Fructosyl peptidyl oxidase

• US 8,962,271 B2
• Filed: 03/27/2014
• Issued: 02/24/2015
• Est. Priority Date: 08/03/2009
• Status: Active Grant

• Current Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG), Ultizyme International Ltd.
• Inventors: Ikebukuro, Kazunori, Koji, Sode
• Primary Examiner(s): FRONDA, CHRISTIAN L
• Application Number:
  US14/227,693
• Publication Number:
  US 20140227728A1
• Time in Patent Office: 334 Days
• Field of Search: None
• US Class Current: 435/25
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin

Abstract

In one form, a fructosyl peptidyl oxidase derived from a budding yeast Phaeosphaeria nodorum for assaying a glycated protein in a sample is provided. The fructosyl peptidyl oxidase has higher activity toward fructosyl valine as well as fructosyl valyl histidine, and may be useful in assaying HbA1c with higher sensitivity and specificity. Still, other forms include unique methods, techniques, systems and devices involving a fructosyl peptidyl oxidase.

Claims

1. A method for assaying a glycated protein in a sample, comprising:

contacting the sample with one of;

a fructosyl peptidyl oxidase comprising an amino acid sequence having at least 70% identity with the amino acid sequence set forth in SEQ ID NO;

1 and a Km value toward fructosyl valyl histidine of 1 mM or less;

a fructosyl peptidyl oxidase comprising an amino acid sequence having at least 70% identity with the amino acid sequence set forth in SEQ ID NO;

1 and a Vmax/Km value toward fructosyl valyl histidine of 10 U/mg·

mM or more; and

a fructosyl peptidyl oxidase comprising an amino acid sequence having at least 70% identity with the amino acid sequence set forth in SEQ ID NO;

1 and residual activity of 50% or more when heat-treated at 50°

C. for 10 minutes; and

measuring the amount of the glycated protein oxidized by the fructosyl peptidyl oxidase.

3. The method of claim 1 wherein said contacting further comprises contacting the sample with an electron transfer mediator.

4. The method of claim 3 wherein the electron transfer mediator is N,N-bis-hydroxyethyl-4-nitrosoaniline.

5. The method of claim 4 wherein said contacting further comprises contacting the sample with one or more reagents selected from saponin, bilirubin oxidase and proteinase N.

6. The method of claim 3 wherein said contacting further comprises contacting the sample with one or more reagents selected from saponin, bilirubin oxidase and proteinase N.

7. The method of claim 1 wherein the fructosyl peptidyl oxidase is immobilized on an electrode.

8. The method of claim 7 wherein the fructosyl peptidyl oxidase is immobilized on the electrode using an azide-unit pendant water-soluble photopolymer (AWP).

9. The method of claim 8, further comprising N,N-bis-hydroxyethyl-4-nitrosoaniline immobilized on the electrode.

10. The method of claim 7, further comprising N,N-bis-hydroxyethyl-4-nitrosoaniline immobilized on the electrode.

11. The method of claim 1 wherein the fructosyl peptidyl oxidase comprises an amino acid sequence having at least 80% identity with the amino acid sequence set forth in SEQ ID NO:

1.

2. A method for assaying HbA1c, comprising:

digesting HbA1c in a sample to generate fructosyl valine or fructosyl valyl histidine;

contacting the fructosyl valine or fructosyl valyl histidine with one of;

a fructosyl peptidyl oxidase comprising an amino acid sequence having at least 70% identity with the amino acid sequence set forth in SEQ ID NO;

1 and a Km value toward fructosyl valyl histidine of 1 mM or less;

a fructosyl peptidyl oxidase comprising an amino acid sequence having at least 70% identity with the amino acid sequence set forth in SEQ ID NO;

1 and a Vmax/Km value toward fructosyl valyl histidine of 10 U/mg·

mM or more; and

a fructosyl peptidyl oxidase comprising an amino acid sequence having at least 70% identity with the amino acid sequence set forth in SEQ ID NO;

1 and residual activity of 50% or more when heat-treated at 50°

C. for 10 minutes; and

measuring the amount of oxidized fructosyl valine or fructosyl valyl histidine.

12. The method of claim 2 wherein said contacting further comprises contacting the sample with an electron transfer mediator.

13. The method of claim 12 wherein the electron transfer mediator is N,N-bis-hydroxyethyl-4-nitrosoaniline.

14. The method of claim 13 wherein said contacting further comprises contacting the sample with one or more reagents selected from saponin, bilirubin oxidase and proteinase N.

15. The method of claim 12 wherein said contacting further comprises contacting the sample with one or more reagents selected from saponin, bilirubin oxidase and proteinase N.

16. The method of claim 2 wherein the fructosyl peptidyl oxidase is immobilized on an electrode.

17. The method of claim 16 wherein the fructosyl peptidyl oxidase is immobilized on the electrode using an azide-unit pendant water-soluble photopolymer (AWP).

18. The method of claim 17, further comprising N,N-bis-hydroxyethyl-4-nitrosoaniline immobilized on the electrode.

19. The method of claim 16, further comprising N,N-bis-hydroxyethyl-4-nitrosoaniline immobilized on the electrode.

20. The method of claim 2 wherein the fructosyl peptidyl oxidase comprises an amino acid sequence having at least 80% identity with the amino acid sequence set forth in SEQ ID NO:

1.