Method of measuring HbA1c

• US 8,008,085 B2
• Filed: 01/30/2008
• Issued: 08/30/2011
• Est. Priority Date: 01/30/2007
• Status: Active Grant

• Current Assignee: ARKRAY, Inc
• Original Assignee: ARKRAY, Inc
• Inventors: Inamura, Norio, Yonehara, Satoshi
• Primary Examiner(s): WALLENHORST, MAUREEN
• Application Number:
  US12/377,232
• Publication Number:
  US 20100178659A1
• Time in Patent Office: 1,308 Days
• Field of Search: 436/8, 436/15, 436/63, 436/66, 436/67, 436/79, 436/110, 436/119, 436/124, 436/164, 436/166, 436/174, 422/61, 422/430, 435/23, 435/28, 435/29, 252/408.1
• US Class Current: 436/67
• 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 measuring HbA1c, wherein the method comprises:

(A) collecting a hemoglobin-containing material from a subject and storing the collected hemoglobin-containing material for a period of at least one day;

(B) reducing carbon dioxide bonded to hemoglobin in the hemoglobin-containing material stored in (A) so as to produce a hemoglobin-containing sample;

(C) cleaving the hemoglobin in the hemoglobin-containing sample produced in (B) by applying a protease treatment to the hemoglobin-containing sample;

(D) treating a glycated part of a hemoglobin fragment obtained from cleaving the hemoglobin in the hemoglobin-containing sample in (C) with fructosyl amine oxidase; and

(E) determining an amount of HbA1c in the hemoglobin-containing sample by measuring a redox reaction between the glycated part and the fructosyl amine oxidase and calculating the amount of HbA1c based on a measurement of the redox reaction.

2. The method of measuring HbA1c according to claim 1, wherein in (B), a strong electrolyte substance having a positive ion and a negative ion is added to the hemoglobin-containing material stored in (A), the positive ion being at least one selected from the group consisting of K⁺, Na⁺, and Mg²⁺, and the negative ion being at least one selected from the group consisting of Cl⁻

, SO₄²⁻

, and NO₃⁻

, wherein in (C), the protease treatment is performed in the presence of the strong electrolyte substance.

3. The method of measuring HbA1c according to claim 2, wherein in (C), the protease treatment is conducted in a reaction solution, and a concentration of the strong electrolyte substance in the reaction solution is 10 to 3000 mmol/L.

4. The method of measuring HbA1c according to claim 2, 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.

5. The method of measuring HbA1c according to claim 1, wherein in (E),

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

6. The method of measuring HbA1c 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 comprises:

(A) collecting a hemoglobin-containing material from a subject and storing the collected hemoglobin-containing material for a period of at least one day in a state in which carbon dioxide generation is inhibited so as produce a hemoglobin-containing sample;

(B) cleaving hemoglobin in the hemoglobin-containing sample produced in (A) by applying a protease treatment to the hemoglobin-containing sample;

(C) treating a glycated part of a hemoglobin fragment obtained from cleaving the hemoglobin in the hemoglobin-containing sample in (B) with fructosyl amine oxidase; and

(D) determining an amount of HbA1c in the hemoglobin-containing sample by measuring a redox reaction between the glycated part and the fructosyl amine oxidase and calculating the amount of HbA1c based on a measurement of the redox reaction.

8. The method of measuring HbA1c according to claim 7, wherein in (A), the hemoglobin-containing material is stored in the presence of a glycolytic inhibitor, and in (B), the protease treatment is conducted in a reaction solution, and a concentration of the glycolytic inhibitor in the reaction solution is 0.01 to 10 mol/L.

9. The method of measuring HbA1c according to claim 7, wherein in (D), a HbA1c ratio is calculated from the total amount of the hemoglobin in the hemoglobin-containing sample and the amount of HbA1c in the hemoglobin-containing sample.

10. The method of measuring HbA1c according to claim 7, wherein the hemoglobin-containing sample is a whole blood sample or a blood cell sample.

11. The method of measuring HbA1c according to claim 7, wherein in (A), the hemoglobin-containing material is stored in the presence of a glycolytic inhibitor.

12. The method of measuring HbA1c according to claim 11, wherein the glycolytic inhibitor is at least one selected from the group consisting of sodium fluoride and potassium fluoride.