Methods for the detection of glycated hemoglobin

• US 8,715,942 B2
• Filed: 11/18/2008
• Issued: 05/06/2014
• Est. Priority Date: 11/20/2007
• Status: Active Grant

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Inventors: Chapoteau, Eddy, Edwards, Richard, Swirski, Chester, Zazulak, Wolodymyr
• Primary Examiner(s): Counts, Gary W
• Application Number:
  US12/743,615
• Publication Number:
  US 20100273181A1
• Time in Patent Office: 1,995 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin

Abstract

Provided herein are methods for detecting glycated hemoglobin in, for example, human whole blood, that are not affected by the presence of variation in amino acid sequence that can exist in hemoglobin β chains. The methods detect all glycated hemoglobin in a sample, regardless of the form of the hemoglobin that has been glycated, and thus detect glycated human Hemoglobin A, Hemoglobin S, and Hemoglobin C.

Claims

1. A method for detecting glycated human hemoglobin comprising:

contacting a blood sample to be tested for glycated human hemoglobin with an endopeptidase;

contacting the endopeptidase-treated blood sample with proline-specific endopeptidase;

and detecting glycated β

-chain amino-terminal pentapeptide of human hemoglobin.

2. The method of claim 1 wherein the blood sample is human whole blood.

3. The method of claim 1 comprising determining the percentage of glycated human hemoglobin in the sample.

4. The method of claim 1 wherein the endopeptidase is an acid endopeptidase selected from the group consisting of pepsin, aspergillopepsin II, cathepsin D, saccharopepsin, mucorpepsin, chymosin, gastricsin, and physirolisin.

5. The method of claim 4 wherein the acid endopeptidase is pepsin.

6. The method of claim 5 comprising contacting the sample with about 5 U/mL to about 10,000 U/mL pepsin at a pH in the range of about 1.0 to about 5.0.

7. The method of claim 1 comprising contacting the endopeptidase-treated blood sample with about 0.1 U/mL to about 30 U/mL proline specific endopeptidase at a pH in the range of about 5.0 to about 9.0.

8. The method of claim 1 comprising detecting the glycated β

-chain amino-terminal pentapeptide of human hemoglobin with a method that comprises the use of high performance liquid chromatography or that comprises the binding of at least one antibody to the glycated pentapeptide.

9. The method of claim 1 comprising detecting the glycated β

-chain amino-terminal pentapeptide of human hemoglobin with an agglutination inhibition immunoassay.

10. The method of claim 9 wherein the agglutinator reagent used in the agglutination inhibition immunoassay is a glycated val-his-leu-thr-tyr-cys (SEQ ID NO:

4) sexapeptide conjugated to polyaspartamide.

11. The method of claim 9 comprising contacting the endopeptidase-treated blood sample with proline specific endopeptidase either simultaneously with, prior to, or following contacting the blood sample with an agglutinator reagent.

12. The method of claim 11 comprising contacting the endopeptidase-treated blood sample with a mixture of proline specific endopeptidase and the agglutinator reagent.

13. The method of claim 12 wherein the mixture of proline specific endopeptidase and the agglutinator reagent comprises about 0.1 g/dL to about 5.0 g/dL bovine serum albumin and about 0.5 mmol/L to about 10.0 mmol/L ethylenediamine tetraacetic acid and has a pH in the range of about 6.0 to about 8.0.

14. The method of claim 11 further comprising contacting the endopeptidase, proline specific endopeptidase, and agglutinator reagent-treated blood sample with an antibody that specifically binds to both glycated β

-chain amino-terminal pentapeptide of human hemoglobin and the agglutinator reagent.

15. The method of claim 14 wherein at least one antibody molecule is bound to one or more latex particles.

16. A method for detecting glycated human hemoglobin comprising:

contacting a blood sample to be tested for glycated human hemoglobin with an endopeptidase either simultaneously with, prior to, or following contacting the blood sample with an agglutinator reagent;

contacting the endopeptidase and agglutinator reagent-treated blood sample with proline-specific endopeptidase; and

detecting glycated β

-chain amino-terminal pentapeptide of human hemoglobin with an agglutination inhibition immunoassay.

17. The method of claim 16 wherein the endopeptidase is an acid endopeptidase selected from the group consisting of pepsin, aspergillopepsin II, cathepsin D, saccharopepsin, mucorpepsin, chymosin, gastricsin, or physirolisin.

18. The method of claim 17 wherein the acid endopeptidase is pepsin.

19. The method of claim 18 comprising contacting the sample with a mixture of pepsin and the agglutinator reagent.

20. The method of claim 19 wherein the mixture of pepsin and the agglutinator reagent comprises about 3 U/mL to about 100 U/mL pepsin and has a pH in the range of about 1.0 to about 4.0.

21. The method of claim 16 further comprising contacting the endopeptidase and agglutinator reagent-treated blood sample with proline specific endopeptidase either simultaneously with or before contacting the blood sample with an antibody that specifically binds to both glycated β

-chain amino-terminal pentapeptide of human hemoglobin and the agglutinator reagent.

22. The method of claim 21 comprising contacting the endopeptidase and agglutinator reagent-treated blood sample with a mixture of proline specific endopeptidase and the antibody.

23. The method of claim 22 wherein the mixture comprises about 2.0 U/mL to about 30.0 U/mL proline specific endopeptidase and has a pH in the range of about 7.0 to about 8.0.

24. The method of claim 21 wherein at least one antibody molecule is bound to one or more latex particles.