Kit comprising ATP-diphosphohydrolase for detecting bacterial ATP in a sample

Patent metadata

Field	Value
Publication number	US-10415073-B2
Application number	US-201515526087-A
Country	US
Kind code	B2
Filing date	Nov 9, 2015
Priority date	Nov 13, 2014
Publication date	Sep 17, 2019
Grant date	Sep 17, 2019

Abstract

A kit for detecting bacterial ATP in a sample is provided. The kit comprises an aqueous composition having a pH of about 6.0 to 7.2. The aqueous composition comprises effective amounts of a polyol, a buffer reagent, a protein, and ATP-diphosphohydrolase. A method of using the kit to detect bacterial ATP is also provided.

First claim

The invention claimed is:

1. A kit, comprising:
an aqueous composition having a pH of about 6.0 to about 7.2, the composition comprising effective amounts of a polyol, a buffer reagent, a protein, and ATP-diphosphohydrolase, wherein the ATP-diphosphohydrolase is present in the aqueous composition at a concentration of about 250 Units per liter to about 2500 Units per liter.

2. The kit of claim 1 , further comprising a somatic cell extractant.

3. The kit of claim 1 , with the proviso that the composition does not include an effective amount of a microbicidal compound.

4. The kit of claim 1 , with the proviso that the composition does not include an effective amount of dithiothreitol, dithioerythritol, or □-mercaptoethanol.

5. The kit of claim 1 , further comprising a luciferase enzyme activity.

6. The kit of claim 5 , wherein the luciferase enzyme activity is isolated from the aqueous composition.

7. The kit of claim 1 , wherein the pH of the aqueous composition is about 6.4 to about 7.0.

8. The kit of claim 1 , wherein the polyol is selected from the group consisting of sorbitol, xylitol, glycerol, and mixtures thereof.

9. The kit of claim 8 , wherein the polyol comprises sorbitol, wherein the sorbitol is present in the aqueous composition at a concentration of about 1 mole/liter to about 1.6 moles/liter.

10. The kit of claim 1 , wherein aqueous composition comprises an effective amount of the somatic cell extractant.

11. The kit of claim 2 , wherein the somatic cell extractant comprises a nonionic surfactant.

12. The kit of claim 11 , wherein the somatic cell extractant is selected from the group consisting of polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether and a polyethylene glycol monoalkyl ether.

13. The kit of claim 1 , wherein the buffer reagent comprises a mixture of HEPES and Tris succinate.

14. The kit of claim 1 , wherein the buffer reagent is present in the aqueous composition at a concentration of about 0.5 mM to about 200 mM.

15. The kit of claim 1 , wherein the protein is selected from a group consisting of serum albumin and a purified collagen.

16. The kit of claim 1 , further comprising a bacterial cell extractant.

17. A method of retaining greater than or equal to 90% of an initial ATP-diphosphohydrolase enzyme activity in an aqueous composition stored above 0° C. for at least 7 days, the method comprising:
forming an aqueous composition having a pH of about 6.0 to 7.2, the composition comprising effective amounts of a polyol, a buffer reagent, a protein, and ATP-diphosphohydrolase, wherein the ATP-diphosphohydrolase is present in the aqueous composition at a concentration of about 250 units per liter to about 2500 Units per liter; and
storing the aqueous composition at a temperature between 1-25° C., inclusive, for a period of at least 7 days;
wherein the aqueous composition has an initial ATP-diphosphohydrolase activity at a first time point;
wherein the combination of the pH and the effective amounts of the polyol, the buffer reagent, the protein, and the ATP-diphosphohydrolase enable retention of greater than or equal to 90% of the initial ATP-diphosphohydrolase activity after the composition is held at about 25° C. from the first time point until a second time point that is at least 7 days after the first time point;
wherein the ATP-diphosphohydrolase activity is measured at about 25° C. in a bioluminescent coupled assay at pH 7.75 with non-rate-limiting concentrations of ATP, luciferin, and luciferase.

18. The method of claim 17 , wherein the combination of the pH and the effective amounts of the polyol, the buffer reagent, the protein, and the ATP-diphosphohydrolase enable retention of greater than or equal to 95% of the initial ATP-diphosphohydrolase activity after the composition is held at about 25° C. from the first time point until the second time point.

19. A method of quantifying bacterial ATP in a sample, the method comprising:
forming a first mixture comprising a sample and an aqueous composition;
wherein the aqueous composition has a pH of about 6.8 to about 7.2 and comprises effective amounts of a polyol, a buffer reagent, a protein, and ATP-diphosphohydrolase, wherein the ATP-diphosphohydrolase is present in the aqueous composition at a concentration of about 250 units per liter to about 2500 Units per liter;
holding the first mixture at a predetermined temperature for a first period of time;
combining the first mixture with a bacterial cell extractant, luciferase, luciferin, and a buffer reagent to form a second mixture with a pH of about 7.75; and
measuring a luciferase-catalyzed bioluminescent reaction.

Assignees

• 3M Innovative Properties Co

Inventors

• Driscoll Mark B

Classifications

• C12Q1/42

  involving phosphatase · CPC title

• C12Q1/008 Primary

  for determining co-enzymes or co-factors, e.g. NAD, ATP · CPC title

• C12Q1/34

  involving hydrolase · CPC title

• G01N2333/914

  Hydrolases (3) · CPC title

• C12Q1/66

  involving luciferase · CPC title