Methods for assaying percentage of glycated hemoglobin

• US 20080096230A1
• Filed: 07/25/2007
• Published: 04/24/2008
• Est. Priority Date: 07/25/2006
• Status: Active Grant

• Current Assignee: Diazyme Laboratories, Inc. (General Atomics, Inc.)
• Original Assignee: General Atomics, Inc.
• Inventors: Yuan, Chong-Sheng, Datta, Abhijit, Liu, Limin
• Granted Patent: US 7,855,079 B2
• Time in Patent Office: Days
• US Class Current: 435/7.8
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin
• CPC Class Codes: Y10T 436/104998 Glucose, ketone, nitrate standard or control
• CPC Class Codes: Y10T 436/105831 Protein or peptide standard or control [e.g., hemoglobin, etc.]
• CPC Class Codes: Y10T 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

The invention provides enzymatic methods for direct determination of percentage of glycated hemoglobin in blood samples without the need of a separated measurement of total hemoglobin content in blood samples. The methods utilizes one or two different types of oxidizing agents which selectively oxidize low-molecular weight reducing substances and high-molecular weight (mainly hemoglobin) reducing substances in blood samples, coupled with enzymatic reactions catalyzed by proteases, fructosyl amino acid oxidase, and peroxidase. The invention provides kits for performing the methods of the invention.

Claims

1. A method for directly assaying percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in a blood sample without measuring the total hemoglobin in the blood sample in a separate process, said method comprising:

a) contacting protein fragments containing glycated peptides or glycated amino acids with a fructosyl amino acid oxidase to generate hydrogen peroxide (H₂O₂), wherein the protein fragments are generated by contacting the blood sample with

     1) a lysing buffer which releases hemoglobin from red blood cells in the blood sample;

     2) a first oxidizing agent which selectively oxidizes low molecular weight reducing substances;

     3) a second oxidizing agent which selectively oxidizes high molecular weight reducing substances, and

     4) a protease which digests glycated hemoglobin into glycated peptides or glycated amino acids;

b) contacting H₂O₂ generated in step a) with a color forming substance in the presence of a peroxidase to generate a measurable signal; and

c) determining percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in the sample by applying the signal generated in step b) to a calibration curve without measuring the total hemoglobin in the blood sample separately.

2. The method of claim 1, wherein the first oxidizing agent is Dess-Martin periodinane or N-ethylmaleimide, and wherein the second oxidizing agent is a tetrazolium salt.

3. The method of claim 1, wherein the lysing buffer contains the first oxidizing agent or the second oxidizing agent.

4. The method of claim 1, wherein the lysing buffer contains the first oxidizing agent and the second oxidizing agent.

5. The method of claim 1, wherein the lysing buffer contains the first oxidizing agent, the second oxidizing agent, and the protease.

6. The method of claim 1, wherein the lysing buffer contains the protease.

7. The method of claim 1, wherein the first oxidizing agent and the second oxidizing agent are formulated in a single composition.

8. The method of claim 1, wherein the first oxidizing agent and the second oxidizing agent are formulated in a separate composition.

9. The method of claim 1, wherein the protease is formulated in a single composition with the first oxidizing agent or the second oxidizing agent.

10. The method of claim 1, wherein the first oxidizing agent, the second oxidizing agent, and the protease are formulated in a single composition.

11. The method of claim 1, wherein the fructosyl amino acid oxidase, the peroxidase, and the color forming substance are formulated in a single composition.

12. The method of claim 2, wherein tetrazolium salt is 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt or 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt.

13. The method of claim 1, wherein the color forming substance is N-Carboxymethylaminocarbonyl)-4,4′

-bis(dimethylamino)-diphenylamine sodium salt (DA-64), N,N,N′

N′

,N″

,N″

-Hexa(3-sulfopropyl)-4,4′

,4″

,-triamino-triphenylmethane hexasodium salt (TPM-PS), or 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenothiazine sodium salt (DA-67).

14. The method of claim 1, wherein the blood sample is a whole blood or collected blood cells.

15. The method of claim 1, wherein the protease is an endo-type protease or an exo-type protease.

16. The method of claim 1, wherein the protease is selected from the group consisting of proteinase K, pronase E, ananine, thermolysin, subtilisin and cow pancreas proteases.

17. The method of claim 1, wherein the protease is a neutral protease of Aspergillus or Bacillus origin.

18. The method of claim 2, wherein the protease generates a glycated peptide from about 2 to about 30 amino acid residues.

19. The method of claim 1, wherein the protease generates glycated glycine, glycated valine or glycated lysine residue or a glycated peptide comprising glycated glycine, glycated valine or glycated lysine residue.

20. The method of claim 1, wherein the peroxidase is horseradish peroxidase.

21. The method of claim 1, wherein the protein fragments containing the glycated peptide or glycated amino acid are contacted with the fructosyl amino acid oxidase and the peroxidase sequentially or simultaneously.

22. The method of claim 1, wherein the fructosyl amino acid oxidase comprises the amino acid sequence set forth in SEQ ID NO:

1

23. The method of claim 1, which is used in the prognosis or diagnosis of a disease or disorder.

24. The method of claim 23, wherein the disease or disorder is diabetes.

25. A kit for directly assaying percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in a blood sample without the need of a separated measurement of total hemoglobin content in blood samples, said kit comprising:

a) a lysing buffer which lyses blood cells to release hemoglobin;

b) a first oxidizing agent which selectively oxidizes low molecular weight reducing substances;

c) a second oxidizing agent which selectively oxidizes high molecular weight reducing substances;

d) a protease which hydrolyzes hemoglobin into protein fragments containing glycated peptides or glycated amino acids;

e) a fructosyl amino acid oxidase which reacts with glycated peptides and glycated amino acids to generate hydrogen peroxide (H₂O₂);

f) a peroxidase and a color forming substance;

g) calibrator(s) with known percentage of glycated hemoglobin or known percentage of glycated hemoglobin A1c for use in constructing a calibration curve; and

h) instructions to perform the method of claim 1.

26. The kit of claim 25, wherein the first oxidizing agent and the second oxidizing agent are contained in the lysing buffer.

27. The kit of claim 25, wherein the first oxidizing agent and the second oxidizing agent are contained in the same buffer with the protease.

28. The kit of claim 25, wherein the fructosyl amino acid oxidase, the peroxidase, and the color forming substance are formulated in a single composition.

33. A kit for directly assaying percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in a blood sample without the need of a separated measurement of total hemoglobin content in blood samples, said kit comprising:

a) a lysing buffer which lyses blood cells to release hemoglobin;

b) an oxidizing agent, wherein the oxidizing agent is a tetrazolium salt;

c) a protease which hydrolyzes hemoglobin into protein fragments containing glycated peptides or glycated amino acids;

d) a fructosyl amino acid oxidase which reacts with glycated peptides and glycated amino acids to generate hydrogen peroxide (H₂O₂);

e) a peroxidase and a color forming substance;

f) calibrator(s) with known percentage of glycated hemoglobin or known percentage of glycated hemoglobin A1c for use in constructing a calibration curve; and

g) instructions to perform the method of claim 28.

34. The kit of claim 33, wherein the oxidizing agent is contained in the lysing buffer.

35. The kit of claim 33 or 34, wherein the protease is contained in the lysing buffer.

36. The kit of claim 33, wherein the oxidizing agent and the protease are formulated in a single composition.

29. A method for directly assaying percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in a blood sample without measuring the total hemoglobin in the blood sample in a separate process, said method comprising:

a) contacting protein fragments containing glycated peptides or glycated amino acids with a fructosyl amino acid oxidase to generate hydrogen peroxide (H₂O₂), wherein the protein fragments are generated by contacting the blood sample with

     1) a lysing buffer which releases hemoglobin from red blood cells in the blood sample;

     2) an oxidizing agent which is a tetrazolium salt, and

     3) a protease which digests glycated hemoglobin into glycated peptides or glycated amino acids;

b) contacting 1H₂O₂ generated in step a) with a color forming substance in the presence of a peroxidase to generate a measurable signal; and

c) determining percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in the sample by applying the signal generated in step b) to a calibration curve without measuring the total hemoglobin in the blood sample separately.

30. The method of claim 29, wherein the lysing buffer contains the oxidizing agent.

31. The method of claim 29 or 30, wherein the lysing buffer contains the protease.

32. The method of claim 29, wherein the oxidizing agent and the protease are formulated in a single composition.