Methods for assaying percentage of glycated hemoglobin

1. A method for directly assaying 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 (H 2 O 2 ), 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, wherein the protease is a neutral protease of Aspergillus or Bacillus origin and generates glycated glycine, glycated valine, glycated lysine residue, or a glycated peptide comprising glycated glycine, glycated valine or glycated lysine residue; wherein the fructosyl amino acid oxidase comprises the amino acid sequence set forth in SEQ ID NO:1;

b) contacting H 2 O 2 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 glycated hemoglobin A1c by correlating the measured value in step b) to a percentage of glycated hemoglobin A1c in a calibration curve without measuring the total hemoglobin in the blood sample separately.

2. The method of claim 1 , wherein the lysing buffer contains the oxidizing agent.

3. The method of claim 1 or 2 , wherein the lysing buffer contains the protease.

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

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

6. The method of claim 1 , 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.

7. 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).

8. The method of claim 1 , wherein the blood sample is a whole blood.

9. The method of claim 1 , wherein the protease generates a glycated peptide from about 2 to about 30 amino acid residues.

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

11. 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.

12. The method of claim 1 , further comprising using the percentage of glycated hemoglobin A1c for prognosing or diagnosing a disease or disorder.

13. A kit for directly assaying 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, wherein the protease is a neutral protease of Aspergillus or Bacillus origin, wherein the protease generates glycated glycine, glycated valine, glycated lysine residue, or a glycated peptide comprising glycated glycine, glycated valine or glycated lysine residue;

d) a fructosyl amino acid oxidase which reacts with glycated peptides and glycated amino acids to generate hydrogen peroxide (H 2 O 2 ), wherein the fructosyl amino acid oxidase comprises the amino acid sequence set forth in SEQ ID NO:1;

e) a peroxidase and a color forming substance;

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

g) instructions to perform the method of claim 1 .

14. The kit of claim 13 , wherein the oxidizing agent is contained in the lysing buffer.

15. The kit of claim 13 or 14 , wherein the protease is contained in the lysing buffer.

16. The kit of claim 13 , wherein the oxidizing agent and the protease are formulated in a single composition.

17. The kit of claim 13 , wherein the fructosyl amino acid oxidase, the peroxidase, and the color forming substance are formulated in a single composition.

18. The kit of claim 13 , 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.

19. The kit of claim 13 , 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).

20. The kit of claim 13 , wherein the protease generates a glycated peptide from about 2 to about 30 amino acid residues.

21. The kit of claim 13 , wherein the peroxidase is horseradish peroxidase.