Provided is a glycated hemoglobin oxidase with small measurement error or without deviation of the measured value from the true value regarding a sample containing glycated abnormal hemoglobin. Provided are a glycated hemoglobin oxidase comprising an amino acid sequence in which the amino acid at the position corresponding to position 113, 109, 106, or 102 of the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid other than a positively-charged amino acid, such as glutamic acid, alanine, or aspartic acid as well as a method and a reagent kit for measurement of glycated hemoglobin using such glycated hemoglobin oxidase. The glycated hemoglobin is capable of reacting with various genotypes and enables highly accurate measurement of glycated hemoglobin in a sample containing glycated abnormal hemoglobin.

Provided is a composition by which glycated hemoglobin can be measured even in the presence of a stronger surfactant than a conventional case. Also provided is a buffer and/or stabilizer which maintains the residual activity of an amadoriase or lowers a reduction of residual activity. The present invention provides a composition for use in measuring glycated hemoglobin containing an amadoriase having substitution of one or more amino acid residues at a position(s) corresponding to an amino acid(s) selected from the group consisting of position 262, position 257, position 249, position 253, position 337, position 340, position 232, position 129, position 132, position 133, position 44, position 256, position 231 and position 81 of an amadoriase derived from the genus Coniochaeta and represented by SEQ ID No: 1 or 3, and having residual activity even in the presence of a surfactant. The present invention also provides a composition and kit for use in measuring glycated hemoglobin, comprising a specific stabilizer and/or a buffer. The present invention can provide an enzyme and a composition for use in measuring glycated hemoglobin, excellent in storage stability even if they are exposed to a surfactant.

A method is provided for measuring glycated hemoglobin in a hemoglobin-containing sample which comprises: adding an enzyme that catalyzes a reaction of oxidizing glycated amino acid or glycated peptide to generate hydrogen peroxide without oxidizing the glycated hemoglobin, to the hemoglobin-containing sample to generate hydrogen peroxide; eliminating the generated hydrogen peroxide; adding an enzyme that catalyzes a reaction of oxidizing glycated hemoglobin to generate hydrogen peroxide thereto to generate hydrogen peroxide; and measuring the generated hydrogen peroxide.

Provided is a composition by which glycated hemoglobin can be measured even in the presence of a stronger surfactant than a conventional case. Also provided is a buffer and/or stabilizer which maintains the residual activity of an amadoriase or lowers a reduction of residual activity. The present invention provides a composition for use in measuring glycated hemoglobin containing an amadoriase having substitution of one or more amino acid residues at a position(s) corresponding to an amino acid(s) selected from the group consisting of position 262, position 257, position 249, position 253, position 337, position 340, position 232, position 129, position 132, position 133, position 44, position 256, position 231 and position 81 of an amadoriase derived from the genus Coniochaeta and represented by SEQ ID No: 1 or 3, and having residual activity even in the presence of a surfactant. The present invention also provides a composition and kit for use in measuring glycated hemoglobin, comprising a specific stabilizer and/or a buffer. The present invention can provide an enzyme and a composition for use in measuring glycated hemoglobin, excellent in storage stability even if they are exposed to a surfactant.

The present invention relates to a kit for quantitative analysis of glycated hemoglobin (HbA1c), and the kit for quantitative analysis of HbA1c according to the present invention has excellent long-term stability of an enzyme reagent and thus has an effect of easily overcoming the disadvantages of the conventional reagents used in enzyme assays (e.g., storage, accuracy, portability, convenience of use, etc.).

A method for determining the amount of glycated hemoglobin (HbA1c), in whichif requiredthe erythrocytes in a sample are hemolyzed, the haemoglobin that is then releasedif requiredis contacted with a proteolytic agent and the glycated hemoglobin degradation products obtained in this way or otherwise are quantified is disclosed. In order to provide such a process and reagents employable therein that has/have the property of sufficient stability of the chemical compounds that are essential to the reaction, the provision of the requisite proteolytic agent in the form of an inactivated protease is proposed, which is then only reactivated in situ. For the stabilization of the hemoglobin, which is unfolded at a very low pH in the range from 1 to 3, at least one suitable stabilizer should be present in the hemolysis solution, and, where a leuco dye is used in connection with the determination of the amount of HbA1c, it is proposed that the latter be stabilized with particular phosphine compounds and/or thio compounds.

A system for determining a concentration of hemoglobin A1C includes a first electrochemical test strip, the first electrochemical test strip providing for an HbA1C concentration; and a second electrochemical test strip, the second electrochemical test strip providing for the total amount of hemoglobin.

This invention provides an amadoriase having improved specific activity on a glycated substrate, compared with conventional amadoriase. Provided is an amadoriase comprising a substitution of the amino acid at the position corresponding to position 64 of the amino acid sequence as shown in SEQ ID NO: 1 with an amino acid selected from the group consisting of glycine, serine, methionine, leucine, threonine, valine, and isoleucine, a method for measurement of HbA1c, and a reagent kit for measurement of HbA1c using such amadoriase. Such method and kit for measurement enable rapid, simple, and accurate quantification of HbA1c.

A method for determining the amount of glycated haemoglobin (HbA1c), in whichif requiredthe erythrocytes in a sample are haemolysed, the haemoglobin that is then releasedif requiredis contacted with a proteolytic agent and the glycated haemoglobin degradation products obtained in this way or otherwise are quantified is disclosed. In order to provide such a process and reagents employable therein that has/have the property of sufficient stability of the chemical compounds that are essential to the reaction, for the stabilization of the haemoglobin which is unfolded at a very low pH in the range from 1 to 3, at least one suitable stabilizer is present in the haemolysis solution. Where a leuco dye is used in connection with the determination of the amount of HbA1c, it is proposed that the latter be stabilized with particular phosphine compounds and/or thio compounds, and, in particular embodiments, the requisite proteolytic agent is to be provided in the form of an inactivated protease which is then only reactivated in situ.

A method for measuring a glycated protein in a sample, the method comprising (1) a step of allowing a sample in which a degradation product has been generated from a glycated protein by a protease to react with an oxidoreductase in the presence of an electron mediator to generate a reduced electron mediator; and (2) a step of detecting the reaction state in the step (1) by an electrochemical technique using an interdigitated electrode.