# US 11,078,517 B2 · Hemoglobin A1c measurement method and measurement kit

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- Number: US 11,078,517 B2
- Document ID: US11078517B2
- Status: Granted
- Kind: B2
- Application number: 15/031,037
- Filed: 2014-10-24
- Published: 2016-09-01
- Granted: 2021-08-03
- Earliest claimed: 2013-10-25
- Assignees: Kikkoman Corporation
- Applicants: Kikkoman Corporation
- Inventors: Yosuke Masakari; Atsushi Ichiyanagi
- Web page: https://patentgrep.com/patent/US11078517B2

## CPC

- C12Q 1/26: involving oxidoreductase
- C12N 9/0032: {with oxygen as acceptor (1.5.3)}
- C12Q 1/28: involving peroxidase
- C12Y 105/03: with oxygen as acceptor (1.5.3)
- G01N 21/78: producing a change of colour
- G01N 33/725: {using peroxidative activity}
- C12Y 111/01: Peroxidases (1.11.1)
- G01N 2333/805: Haemoglobins; Myoglobins
- G01N 2333/90672: with oxygen as acceptor (1.5.3) in general
- G01N 2333/908: acting on hydrogen peroxide as acceptor (1.11)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

This invention provides an amadoriase that acts on the β-chain of hemoglobin A1c (HbA1c) and generates hydrogen peroxide, a method for measurement of HbA1c using such amadoriase, and a reagent kit for measurement of HbA1c using such amadoriase. The method for measurement of HbA1c involves the use of an amadoriase that has specific activity of 0.1 U/mg or greater to αF6P and oxidizes the HbA1c β-chain amino terminus so as to generate hydrogen peroxide, and the reagent kit for measurement of HbA1c comprises such amadoriase. The method and the kit for measurement of HbA1c enable quantification of HbA1c to be performed rapidly, simply, and accurately.

## Claims

### Claim 1

A reagent kit for measurement of hemoglobin A1c in a sample comprising ingredients (1) and (2) below:
(1) a modified amadoriase that acts directly on hemoglobin A1c and generates hydrogen peroxide; and
(2) a reagent for measurement of hydrogen peroxide,
wherein the modified amadoriase is selected from:
(i) a modified amadoriase comprising the amino acid sequence of SEQ ID NO: 1, and wherein the amino acid at position 62 is substituted with aspartic acid; the amino acid at position 63 is substituted with histidine; the amino acid at position 102 is substituted with lysine; the amino acid at position 106 is substituted with lysine; the amino acid at position 110 is substituted with leucine; the amino acid at position 113 is substituted with lysine; and the amino acid at position 355 is substituted with serine, and
(ii) a modified amadoriase comprising an amino acid sequence that is at least 90% identical to the amadoriase of (i).

### Claim 2 (depends on 1)

The method of claim 1, wherein the modified amadoriase is obtained from the genus Coniochaeta.

### Claim 3

A reagent kit for measurement of hemoglobin A1c in a sample comprising ingredients (1) and (2) below:
(1) a modified amadoriase that acts directly on hemoglobin A1c and generates hydrogen peroxide; and
(2) a reagent for measurement of hydrogen peroxide,
wherein the modified amadoriase is selected from:
(i) a modified amadoriase comprising the amino acid sequence of SEQ ID NO: 1, and wherein the amino acid at position 62 is substituted with aspartic acid; the amino acid at position 63 is substituted with histidine; the amino acid at position 102 is substituted with lysine; the amino acid at position 106 is substituted with lysine; the amino acid at position 110 is substituted with leucine; the amino acid at position 113 is substituted with lysine; and the amino acid at position 355 is substituted with serine, and
(ii) a modified amadoriase comprising an amino acid sequence that is at least 90% identical to the amadoriase of (i).

### Claim 4 (depends on 1)

The method of claim 1, wherein the modified amadoriase further comprises asparagine at the position corresponding to position 68 of SEQ ID NO: 1 and threonine at the position corresponding to position 356 of SEQ ID NO: 1.