Reagents and methods for use in bioluminescence

• US 5,770,391 A
• Filed: 11/30/1995
• Issued: 06/23/1998
• Est. Priority Date: 06/01/1993
• Status: Expired due to Term

• Current Assignee: Celsis International Limited (Pathology Associates, Inc.)
• Original Assignee: Celsis International, PLC
• Inventors: Grant, Peter Leonard, Foote, Nicholas Peter Martin
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/564,083
• Time in Patent Office: 936 Days
• Field of Search: 435/6, 435/7.1, 435/8-26, 435/810, 435/4, 435/28, 436/501
• US Class Current: 435/8
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: C12Q 1/66 involving luciferase

Abstract

The subject invention pertains to a combination of enzyme activities comprising an ATP-degrading enzyme and one or more enzymes capable of degrading substances, other than ATP, that are substrates for the light-emitting reaction of firefly luciferase. These activities can be used to treat a growth medium in order to reduce background to negligible amounts, in a subsequent bioluminescence assay.

Claims

1. A purified composition comprising an apyrase enzyme and one or more enzymes capable of degrading substances, other than ATP, wherein said substances are, or can undergo conversion to become, substrates utilized in the light-emitting forward reaction of firefly luciferase, wherein said one or more enzymes capable of degrading substance other than ATP do not include luciferase and wherein each of said one or more enzymes capable of degrading substances other than ATP reduce the light emission produced from said light-emitting reaction of firefly luciferase.

2. The composition of claim 1, in which a product of reaction of said one or more enzymes capable of degrading substances other than ATP with a non-ATP substrate is ATP.

3. The composition according to claim 1, in which said one or more enzymes capable of degrading substances other than ATP have FAD-degrading activity.

4. The composition according to claim 1, comprising an ATPase and an enzyme having phosphodiesterase I activity.

5. The composition according to claim 1, which comprises an ATPase and an enzyme having phosphatase activity.

6. The composition according to claim 1, in which ATPase is potato apyrase.

7. The composition according to claim 1, in freeze-dried form.

8. The composition according to claim 1, which additionally comprises Mg²⁺ or other divalent metal ion.

9. The composition according to claim 1, which additionally comprises one or more bioluminescence reagents selected from the group consisting of luciferin and luciferase.

10. The composition according to claim 7, in which the components are provided in kit form, in two or more containers.

11. A method for treating a growth medium, which comprises adding to the medium the enzyme activities defined in claim 1.

13. The composition according to claim 8, in which the components are provided in kit form, in two or more containers.

12. A method for reducing the background bioluminescence in a bioluminescence assay which comprises the steps of adding to the sample an ATP-degrading enzyme and one or more enzymes capable of degrading substances, other than ATP, wherein said substances are, or can undergo conversion to become, substrates utilized in the light-emitting forward reaction of firefly luciferase, wherein said one or more enzymes capable of degrading substances other than ATP do not include luciferase and wherein each of said one or more enzymes capable of degrading substances other than ATP reduce the light emission produced from said light-emitting reaction of firefly luciferase, and subsequently assaying the sample for bioluminescent light emission.