Assay for quantifying polyphosphates

Patent metadata

Field	Value
Publication number	US-11519917-B2
Application number	US-201816604269-A
Country	US
Kind code	B2
Filing date	Apr 13, 2018
Priority date	Apr 13, 2017
Publication date	Dec 6, 2022
Grant date	Dec 6, 2022

Abstract

Provided herein are compositions and methods for quantifying polyphosphates. In particular, provided herein are solution and substrate based assays for quantifying polyphosphates in complex samples.

First claim

We claim:

1. A method of detecting polyphosphate in a sample, comprising:
a) contacting a sample comprising polyphosphate with an agent that inhibits binding of said polyphosphate to proteins in said sample;
b) contacting said sample with a solid support coated in a polymer under conditions such that said polyphosphate binds to said polymer;
c) washing said solid support to remove proteins in said sample;
d) contacting said solid support with a polyphosphate binding protein under conditions such that said polyphosphate binding protein binds to polyphosphate; and
e) quantifying the level of polyphosphate binding protein bound to said polyphosphate.

2. A method of detecting polyphosphate in a sample, comprising:
a) contacting a sample comprising polyphosphate with an agent that inhibits binding of said polyphosphate to proteins in said sample and a known amount of labeled polyphosphate;
b) contacting said sample with a solid support coated in a polymer under conditions such that said polyphosphate binds to said polymer;
c) washing said solid support to remove proteins in said sample; and
d) quantifying the level of labeled polyphosphate.

3. The method of claim 1 , wherein said polymer is polyethylenimine.

4. The method of claim 1 , wherein said agent is a salt.

5. The method of claim 4 , wherein said salt is LiCl.

6. The method of claim 1 , wherein said solid support comprises a sample well.

7. The method of claim 1 , wherein said polyphosphate binding protein is E. coli polyP-binding protein (PPXbd).

8. The method of claim 1 , wherein said polyphosphate binding protein is labeled.

9. The method of claim 8 , wherein said label is biotin.

10. The method of claim 1 , wherein said labeled polyphosphate is labeled with biotin.

11. The method of claim 1 , wherein said quantifying comprises contacting said sample with streptavidin-peroxidase and a substrate for said peroxidase.

12. The method claim 1 , wherein said washing said sample comprises washing with a buffer comprising urea and a salt.

13. The method of claim 12 , wherein said quantifying comprising comparing the amount of substrate to a standard curve.

14. The method of claim 1 , wherein said sample is a biological sample or an environmental sample.

15. The method of claim 14 , wherein said environmental sample is a water sample.

16. The method of claim 14 , wherein said biological sample is selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, lavage fluid, and stool.

17. The method of claim 1 , wherein said polyphosphate is 20 to 5000 units in length.

18. The method of claim 16 , wherein the sample is from a human subject.

19. The method of claim 18 , wherein said subject is diagnosed with or suspected of having a disease or condition selected from the group consisting of deep vein thrombosis, sepsis, trauma, cancer, a clotting disorder, an inflammatory disease, and a fibrotic disease.

Assignees

• Univ Michigan Regents
• Univ Illinois

Inventors

• Morrissey James H
• Smith Stephanie A
• Baker Catherine J
• Hemp Rachel
• Travers Richard J

Classifications

• G01N33/58 Primary

  involving labelled substances (G01N33/53 takes precedence) · CPC title

• C01B25/40 Primary

  Polyphosphates · CPC title

• G01N33/86

  involving blood coagulating time {or factors, or their receptors} · CPC title

• G01N2333/245

  Escherichia (G) · CPC title

• C01B25/38

  Condensed phosphates · CPC title