METHOD OF MEASURING HbA1c

• US 20100178659A1
• Filed: 01/30/2008
• Published: 07/15/2010
• Est. Priority Date: 01/30/2007
• Status: Active Grant

• Current Assignee: ARKRAY, Inc
• Original Assignee: ARKRAY, Inc
• Inventors: Inamura, Norio, Yonehara, Satoshi
• Granted Patent: US 8,008,085 B2
• Time in Patent Office: Days
• US Class Current: 435/23
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/805 Haemoglobins; Myoglobins
• CPC Class Codes: G01N 33/721 Haemoglobin
• CPC Class Codes: Y10T 436/19 Halogen containing
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

A method of measuring HbA1c is provided that, even with a whole blood sample after storage, measurement accuracy substantially equal to a whole blood sample right after collection can be maintained. Whole blood is stored in a presence of a glycolytic inhibitor and protease is added to the stored whole blood sample to cleave hemoglobin in the whole blood sample. Then a glycated part of a hemoglobin fragment thereby obtained is treated with fructosyl amine oxidase. Thereafter, a glycation degree of HbA1c is determined by measuring a redox reaction between the glycated part and the fructosyl amine oxidase. Further, instead of storage of the whole blood in a presence of the glycolytic inhibitor, a strong electrolyte substance such as KCl, K₂SO₄, KNO, NaCl, Na₂SO₄, NaNO, MgCl₂, MgSO₄, Mg(NO)₂, etc. is added to the whole blood after storage and a protease treatment is performed in a presence of the strong electrolyte substance. According to these methods, fluctuation in a measurement value of HbA1c due to storage of the whole blood can be avoided.

Claims

1. A method of producing a hemoglobin-containing sample used for a method of measuring HbA1c, wherein the method includes:

(A1) a process of storing a hemoglobin-containing material in a state in which carbon dioxide generation is inhibited;

or

(A2) a process of reducing carbon dioxide bonded to hemoglobin in a hemoglobin-containing material after storage.

2. The method of producing according to claim 1, wherein in the process (A1), the hemoglobin-containing material is stored in a presence of a glycolytic inhibitor.

3. The method of producing according to claim 2, wherein the glycolytic inhibitor is at least one of sodium fluoride and potassium fluoride.

4. The method of producing according to claim 1, wherein in the process (A2), a strong electrolyte substance having a positive ion at least one selected from the group consisting of K⁺, Na⁺, and Mg²⁺ and a negative ion at least one selected from the group consisting of Cl⁻

, SO₄²⁻

, and NO₃⁻

is added to the hemoglobin-containing material after storage.

5. The method of producing according to claim 4, wherein the strong electrolyte substance is at least one selected from the group consisting of KCl, K₂SO₄, KNO₃, NaCl, Na₂SO₄, NaNO₃, MgCl₂, MgSO₄, and Mg(NO₃)₂.

6. The method of producing according to claim 1, wherein the hemoglobin-containing sample is a whole blood sample or a blood cell sample.

7. A method of measuring HbA1c, wherein the method includes:

(A) a process of preparing a hemoglobin-containing sample after storage by a method of producing according to claim 1;

(B) a process of cleaving hemoglobin in the hemoglobin-containing sample by applying a protease treatment to the hemoglobin-containing sample after storage;

(C) a process of treating a glycated part of a hemoglobin fragment obtained by the process (B) with fructosyl amine oxidase; and

(D) a process of determining a HbA1c amount by measuring a redox reaction between the glycated part and the fructosyl amine oxidase.

8. The method of measuring HbA1c according to claim 7, wherein the process (A) is a process of preparing the hemoglobin-containing sample after storage by storing a hemoglobin-containing material in a presence of a glycolytic inhibitor, and in the process (B), a concentration of the glycolytic inhibitor in a reaction solution for the protease treatment is 0.01 to 10 mol/L.

9. The method of measuring HbA1c according to claim 7, wherein the process (A) is a process of preparing the hemoglobin-containing sample after storage by adding a strong electrolyte substance having a positive ion at least one selected from the group consisting of K⁺, Na⁺, and Mg²⁺ and a negative ion at least one selected from the group consisting of Cl⁻

, SO₄²⁻

, and NO₃⁻

to a hemoglobin-containing material after storage, and in the process (B), the protease treatment is performed in a presence of the strong electrolyte substance.

10. The method of measuring HbA1c according to claim 9, wherein in the process (B), a concentration of the strong electrolyte substance in a reaction solution for the protease treatment is 10 to 3000 mmol/L.

11. The method of measuring HbA 1c according to claim 7, wherein in the process (D),

the HbA1c amount in hemoglobin in the hemoglobin-containing sample is calculated by measuring the redox reaction between the glycated part and the fructosyl amine oxidase, and

a HbA1c ratio is calculated from an amount of hemoglobin and the amount of HbA1c in the hemoglobin-containing sample.

12. The method of measuring HbA1c according to claim 8, wherein the hemoglobin-containing sample is a whole blood sample or a blood cell sample.

13. A storage reagent used for a method of producing a hemoglobin-containing sample according to claim 1 for storing a hemoglobin-containing material, wherein the storage reagent comprises a glycolytic inhibitor.

14. The storage reagent according to claim 13, wherein the glycolytic inhibitor is at least one of sodium fluoride and potassium fluoride.

15. The storage reagent according to claim 13, wherein the storage reagent inhibits carbon dioxide generation during storage of the hemoglobin-containing material.

16. The storage reagent according to claim 13, wherein the hemoglobin-containing sample is a whole blood sample or a blood cell sample.

17. A treatment reagent used for a method of producing a hemoglobin-containing sample according to claim 1 for treating a hemoglobin-containing material after storage, wherein the treatment reagent comprises:

a strong electrolyte substance having a positive ion at least one selected from the group consisting of K⁺, Na⁺, and Mg²⁺ and a negative ion at least one selected from the group consisting of Cl⁻

, SO₄²⁻

, and NO₃⁻

.

18. The treatment reagent according to claim 17, wherein the strong electrolyte substance is at least one selected from the group consisting of KCl, K₂SO₄, KNO₃, NaCl, Na₂SO₄, NaNO₃, MgCl₂, MgSO₄, and Mg(NO₃)₂.

19. The treatment reagent according to claim 17, wherein the treatment reagent further includes protease.

20. The treatment reagent according to claim 17, wherein the treatment reagent reduces carbon dioxide bonded to hemoglobin in the hemoglobin-containing material after storage.

21. The treatment reagent according to claim 17, wherein the hemoglobin-containing sample is a whole blood sample or a blood cell sample.

22. A measurement kit used for a method of measuring HbA1c according to claim 7, wherein the measurement kit is a reagent kit comprising:

a first reagent containing fructosyl amine oxidase and a strong electrolyte substance; and

a second reagent containing protease.

23. The measurement kit according to claim 22, wherein the first reagent further contains oxidase and the second reagent further contains a substrate developing a color by oxidation.