measuring the luminescence of said sample comprising:

(a) mixing a sample suspected of containing luciferase with a reaction mixture containing luciferin, adenosine triphosphate ("ATP"), cofactors necessary for luciferase catalytic activity, and adenosine monophosphate ("AMP"), the amounts of ingredients in said reaction mixture being selected to produce luminescence having a duration of at least one hour and an intensity that varies substantially linearly with time; and

(b) measuring said luminescence produced by said reaction mixture containing said sample.

2. The method of claim 1, wherein the biological sample comprises a cell that produces luciferase.

3. The method of claim 1 wherein 100 ml of the reaction mixture contains from about 0.2 to about 30 mg luciferin, from about 10 to about 300 mg adenosine triphosphate, from about 0.2 to about 30 mg adenosine monophosphate, from about 200 to about 2000 mg dithiothreitol, and from about 10 to about 50 mg ethylenediaminetetraacetic acid.

4. The method of claim 1, wherein said reaction mixture contains a protease inhibitor, a free radical scavenger, a chelating agent, and a detergent to form an admixture.

5. The method of claim 4, wherein the chelating agent is ethylenediaminetetraacetic acid.

6. The method of claim 4, wherein the protease inhibitor is phenylacetic acid or oxalic acid.

7. The method of claim 4, wherein the detergent is nonylphenoxypolyethoxyethanol.

8. The method of claim 4, wherein the free radical scavenger is dithiothreitol.

9. A method for detecting the presence of luciferase in a biological sample comprising:

mixing a sample suspected of containing luciferase with a reaction mixture containing luciferin, a protease inhibitor, adenosine triphosphate, cofactors necessary for luciferase catalytic activity, adenosine monophosphate, and nonylphenoxypolyethoxyethanol to form an admixture the amounts of ingredients in said reaction mixture being selected to produce luminescence having a duration of at least five hours and an intensity that varies substantially linearly with time; and

measuring the resulting luminescence of the mixed sample and reaction mixture.

10. The method of claim 9 wherein said protease inhibitor is at least one acid selected from the group consisting of phenylacetic acid and oxalic acid.

11. The method of claim 9 wherein said reaction mixture further includes a chelating agent.

12. The method of claim 11 wherein the chelating agent is ethylenediaminetetraacetic acid.

13. The method of claim 12 wherein said reaction mixture further includes a free radical scavenger.

14. The method of claim 13 wherein said free radical scavenger is dithiothreitol.

15. The method of claim 14 wherein 100 ml of the admixture contains from about 0.2 to about 30 mg luciferin, from about 10 to about 300 mg adenosine triphosphate, from about 0.2 to about 30 mg adenosine monophosphate, from about 200 to about 2000 mg dithiothreitol, from about 10 to about 50 mg ethylenediamine-tetraacetic acid, from about 1 to about 10 mg phenylacetic acid, and from about 0.2 to about 5 mg oxalic acid.

16. A method for detecting the presence of luciferase in a biological sample comprising:

creating a reaction admixture containing luciferin, adenosine triphosphate, cofactors necessary for luciferase catalytic activity, a protease inhibitor, adenosine monophosphate, a detergent, a free radical scavenger, and a chelating agent the amounts of ingredients in said admixture being selected to produce luminscence having a duration of at least five hours and an intensity that varies substantially linearly with time,

suspending a biological sample suspected of containing luciferase in a medium containing Mg⁺²,

mixing said reaction admixture with said biological sample such that the ratio of Mg⁺² to said chelating agent is less than about 0.8, and

measuring the luminescence.

17. The method of claim 16 wherein said chelating agent is ethylenediaminetetraacetic acid.

18. The method of claim 16 wherein said protease inhibitor is at least one acid selected from a group consisting of phenylacetic acid and oxalic acid.

19. The method of claim 16 wherein said detergent is nonylphenoxypolyethoxyethanol.

20. The method of claim 16 wherein said free radical scavenger is dithiothreitol.

21. A method for detecting the presence of luciferase in a biological sample comprising:

creating an admixture containing luciferin, adenosine triphosphate, cofactors necessary for luciferase catalytic activity, phenylacetic acid, oxalic acid, adenosine monophosphate, dithiothreitol, ethylenediaminetetraacetic acid, and nonylphenoxypolyethoxyethanol, said admixture being substantially free of Coenzyme A, the amounts of ingredients in said admixture being selected to produce luminescence having a duration of at least five hours and an intensity that varies substantially linearly with time,

suspending a biological sample suspected of containing luciferase in a medium containing Mg⁺²,

mixing said admixture with said biological sample, such that the ratio of Mg⁺² to ethylenediaminetetraacetic acid is between about 0.4 and about 0.8, and

measuring the luminescence.

22. A method for detecting the presence of luciferase in a biological sample comprising:

creating a reaction admixture containing luciferin, adenosine triphosphate, cofactors necessary for luciferase catalytic activity, a protease inhibitor, adenosine monophosphate, a detergent, a free radical scavenger, and ethylenediaminetetraacetic acid, the amounts of ingredients in said admixtuxe being selected to produce luminescence having a duration of at least five hours and an intensity that varies substanially linearly with time,

suspending a biological sample suspected of containing luciferase in a medium containing Mg⁺²,

mixing said reaction admixture with said medium containing the biological sample, said admixture containing sufficient ethylenediaminetetraacetic acid to remove said Mg⁺², and

measuring the luminescence.

23. A method for detecting the presence of luciferase in a biological sample by measuring the luminescence of said sample comprising;

mixing a sample suspected of containing luciferase with a reaction mixture containing from about 0.2 to about 30 mg luciferin, from about 10 to about 300 mg adenosine triphosphate ("ATP"), from about 0.2 to about 35 mg adenosine monophosphate ("AMP"), from about 200 to about 2000 mg dithiothreitol ("DDT"), from about 10 to about 50 mg ethylenediaminetetraacetic acid ("EDTA"), cofactors necessary for luciferase catalytic activity, and nonylphenoxypolyethoxyethanol the amounts of ingredients in said reaction mixture producing a luminescence having a duration of at least five hours and an intensity that varies substantially linearly with time,

suspending a biological sample suspected of containing luciferase in a medium containing Mg⁺²,

mixing said reaction admixture with said biological sample such that the ratio of Mg⁺² to said chelating agent is less than about 0.8, and

measuring said luminescence produced by said reaction mixture containing said sample.

24. A method for detecting the presence of luciferase in a biological sample by measuring the luminescence of said sample comprising:

(a) mixing a sample suspected of containing luciferase with a reaction mixture containing luciferin, adenosine triphosphate ("ATP"), cofactors necessary for luciferase catalytic activity, and adenosine monophosphate ("AMP"), the amounts of ingredients in said reaction mixture being selected to produce luminescence having a duration of at least five hours and an intensity that varies substantially linearly with time; and

(b) measuring said luminescence produced by said reaction mixture containing said sample.

25. A method for detecting the presence of luciferase in a biological sample by measuring the luminescence of said sample comprising:

(a) mixing a sample suspected of containing luciferase with a reaction mixture containing luciferin, adenosine triphosphate ("ATP"), cofactors necessary for luciferase catalytic activity, and adenosine monophosphate ("AMP"), the amounts of ingredients in said reaction mixture being selected to produce luminescence having a duration of at least eight hours and an intensity that varies substantially linearly with time; and

(b) measuring said luminescence produced by said reaction mixture containing said sample.