Methods and compositions for determination of glycated proteins

• US 7,153,666 B2
• Filed: 07/17/2003
• Issued: 12/26/2006
• Est. Priority Date: 07/17/2003
• Status: Active Grant

• Current Assignee: Diazyme Laboratories, Inc. (General Atomics, Inc.)
• Original Assignee: General Atomics, Inc.
• Inventors: Wang, Yuping, Yuan, Chong-Sheng, Datta, Abhijit
• Primary Examiner(s): Prouty, Rebecca E.
• Assistant Examiner(s): Walicka, Malgorzata A.
• Application Number:
  US10/622,893
• Publication Number:
  US 20050014935A1
• Time in Patent Office: 1,258 Days
• Field of Search: 435/189, 530/350, 530/387.3, 530/387.9, 424/94.4, 536/23.2, 536/23.4
• US Class Current: 435/23
• CPC Class Codes: C07K 2319/40 containing a tag for immunodetection, or an epitope for immunisation
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C12N 9/0032 with oxygen as acceptor (1.5.3)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2400/00 Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• CPC Class Codes: G01N 2800/042 Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin

Abstract

This invention relates generally to the field of glycated protein detection. In particular, the invention provides chimeric proteins, nucleic acids encoding the chimeric proteins, methods and kits for assaying for a glycated protein in a sample, using inter alia, an amadoriase.

Claims

1. An isolated chimeric protein having enzymatic activity of an amadoriase, which chimeric protein comprises, from N-terminus to C-terminus:

a) a first peptidyl fragment comprising a bacterial leader sequence comprising the amino acid sequence set forth in SEQ ID NO;

1;

b) a second peptidyl fragment comprising an amadoriase comprising the amino acid sequence set forth in SEQ ID NO;

3; and

c) a third peptidyl fragment comprising a second bacterial leader sequence comprising the amino acid sequence set forth in SEQ ID NO;

4.

2. The isolated chimeric protein of claim 1, wherein the first and second peptidyl fragments are linked via a cleavable linkage.

3. The isolated chimeric protein of claim 1, which further comprises, at its C-terminus, a fourth peptidyl fragment comprising a peptide tag.

4. The isolated chimeric protein of claim 3, wherein the peptide tag is selected from the group consisting of FLAG, HA, HAl, c-Myc, 6-His, AUl, EE, T7, 4A6, {acute over (ε

)}, B, gE and Tyl tag.

5. The isolated chimeric protein of claim 1, which comprises the amino acid sequence set forth in SEQ ID NO:

5

6. A chimeric protein produced by growing a recombinant cell containing a nucleic acid comprising a nucleotide sequence encoding the chimeric protein of claim 1 such that the encoded chimeric protein is expressed by the cell, and recovering the expressed chimeric protein.

7. A method for assaying for a glycated protein in a sample, which method comprises:

a) contacting a sample to be assayed with a protease to generate a glycated peptide or a glycated amino acid from a glycated protein, if contained in said sample;

b) contacting said generated glycated peptide or glycated amino acid with a chimeric protein of claim 1 to oxidize said glycated peptide or glycated amino acid; and

c) assessing oxidation of said glycated peptide or glycated amino acid by said chimeric protein to determine the presence and/or amount of said glycated protein in said sample.

8. The method of claim 7, wherein the sample is a blood sample.

9. The method of claim 8, wherein the blood sample is a plasma, serum, red blood cell or whole blood sample.

10. The method of claim 7, wherein the glycated protein to be assayed is glycoalbumin or glycohemoglobin.

11. The method of claim 7, wherein the protease is an endo-type protease or an exo-type protease.

12. The method of claim 11, wherein the endo-type protease is selected from the group consisting of trypsin, α

-chymotrypsin, subtilisin, proteinase K, papain, cathepsin B, pepsin, thermolysin, protease XVII, protease XXI, lysylendopeptidase, prolether and bromelain F.

13. The method of claim 11, wherein the exo-type protease is an aminopeptidase or a carboxypeptidase.

14. The method of claim 7, wherein the protease is selected from the group consisting of proteinase K, pronase E, ananine, thermolysin, subtilisin and cow pancreas proteases.

15. The method of claim 7, wherein the protease generates a glycated peptide from about 2 to about 30 amino acid residues.

16. The method of claim 7, 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.

17. The method of claim 7, wherein the chimeric protein comprises the amino acid sequence set forth in SEQ ID NO:

5.

18. The method of claim 7, wherein the chimeric protein is encoded by the nucleotide sequence set forth in SEQ ID NO:

6.

19. The method of claim 7, wherein the oxidation of the glycated peptide or glycated amino acid is assessed by assessing consumption of the glycated peptide or glycated amino acid, or O₂ in the oxidation reaction or the formation of the oxidized glucose (glucosone), H₂O₂ or the amino acid in the oxidation reaction.

20. The method of claim 19, wherein the O₂ consumption is assessed by an oxygen electrode.

21. The method of claim 19, wherein the H₂ formation is assessed by a peroxidase.

22. The method of claim 21, wherein the peroxidase is horseradish peroxidase.

23. The method of claim 21, wherein the H₂O₂ formation is assessed by a peroxidase and Trinder reaction.

24. The method of claim 21, wherein the glycated peptide or glycated amino acid is contacted with the chimeric protein and the peroxidase sequentially or simultaneously.

25. The method of claim 19, wherein the glucosone formation is assessed by a glucose oxidase.

26. The method of claim 19, wherein the glucosone formation is assessed by a combination of glucose 6-phosphate dehydrogenase and hexokinase.

27. The method of claim 7, wherein the protease is inactivated before or current with the contact between the glycated peptide or glycated amino acid and the chimeric protein.

28. The method of claim 27, wherein the protease is inactivated by an inhibitor of the protease, or by a heat treatment if the protease is inactivated before the contact between the glycated peptide or glycated amino acid and the chimeric protein.

29. The method of claim 7, wherein ascorbate interference is countered using a copper (II) compound, a cholic acid or a bathophenanthroline disulphonic acid or a mixture thereof.

30. The method of claim 7, wherein bilirubin interference is countered using a ferrocyanide salt.

31. The method of claim 7, which is used in the prognosis or diagnosis of a disease or disorder.

32. The method of claim 31, wherein the disease or disorder is diabetes.

33. A kit for assaying for a glycated protein in a sample, which kit comprises:

a) a protease to generate glycated peptide or glycated amino acid from a glycated protein, if contained in a sample;

b) a chimeric protein of claim 1 to oxidize said glycated peptide or glycated amino acid; and

c) means for assessing oxidation of said glycated peptide or glycated amino acid by said chimeric protein to determine the presence and/or amount of said glycated protein in said sample.

34. The kit of claim 33, wherein the means for assessing oxidation of said glycated peptide or glycated amino acid by said chimeric protein comprises peroxidase.

35. The kit of claim 34, wherein the chimeric protein and the peroxidase are formulated in a single composition.