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.

Provided are an amadoriase that is less likely to be influenced by oxygen concentration and a method and a reagent kit for measurement of HbA1c using such amadoriase. Provided are an amadoriase that is obtained via substitution of one or more amino acid residues at a position or positions corresponding to the position(s) selected from the group consisting of positions 280, 267, 269, 54, and 241 of the amadoriase derived from the genus Coniochaeta, a method for measurement of HbA1c, a reagent kit for measurement, and a sensor using such amadoriase. The modified amadoriase according to the invention has a lowered oxidase activity and an enhanced dehydrogenase activity, and this enables the use of an electron mediator, and this reduces the influence of oxygen concentration. Thus, HbA1c can be measured with high sensitivity.

Provided are an amadoriase that is less likely to be influenced by oxygen concentration and a method and a reagent kit for measurement of HbA1c using such amadoriase. Provided are an amadoriase that is obtained via substitution of one or more amino acid residues at a position or positions corresponding to the position(s) selected from the group consisting of positions 280, 267, 269, 54, and 241 of the amadoriase derived from the genus Coniochaeta, a method for measurement of HbA1c, a reagent kit for measurement, and a sensor using such amadoriase. The modified amadoriase according to the invention has a lowered oxidase activity and an enhanced dehydrogenase activity, and this enables the use of an electron mediator, and this reduces the influence of oxygen concentration. Thus, HbA1c can be measured with high sensitivity.

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.

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.

This invention provides an amadoriase having high reactivity with -fructosyl hexapeptide (F6P) derived from the -chain amino terminus of glycated hemoglobin (HbAlc), which enables satisfactory quantification of F6P cleaved from HbAlc. A novel amadoriase is derived from the amadoriase of the genus Coniochaeta by substitution of one or more amino acids at positions selected from the group consisting of positions 62, 63, 102, 106, 110, 113, 355, and 419. Such amadoriase enables rapid, simple, accurate, and satisfactory quantification of F6P cleaved from HbAlc. With the use of such amadoriase, a method for measurement of HbAlc by an enzymatic method and a kit for measurement of HbAlc that are based on the same principle as used in the conventional standard method and excellent in terms of the relevance with the standard method.

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.

This invention provides an amadoriase that can react with a wide variety of glycated peptides generated upon hydrolysis of the chain of hemoglobin A1c (HbA1c) to generate hydrogen peroxide, a method for measurement of HbA1c using such amadoriase, and a reagent kit for measurement of HbA1c using such amadoriase. Provided is an amadoriase obtained by substitution of one or more amino acids at positions corresponding to amino acids selected from the group consisting of amino acids 62, 63, 102, 106, 110, 113, and 355 in the amino acid sequence of the amadoriase derived from the genus Coniochaeta or the like which amadoriase is capable of oxidizing a wide variety of glycated peptides to generate hydrogen peroxide. Further provided is a method for measurement of HbA1c comprising using such amadoriase as well as a reagent kit for measurement of HbA1c comprising such amadoriase. This invention can provide a method for measurement of HbA1c that enables quantification of HbA1c to be performed rapidly, simply, and accurately with the use of a small amount of a protease and a kit used for such measurement. This invention can also provide a method for measurement of HbA1c that enables quantification of HbA1c to be performed rapidly, simply, and accurately, with high sensitivity with the use of a small amount of a protease and a kit used for such measurement.

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 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.