TW201312118A - Multilayer test film for measuring glycosylated hemoglobin and measuring method using it - Google Patents

Abstract

The present invention is to provide a multilayer test film for colorimetric quantifying a glycosylated hemoglobin, which is hard to be affected by a glycosylated amino acid and/or glycosylated peptide that are not from the glycosylated hemoglobin being probably existed in the sample to be tested, and also provides a measuring method. A multilayer test film for colorimetric quantifying the glycosylated hemoglobin, which at least laminates a (a) layer and a (b) layer represented as follow from a surface of dispensing the sample to be tested in order of the (a) layer and the (b) layer. Besides, the multilayer test film does not comprise a blood separating layer. (a) layer: a polymer substrate lat least bearing a glycosylated amino acid oxidase; (b) layer: a polymer substrate at least bearing a proteinase.

Description

Multilayer test piece for measuring glycated hemoglobin, and measuring method using same

The present invention relates to a multilayer test piece and a measuring method for colorimetric quantification of glycated hemoglobin content in a test sample correctly, simply and quickly at a diagnostic site.

Measuring glycated proteins is very important in the diagnosis, prevention, and treatment of diabetes. The glycated protein is produced by non-enzymatic reaction of glucose in the living body with an amine group of a protein in blood (mainly an α-amino group of a terminal amino acid or a γ-amino group of a lysine residue). Since the degree of glycation of the protein is directly proportional to the blood glucose concentration (hereinafter referred to as blood glucose level), for example, in the case of glycated hemoglobin, the blood sugar control state can be grasped within about 2 to 3 months by measuring the glycated protein. In the case of glycated albumin, the blood sugar control state in about 2 to 3 weeks can be grasped by measuring the glycated protein.

In particular, heme A1c, which is a kind of glycated hemoglobin, is considered to be the most important in the diagnosis of diabetes.

As a method of measuring the glycated protein, a high performance liquid chromatography (HPLC) method, an immunoassay, an enzymatic method, and the like are known. In the prior art, high-performance liquid chromatography (HPLC) and immunoassay occupy most of them, but in recent years, since a large number of samples can be measured quickly, in large quantities, correctly, and inexpensively by enzymatic methods, they have also been frequently adopted.

For the enzymatic method, for example, a technique of performing colorimetric quantification of glycated proteins by the following procedure is known (for example, refer to Patent Documents 1 to 4).

(1) A step of isolating red blood cells in a sample to be tested to extract glycated hemoglobin.

(2) A step of reacting the glycated hemoglobin with a protease to excise a glycated amino acid and/or a glycated peptide from the glycated β-strand N-terminus of glycated heme.

(3) a step of reacting the above glycated amino acid and/or glycated peptide with a glycated amino acid oxidase to form hydrogen peroxide.

(4) a step of reacting the above hydrogen peroxide with a redox-based color developing reagent in the presence of a peroxidase to develop a color.

(5) A step of colorimetric quantification of the glycated hemoglobin content in the test sample according to the above color development.

However, the prior art involved has a problem of an increase in apparent measurement due to the influence of glycated amino acids and/or glycated peptides which may be present in the sample to be tested but not from the glycated protein to be measured.

On the other hand, in order to solve the above problems, a method of measuring a glycated protein content (for example, Patent Documents 5 to 9) has been invented, which is characterized by including a pretreatment step of making a glycated amine before treating the glycated protein with a protease. The acid oxidase acts on the above-described glycated amino acid and/or glycated peptide which may be present in the sample to be tested but not from the glycated protein, thereby subjecting the above glycated amino acid and/or glycated peptide to decomposition treatment.

However, the prior art involved is a method of diluting a sample to be tested, adding a reagent in a liquid state, and measuring the absorbance of the reaction solution after reacting in the reaction vessel, and a method using a biochemical automatic analyzer is mainstream. However, the biochemical automatic analyzer is difficult to apply to POCT (point of care testing) because it is a large-scale device and is expensive, requires a skilled test technician to operate, and takes a long time from blood collection to reporting results. Immediate inspection at the clinical site)).

[Prior Art Literature] [Patent Literature]

Patent Document 1: International Publication No. 2002-006519

Patent Document 2: Japanese Laid-Open Patent Publication No. 2004-222570

Patent Document 3: Japanese Laid-Open Patent Publication No. 2007-181466

Patent Document 4: Japanese Laid-Open Patent Publication No. 2010-148515

Patent Document 5: Japanese Laid-Open Patent Publication No. 2007-289202

Patent Document 6: Japanese Laid-Open Patent Publication No. 2010-104386

Patent Document 7: Japanese Patent Laid-Open Publication No. 2010-110333

Patent Document 8: Japanese Laid-Open Patent Publication No. 2010-252814

Patent Document 9: Japanese Laid-Open Patent Publication No. 2010-263921

Non-patent literature

Non-Patent Document 1: Clinical Chemistry 43: 12 2390-2396 (1997)

The present invention has been made in the context of technical problems related to the prior art. That is, an object of the present invention is to provide a multilayer test piece and a measuring method for performing colorimetric quantification of glycated hemoglobin content in a test sample correctly, simply and promptly at a diagnosis site. More specifically, it is an object of the present invention to provide a method which is less susceptible to the influence of a glycated amino acid and/or a glycated peptide which may be present in a sample to be tested but which is not derived from a glycated protein, and which is excellent in storage stability. A multilayer test piece and a measuring method for colorimetric quantification of glycated hemoglobin content.

The inventors of the present application have found that by saccharifying an amino acid oxidase on a layer closer to the sample to be tested than the protease, the glycated amino acid oxidase acts before the glycated hemoglobin is cleaved by the protease. The glycated amino acid and/or glycated peptide which may be present in the sample to be tested but not derived from the glycated protein is capable of undergoing decomposition treatment of the above glycated amino acid and/or glycated peptide.

It has also been found that when the protease is carried on the same layer as the glycated amino acid oxidase, the inactivation and/or decomposition of the glycated amino acid oxidase caused by the protease occurs during storage, resulting in a significant decrease in storage stability.

In addition, it has been found that when the peroxidase and the redox-based chromogenic reagent are carried in the same layer, self-coloring of the redox-based chromogenic reagent caused by peroxidase occurs during storage, resulting in a significant decrease in storage stability. . Since the concentration of glycated hemoglobin in the sample to be tested is generally low in concentration compared with the glucose concentration or the like, it is preferred to use a high-sensitivity colorless pigment when measuring glycated hemoglobin, but the colorless pigment is particularly unstable and easily occurs. Color development.

Thus the inventors of the present invention carried the protease and the glycated amino acid oxidase on different layers and carried the glycated amino acid oxidase on a layer closer to the sample to be tested than the protease. Thus, it was found that it is not easily affected by the glycosylated amino acid and/or glycated peptide which may be present in the sample to be tested but not by glycated hemoglobin, and the storage stability of the glycated amino acid oxidase is also remarkably improved. Thus, the present invention has been completed. Further, the present invention has been accomplished by carrying the peroxidase and the redox-based color developing reagent separately on different layers, thereby improving the storage stability of the redox-based color developing reagent.

That is, the present invention has the following constituent elements.

A multilayer test piece for colorimetric quantification of glycated hemoglobin content, characterized in that the multilayer test piece is laminated with at least the following layers (a) and (b), the layer (a) And the layer (b) is laminated in the order of (a) layer and (b) layer from the sample surface to be tested, and the multilayer test piece does not have a blood separation layer, wherein the layer (a): at least carries saccharification A polymer substrate of an amino acid oxidase, and a layer (b): a polymer substrate carrying at least a protease.

2. A multilayer test piece for colorimetric quantification of glycated hemoglobin content, characterized in that said multilayer test piece is laminated with at least the following layers (a) and (b), said (a) The layer and the (b) layer are layered in the order of the layers (a) and (b) to be tested, and the peroxidases are respectively carried on the different layers in the layers (a) and (b). a redox coloring reagent, and the multilayer test piece does not have a blood separation layer, wherein (a) layer: a polymer substrate carrying at least a glycated amino acid oxidase, and (b) a layer: at least a protease Polymer substrate.

3. The multilayer test piece according to 1 or 2, wherein the multilayer test piece further carries a surfactant on at least one of layers (a) and (b).

4. A multilayer test piece for colorimetric quantification of glycated hemoglobin content, characterized in that said multilayer test piece has at least the following layers (a), (b) and (c) laminated, The (a) layer, the (b) layer and the (c) layer are from the (a) layer and the (b) layer and the (c) layer, the (a) layer and the (c) layer, and (b) layer or (c) layer and (a) layer and (b) layer are sequentially laminated, and the multilayer test piece does not have a blood separation layer, wherein (a) layer: at least carries glycated amino acid oxidation The polymer substrate of the enzyme, the layer (b): a polymer substrate carrying at least a protease, and the layer (c): a polymer substrate carrying at least a reagent other than the protease and the glycated amino acid oxidase.

5. A multilayer test piece for colorimetric quantification of glycated hemoglobin content, characterized in that said multilayer test piece has at least the following layers (a), (b) and (c) laminated; The (a) layer, the (b) layer and the (c) layer are from the (a) layer and the (b) layer and the (c) layer, the (a) layer and the (c) layer, and a layered layer of (b) or (c) and (a) and (b), respectively carrying a peroxidase and a different layer in layers (a), (b) and (c) a redox coloring reagent, and the multilayer test piece does not have a blood separation layer, wherein (a) layer: a polymer substrate carrying at least a glycated amino acid oxidase, and (b) a layer: at least a protease Polymer substrate, layer (c): a polymer substrate carrying at least any reagent other than a protease and a glycosylated amino acid oxidase.

6. The multilayer test piece according to 4 or 5, wherein the multilayer test piece further carries a surfactant on at least one of the layers (a), (b) and (c).

The multilayer test piece according to any one of 1 to 6, wherein the protease is selected from at least a protease derived from Bacillus, a protease derived from Aspergillus, and from Streptomyces ( More than one of the protease consisting of Streptomyces and a group consisting of proteases of Tritirachium.

The multilayer test piece according to any one of 1 to 7, wherein the redox-based color developing reagent is a colorless dye having a maximum absorption wavelength of from 600 nm to 800 nm.

The multilayer test piece according to any one of 1 to 8, wherein the surfactant is a nonionic surfactant having a Hydrophile Lipophile Balance Value (HLB Value) of 10-20. .

A method for measuring, wherein the measuring method uses the multilayer test piece according to any one of 1 to 9, wherein the glycated hemoglobin content is subjected to colorimetric quantification through at least the following steps (i) to (iii); Step (i): a step of spotting a sample to be tested on the upper surface of the multilayer test piece; and (ii): measuring reflectance from the surface opposite to the sample to be tested of the multilayer test piece And/or the step of absorbance; and step (iii): calculating from the obtained reflectance and/or absorbance a group selected from the group consisting of a heme content, a glycated hemoglobin content, a glycated hemoglobin content, and a heme content More than one step.

11. The measuring method according to 10, characterized in that the sample to be tested is a whole blood sample.

According to the present invention, it is possible to provide a glycated hemoglobin content which is less susceptible to the influence of glycated amino acids and/or glycated peptides which may be present in the sample to be tested but not by glycated hemoglobin and which is excellent in storage stability. A multilayer test piece for colorimetric quantification was performed.

[detailed description]

Hereinafter, the present invention will be described in detail.

(measuring object)

The measurement object of the present invention is glycated hemoglobin, and from the viewpoint of diagnostic use of diabetes, heme A1c (hereinafter sometimes referred to as HbA1c) which is glycated by the N-terminus of the β-chain of heme is preferred. Further, the glycated amino acid and/or glycated peptide derived from glycated hemoglobin as an object to be tested is sometimes referred to as a saccharide to be tested, and is not derived from a glycated amino acid which is a glycated hemoglobin to be tested. And/or glycated peptides are sometimes also referred to as non-test substance saccharides.

(To be tested)

Examples of the test sample of the present invention include whole blood, blood cells, plasma, serum, myeloid, sweat, urine, tears, saliva, skin, mucous membranes, hair, and the like (ie, taken from a living body). Samples) or foods such as beverage water and seasonings. Further, the biological sample is not limited to a human body, and a biological sample derived from a mammal such as a dog, a cat, or a cow is also an object. Among them, from the viewpoint of diagnostic use of diabetes, whole blood or blood cells are preferred, and from the viewpoint of POCT, whole blood which has not been subjected to treatment before blood cell/plasma or serum separation is more preferable.

The amount of the sample to be tested in the present invention is not particularly limited, and is, for example, preferably 1 μL to 50 μL, more preferably 1 μL to 40 μL, still more preferably 1 μL to 30 μL. If the sample to be tested is less than 1 μL, it may not be possible to spread from the uppermost layer to the lowermost layer of the multilayer test piece. On the other hand, if the sample to be tested is more than 50 μL, for example, when the sample to be tested is blood, the burden on the patient becomes large, which is not preferable.

(measurement principle)

The measuring principle of glycated hemoglobin in the present invention is measured by a so-called enzyme colorimetric method in which an enzymatic reaction (so-called dry chemical treatment method) is carried out by means of moisture in a sample to be tested, thereby enabling a multilayer test. The patches are developed, and then the degree of color development is measured by measuring the reflected light. By using the above measurement principle, the device is small, lightweight, and low in price. In addition, accurate, simple and rapid measurement is possible.

Hereinafter, the reaction in measuring the glycated hemoglobin content in the red blood cells will be described, but this does not constitute any limitation to the present invention.

(1) A step of reacting red blood cells in a sample to be tested with a surfactant to hemolyze to extract glycated hemoglobin.

(2) a step of reacting a non-test substance saccharide with a glycated amino acid oxidase to reduce the influence of the non-test substance saccharide.

(3) a step of reacting the glycated hemoglobin with a protease to excise a saccharide of a subject to be detected from a glycated β-strand N-terminus of glycated heme.

(4) a step of reacting the above-mentioned analyte saccharide with a glycated amino acid oxidase to form hydrogen peroxide.

(5) a step of reacting the above hydrogen peroxide with a redox-based color developing reagent in the presence of a peroxidase to develop a color.

(6) A step of colorimetric quantification of the glycated hemoglobin content in the test sample according to the above color development.

(multilayer test piece)

The multilayer test piece of the present invention is composed of a plurality of polymer substrates (hereinafter sometimes referred to as layers). The number of layers of the multilayer test piece may vary depending on the embodiment, and is preferably 2 to 7 layers, more preferably 2 to 6 layers, still more preferably 2 to 5 layers. In addition, in addition to the polymer substrate carrying the reagent (surfactant, protease, glycosylated amino acid oxidase, peroxidase, redox chromogenic reagent, etc.) required by the present invention, the number of layers is also included. For unfolding the blood, the transparent layer or the like. When the number of layers is one, the protease and the glycated amino acid oxidase have to be carried on the same layer, and it is difficult to carry out the step for reducing the influence of the saccharide of the non-subject. In addition, when the number of layers is one layer, protease and glycated amino acid oxidase, and peroxidase and redox-based color reagents have to be carried in the same layer, which may cause loss of glycosylated amino acid oxidase caused by protease. Self-developing of living, decomposing or redox coloring reagents. That is, the storage stability of the multilayer test piece may be remarkably lowered. On the other hand, when the number of layers is more than 7 layers, it is not preferable because the amount of samples to be tested required to be expanded from the uppermost layer to the lowermost layer becomes large.

The above multilayer test piece can be produced by any procedure. Typically, a plurality of layers can be produced by a conventional procedure in which a plurality of layers are separately immersed in one or more reagent solutions and then dried, and then assembled into a final test piece.

The multilayer test piece described above is characterized in that it does not contain a blood separation layer. Further, the blood separation layer refers to a layer for obtaining plasma or serum by filtering blood cells from whole blood. The object to be measured of the present invention is not glycated albumin contained in plasma or serum, but glycated hemoglobin contained in blood cells. Therefore, measurement of blood cells cannot be measured.

(layer structure)

The layer structure of the above multilayer test piece requires that the protease be carried on a layer (hereinafter, also referred to as an upper layer) which is closer to the sample to be tested than the glycated amino acid oxidase. When the glycated amino acid oxidase is carried on a layer farther from the sample to be tested than the protease (hereinafter, sometimes referred to as the lower layer), it is impossible to reduce the influence of the saccharide of the non-test object. A step of.

Further, the layer structure of the above multilayer test piece requires that the protease and the glycated amino acid oxidase are respectively carried on different layers, and it is further preferred to carry the peroxidase and the redox-based color developing reagent on different layers, respectively. When the protease and the glycated amino acid oxidase are carried in the same layer, the inactivation and decomposition of the glycated amino acid oxidase caused by the protease may occur, when the peroxidase and the redox-based color developing reagent are carried in the same At the time of the layer, the self-developing of the redox coloring reagent may occur. That is, the storage stability of the multilayer test piece may be remarkably lowered. Specific examples of the layer structure are shown below, but this does not constitute any limitation on the present invention.

When the first layer side of the multilayer test piece is the sample side to be tested and the second layer side is the reflected light measuring surface, the following structure in which the glycated amino acid oxidase is carried on the upper layer than the protease can be cited.

1. The first layer: glycosylated amino acid oxidase; and the second layer: protease, peroxidase, redox system color reagent.

2. The first layer: glycosylated amino acid oxidase, peroxidase; and the second layer: protease, redox system color reagent.

3. The first layer: glycosylated amino acid oxidase, redox chromogenic reagent; and the second layer: protease, peroxidase.

4. The first layer: a glycosylated amino acid oxidase, a peroxidase, a redox-based color reagent; and a second layer: a protease.

Among them, it is more preferable to carry the peroxidase and the redox coloring reagent separately in the following structures of different layers.

2. The first layer: glycosylated amino acid oxidase, peroxidase; the second layer: protease, redox color reagent.

3, the first layer: glycosylated amino acid oxidase, redox system color reagent; second layer: protease, peroxidase.

Further preferably, the following structure in which the redox-based color developing reagent is carried on the reflected light measuring surface (second layer) and can be expected to be highly sensitive.

2. The first layer: glycosylated amino acid oxidase, peroxidase; the second layer: protease, redox color reagent.

When the first layer side of the multilayer test piece is the sample side to be tested and the third layer side is the reflected light measuring surface, the following structure in which the glycated amino acid oxidase is carried on the upper layer than the protease is exemplified.

1. The first layer: glycosylated amino acid oxidase; the second layer: protease; and the third layer: peroxidase, redox system color reagent.

2. The first layer: glycosylated amino acid oxidase; the second layer: peroxidase; and the third layer: protease, redox color reagent.

3. The first layer: glycosylated amino acid oxidase; the second layer: redox color reagent; and the third layer: protease, peroxidase.

4. The first layer: peroxidase; the second layer: glycosylated amino acid oxidase; and the third layer: protease, redox chromogenic reagent.

5. The first layer: redox chromogenic reagent; the second layer: glycated amino acid oxidase; and the third layer: protease, peroxidase.

6, the first layer: glycosylated amino acid oxidase; the second layer: protease, peroxidase; and the third layer: redox system color reagent.

7. The first layer: glycosylated amino acid oxidase; the second layer: protease, redox color reagent; and the third layer: peroxidase.

8, the first layer: glycosylated amino acid oxidase; the second layer: peroxidase, redox system color reagent; and the third layer: protease.

9. The first layer: peroxidase; the second layer: glycosylated amino acid oxidase, redox chromogenic reagent; and the third layer: protease.

10. The first layer: a redox-based color developing reagent; the second layer: a glycosylated amino acid oxidase, a peroxidase; and a third layer: a protease.

11. The first layer: glycosylated amino acid oxidase, peroxidase; the second layer: protease; and the third layer: redox system color reagent.

12. The first layer: saccharified amino acid oxidase, redox chromogenic reagent; second layer: protease; and the third layer: peroxidase.

13. The first layer: glycosylated amino acid oxidase, peroxidase; the second layer: redox chromogenic reagent; and the third layer: protease.

14, the first layer: glycosylated amino acid oxidase, redox system color reagent; second layer: peroxidase; and the third layer: protease.

15. The first layer: peroxidase, redox chromogenic reagent; the second layer: glycosylated amino acid oxidase; and the third layer: protease.

Among them, it is more preferable to carry the following structures of the peroxidase and the redox-based color developing reagent on different layers.

2. The first layer: glycosylated amino acid oxidase; the second layer: peroxidase; and the third layer: protease, redox color reagent.

3. The first layer: glycosylated amino acid oxidase; the second layer: redox color reagent; and the third layer: protease, peroxidase.

4. The first layer: peroxidase; the second layer: glycosylated amino acid oxidase; and the third layer: protease, redox chromogenic reagent.

5. The first layer: redox chromogenic reagent; the second layer: glycated amino acid oxidase; and the third layer: protease, peroxidase.

6, the first layer: glycosylated amino acid oxidase; the second layer: protease, peroxidase; and the third layer: redox system color reagent.

7. The first layer: glycosylated amino acid oxidase; the second layer: protease, redox color reagent; and the third layer: peroxidase.

9. The first layer: peroxidase; the second layer: glycosylated amino acid oxidase, redox chromogenic reagent; and the third layer: protease.

10. The first layer: a redox-based color developing reagent; the second layer: a glycosylated amino acid oxidase, a peroxidase; and a third layer: a protease.

11. The first layer: glycosylated amino acid oxidase, peroxidase; the second layer: protease; and the third layer: redox system color reagent.

12. The first layer: saccharified amino acid oxidase, redox chromogenic reagent; second layer: protease; and the third layer: peroxidase.

13. The first layer: glycosylated amino acid oxidase, peroxidase; the second layer: redox chromogenic reagent; and the third layer: protease.

14, the first layer: glycosylated amino acid oxidase, redox system color reagent; second layer: peroxidase; and the third layer: protease.

Further preferably, the redox-based color developing reagent is carried on the reflected light measuring surface (third layer), and the following structure can be expected to perform measurement with high sensitivity.

2. The first layer: glycosylated amino acid oxidase; the second layer: peroxidase; and the third layer: protease, redox color reagent.

4. The first layer: peroxidase; the second layer: glycosylated amino acid oxidase; and the third layer: protease, redox chromogenic reagent.

6, the first layer: glycosylated amino acid oxidase; the second layer: protease, peroxidase; and the third layer: redox system color reagent.

11. The first layer: glycosylated amino acid oxidase, peroxidase; the second layer: protease; and the third layer: redox system color reagent.

Preferably, at least one of the above layers (first layer, second layer, third layer) further carries a surfactant. Further, as long as the protease and the glycated amino acid oxidase and the peroxidase and the redox-based color developing reagent are not present in the same layer, the above reagent (surfactant, protease, glycosylated amino acid oxidase, peroxidase, oxidation) The reducing system color developing reagent or the like can be repeatedly carried on each layer (the first layer, the second layer, and the third layer). In addition, the layers may be loaded with a buffer as needed.

(polymer substrate)

The polymer substrate constituting the multilayer test piece of the present invention is capable of carrying a required amount of the above reagent (a surfactant, a protease, a glycated amino acid oxidase, a peroxidase, a redox-based color developing reagent, etc.). And the sample to be tested can be appropriately spread in the horizontal direction and the vertical direction, and any form and composition of the material can be used. Specific examples of the form and composition of the polymer substrate are shown below, but this does not constitute any limitation on the present invention.

(form)

Examples of the form of the polymer base material include a material that can be self-supporting such as a filter paper, a fiber structure, a porous film (membrane screening test), a film, or a polymer gel, which is not self-supporting. Further, when a material which is not self-supporting such as a polymer gel is used, a material which can be self-supporting such as a filter paper, a fiber structure, a porous film (membrane screening test), or a film is preferably used as a support.

(composition)

Examples of the composition of the polymer base material include polyethylene terephthalate (PET), polybutylene terephthalate (PBT), and polyethylene naphthalate as a polyester resin. Alcohol esters (PEN) and polybutylene naphthalate (PBN), etc.; polyethylene (PE), polypropylene (PP) and polybutene as olefin resins; polyvinyl chloride (PVC) as vinyl resin ), polyvinylidene fluoride and polyvinyl acetate; acrylic resin; acrylate resin; polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF) as fluororesins; polycarbonate resin; Resin polyoxymethylene (POM), polyphenylene ether (PPO), polyether ketone (PEK), polyetheretherketone (PEEK), polyphenylene sulfide (PPS), polyfluorene (PSU), polyether oxime (PES) And polyetherimine (PEI) and the like; nylon and linaloamine as a polyamide resin; cellulose acetate, nitrocellulose, and regenerated cellulose as cellulose.

The shape of the polymer base material is not particularly limited, and examples thereof include a thin plate shape such as a square, a rectangle, a circle, and an ellipse.

The smaller the area of the polymer substrate, the better the area of the polymer substrate, but it is preferably, for example, from 1 mm ² to 1000 mm ² , more preferably from 2 mm ² to 500 mm ² , from the viewpoint of small size, light weight, and low cost of the device. Good for 5mm ² ~ 200mm ² .

The thickness of the polymer substrate (powder layer) is preferably, for example, 10 μm to 2000 μm from the viewpoints of the developability of the sample to be tested and the reactivity of the enzyme (protease, glycated amino acid oxidase, peroxidase). It is more preferably 20 μm to 1000 μm, still more preferably 50 μm to 500 μm.

(detection)

When the reaction is detected, it is most convenient to irradiate light (incident light) to the multilayer test piece which has been developed, and then to detect the reflected light, but other methods may be used. The light source is not particularly limited, and examples thereof include a UV lamp, a xenon lamp, a xenon lamp, a mercury lamp, a xenon lamp, a tungsten lamp, a halogen lamp, a light emitting diode (LED), and a laser. Among them, a light-emitting diode (LED) is preferred from the viewpoints of easiness of control of light wavelength, small size, light weight, and low cost of the device. The angle (incident angle) of the incident light is not particularly limited, and any angle can be adopted. On the other hand, the detection of the reflected light is not particularly limited, and is preferably perpendicular to the detection surface. In addition, if a photodiode or an integrating sphere is used, the detection can be easily performed.

(protease)

As the protease to be used in the present invention, any kind of protease can be used as long as it can excise the saccharide of the substance to be tested with the glycated β-strand N-terminal of glycated heme, and for example, it can be exemplified by animals, plants, and microorganisms. Protease and the like. Specific examples of the protease are shown below, but this does not constitute any limitation on the present invention.

(protease from animals)

Examples of the protease derived from animals include Factor Xa (factor Xa), plasmin, thrombin, pepsin, leucinaminopeptidase, and pancreatin ( Pancreatin), trypsin (trypsin), trypsin, chtmotrypsin A, aminopeptidase M, carboxypeptidase A, carboxypeptidase B Calpain, cathepsin B, cathepsin C, cathepsin D, endoprotinase Arg-C, and the like.

(Protease from plants)

Examples of the plant-derived protease include carboxypeptidase W, kallikrein, ficin, papain, chimopapain, and bromelain. Wait.

(protease from microorganisms)

Examples of the protease derived from a microorganism include Bacillus represented by subtilisin, thermolysin, dispase, and proteinase N. a protease derived from Aspergillus represented by an IP enzyme or the like; a protease derived from Streptomyces represented by pronase or the like; a protein represented by proteinase K or the like a protease derived from Tritirachium; a protease derived from Rhizopus represented by peptidase R; and a Penicillium represented by carboxypeptidase P and PD. a protease derived from Staphylococcus represented by endoprotinase Glu-C or the like; a protease derived from Clostridium represented by clostripain or the like; a protease derived from Lysobacter represented by endoprotinase Lys-C or the like; a metalloendopeipididase or the like as a representative a protease derived from sputum (Grifola); a protease derived from yeast (Yeast) represented by carboxypeptidase Y, proteinase A, or the like; and a representative represented by aminopeptidase T or the like a protease of Thermus; a protease derived from Pseudomonus represented by endoprotinase Asp-N; and a mass spectrometry lysylendopeipidase A protease such as Achromobacter which is represented by achromopodipidase or the like.

Among them, from the viewpoints of stability, reactivity (cut rate of heme), availability, price, and the like, a protease derived from a microorganism is preferred, and a protease derived from Bacillus is more preferably selected. One or more of a group consisting of a protease derived from Aspergillus, a protease derived from Streptomyces, and a protease derived from Tritirachium. As a commercial product, Toyoteam NEP (as a protease derived from Bacillus) NEP, manufactured by Toyobo Co., Ltd., Type-X (made by SIGMA), Type-XXIV (made by SIGMA), thermophilic protease (made by Daiwa Kasei Co., Ltd.), high temperature protease PC10 (made by Daiwa Kasei Co., Ltd.), Aspergillus protease Type-XIII (manufactured by SIGMA), Type-XXIII (manufactured by SIGMA), Type-XIV as a protease derived from Streptomyces, and protease as a protease derived from Tritirachium K (made by Roche) is suitable for use.

Among them, Toyoteam NEP (manufactured by Toyobo Co., Ltd.), Type-X (manufactured by SIGMA Co., Ltd.), Type-XXIV (manufactured by SIGMA Co., Ltd.), which is a protease derived from Bacillus, is used for the measurement of heme Alc. Thermophilic protease (manufactured by Daiwa Kasei Co., Ltd.), high temperature protease PC10 (manufactured by Daiwa Kasei Co., Ltd.), and Type-XIV which is a protease derived from Streptomyces are more suitable for use.

The protease may be a refined product or a crude product as long as the target activity is achieved. Alternatively, it may be a substance produced by genetic manipulation, regardless of the presence or absence of chemical modification. Further, the above proteases may be used singly or in combination of two or more.

The concentration of the protease is not particularly limited, but preferably ^{0.1U / cm 2 ~ 10000U / cm} 2, more preferably ^{1U / cm 2 ~ 1000U / cm} 2. When the protease concentration is less than 0.1 U/cm ² , the measurement time is prolonged due to a decrease in reactivity, which is not preferable. On the other hand, when the protease concentration is higher than 10000 U/cm ² , it sometimes causes an increase in background or a high price.

The pH at which the above protease reaction is carried out may not be adjusted, but is preferably adjusted by a suitable pH adjusting agent such as the following buffer to bring the protease to be used to the most suitable pH.

(glycosylated amino acid oxidase)

The glycated amino acid oxidase (hereinafter sometimes referred to as fructosyl amino acid oxidase FAOD) used in the present invention is referred to in the public literature as the following various names: fructosylamine oxidase, fructosylamino acid oxidation Enzyme, fructosyl methionine oxidase, fructosylamine oxidase, fructosyl amino acid oxidase, fructosyl methionine oxidase, glycosylated amine oxidase, glycosylated amino acid oxidase, glycosylated amino acid oxidase Glycosylamine oxidase, glycosylated amino acid oxidase, glycated amino acid oxidase, amadoriase, ketamine oxidase, ketamine oxidase, and the like.

As the glycated amino acid oxidase to be used in the present invention, any kind of enzyme can be used as long as it is an enzyme which generates a hydrogen peroxide by specifically reacting with the saccharide of the object to be tested or the saccharide of the non-test substance. Specific examples of the glycated amino acid oxidase are shown below, but this does not constitute any limitation on the present invention.

Examples of the glycated amino acid oxidase include an enzyme derived from Gibberella, an enzyme derived from Aspergillus, an enzyme derived from Penicillium, an enzyme derived from Fusarium, and a stick. Enzymes of Corynebacterium, enzymes from Coniochaeta, enzymes from Eupenicillium, enzymes from Achaetomiella, enzymes from Chaetomium, enzymes from coliforms, from Germany An enzyme of Debaryomyces, an enzyme from Curvularia, an enzyme from Neocosmospora, an enzyme from Cryptococcus, an enzyme from phaeosphaeria, An enzyme derived from Candida and an enzyme derived from Acremonium.

Among them, from the viewpoints of stability, reactivity (oxidation rate of glycated amino acid and/or glycated peptide), availability, price, and the like, it is preferably selected from an enzyme derived from Coniochaeta. Enzymes of Eupenicillium, enzymes from Curvularia, enzymes from Neocosmospora, enzymes from Aspergillus, enzymes from Cryptococcus, and from dark More than one group consisting of enzymes of phaeosphaeria, more preferably selected from the group consisting of an enzyme from Coniochaeta, an enzyme from Aspergillus, an enzyme from Cryptococcus, and One or more of the groups consisting of enzymes derived from phaeosphaeria.

Among them, from the viewpoint of measuring heme Alc, it is preferably one or more selected from the group consisting of an enzyme derived from Coniochaeta and an enzyme derived from phaeosphaeria.

The saccharified amino acid oxidase may be a refined product or a crude product as long as the target activity is achieved. Alternatively, it may be a substance produced by genetic manipulation, regardless of the presence or absence of chemical modification. Further, the glycated amino acid oxidase may be used singly or in combination of two or more.

The concentration of the glycated amino acid oxidase is not particularly limited, but preferably ^{0.01U / cm 2 ~ 1000U / cm} 2, more preferably ^{0.1U / cm 2 ~ 100U / cm} 2. When the concentration of the glycated amino acid oxidase is less than 0.01 U/cm ² , the measurement time is prolonged due to a decrease in reactivity, which is not preferable. On the other hand, when the concentration of the glycated amino acid oxidase is higher than 1000 U/cm ² , the background may be increased or the price may be increased.

The pH at which the above glycated amino acid oxidase reaction is carried out may not be adjusted, but is preferably adjusted by a suitable pH adjusting agent such as the following buffer to achieve the most suitable glycated amino acid oxidase to be used. pH.

(peroxidase)

The peroxidase used in the present invention may be any enzyme which can catalyze the reaction between hydrogen peroxide and a redox-based color developing reagent, and examples thereof include plants, bacteria, and basidiomycetes. Peroxidase. Among them, from the viewpoints of purity, availability, price, and the like, a peroxidase derived from mustard, rice, and soybean is preferable, and a peroxidase derived from mustard is more preferable. As a commercially available product, PEO-131 (made by Toyobo Co., Ltd.), PEO-301 (made by Toyobo Co., Ltd.), PEO-302 (made by Toyobo Co., Ltd.), etc. are suitable for use.

The concentration of the peroxidase is not particularly limited, but preferably ^{0.01U / cm 2 ~ 1000U / cm} 2, more preferably ^{0.1U / cm 2 ~ 100U / cm} 2. When the peroxidase concentration is less than 0.01 U/cm ² , the measurement time is prolonged due to a decrease in reactivity, which is not preferable. On the other hand, when the peroxidase concentration is higher than 1000 U/cm ² , it sometimes causes an increase in background or a high price.

The pH at which the above peroxidase reaction is carried out may not be adjusted, but is preferably adjusted by a suitable pH adjusting agent such as the following buffer to achieve the most suitable pH for the peroxidase to be used.

(redox system color reagent)

The redox-based color developing reagent used in the present invention may be any coloring matter as long as it can react with hydrogen peroxide, and examples thereof include a hydrogen donor, a coupling agent, and a leuco body. Further, a representative example using a hydrogen donor and a coupling agent is a Trinder method in which a hydrogen donor and a coupling agent are oxidatively condensed by hydrogen peroxide in the presence of a peroxidase to form a dye.

Specific examples of the redox-based color developing reagent are shown below, but this does not constitute any limitation on the present invention.

(hydrogen donor)

Examples of the hydrogen donor include a phenol, a phenol derivative, an aniline derivative, a naphthol, a naphthol derivative, a naphthylamine, and a naphthylamine derivative. Specific examples thereof include N-ethyl-N-(3-sulfopropyl)aniline (ALPS) and N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS). , N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS), N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS), N- Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5- Dimethylaniline (MAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-ethyl-N-(3- Methylphenyl)-N'-acetamethylenediamine, N-ethyl-N-(3-methylphenyl)-N'-amber ethylenediamine, N-(2-hydroxy-3-sulfonate Propyl)-2,5-dimethylaniline, N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N-sulfopropylaniline, N-sulfonate Propyl-3,5-dimethoxyaniline and the like.

(coupling agent)

As the coupling agent, 4-aminoantipyrine (4AA), an amine antipyrine derivative, vanillin diamine sulfonic acid, 3-methyl-2-benzothiazoline may be mentioned. Ketone hydrazine hydrochloride hydrate (MBTH: methybenzthiazoline hydrazone), sulfonated 3-methyl-2-benzothiazolinone hydrochloride hydrate (SMBTH: sulphonated methybenzthiazoline hydrazone).

(hidden body)

Examples of the leuco body include a triphenylmethane derivative, a phenothiazine derivative, and a diphenylamine derivative. Specifically, 4,4'-benzylidene bis(N,N-dimethylaniline), 4,4'-bis(N-ethyl-N-(3-sulfopropylamino) -2,6-dimethylaniline)methane, 1-(ethylaminothiocarbonyl)-2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis (4 -diethylaminophenyl)imidazole, 4,4'-bis(dimethylamino)diphenylamine, N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine Sodium salt (DA64), 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium salt (DA67), and the like.

Among them, from the reasons of the molar absorption coefficient, the maximum absorption wavelength, and the like, a leuco body is preferred, and N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine is more preferred. Sodium salt (DA64), 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium salt (DA67).

The maximum absorption wavelength of the above redox-based color developing reagent is preferably from 600 nm to 800 nm, more preferably from 650 nm to 750 nm. From the viewpoint of measuring heme A1c, when the maximum absorption wavelength is on the low wavelength side of less than 600 nm, the coloration spectrum of the redox-based color reagent may overlap with the spectrum of heme, which may lower the sensitivity. On the other hand, when the maximum absorption wavelength is on the high wavelength side of more than 800 nm, the detection device may be enlarged.

The concentration of the redox coloring reagent is not particularly limited, but is preferably 0.0001 mg/cm ² to 10 mg/cm ² , more preferably 0.001 mg/cm ² to 1 mg/cm ² . When the concentration of the redox coloring reagent is less than 0.0001 mg/cm ² , the sensitivity may be lowered. On the other hand, when the concentration of the redox coloring reagent is higher than 10 mg/cm ² , the background may be increased or the price may be increased.

(surfactant)

As the surfactant to be used in the present invention, any kind of surfactant, preferably a surfactant of a hemolytic agent and a protease reaction promoter, may be used as long as it functions as a hemolytic agent and/or a protease reaction promoter. Agent. Examples of the surfactant include polyoxyethylene alkylphenyl ether (such as Triton (registered trademark) surfactant) and polyoxyethylene alkyl ether (Brij (registered trademark) surfactant). Non-ionic properties such as polyoxyethylene sorbitan fatty acid ester (Tween (registered trademark) surfactant), polyoxyethylene fatty acid ester, sorbitan fatty acid ester, alkyl glucoside, fatty acid sucrose ester Surfactant. Among them, a polyoxyethylene alkylphenyl ether (Triton (registered trademark) surfactant, etc. is preferred from the viewpoints of reactivity (hemolysis rate) as a hemolytic agent, actionability as a protease reaction promoter, and price. ). Commercially available, Triton X (registered trademark)-100 (manufactured by Nacalai Tesque Co., Ltd.), Triton X (registered trademark)-114 (manufactured by Nacalai Tesque Co., Ltd.), and Nonidet (registered trademark) P-40 (manufactured by Nacalai Tesque Co., Ltd.) are suitable. use. Further, the above surfactants may be used singly or in combination of two or more.

The hydrophile lipophile balance value (HLB Value) of the above surfactant is preferably from 10 to 20, more preferably from 12 to 20, still more preferably from 14 to 20. When the HLB value is less than 10, it is feared that a sufficient hemolysis effect and an effect of promoting a protease reaction are not obtained. On the other hand, surfactants with an HLB value greater than 20 do not exist on the definition of HLB.

The concentration of the above surfactant is not particularly limited, but is preferably 0.0001 mg/cm ² to 10 mg/cm ² , more preferably 0.001 mg/cm ² to 1 mg/cm ² . When the surfactant concentration is less than 0.0001 mg/cm ² , there is a fear that a sufficient hemolysis effect and a protease reaction promoting effect are not obtained. On the other hand, when the surfactant concentration was higher than 10 mg/cm ² , no improvement in effect was observed.

When the whole sample is in the test sample, the hydrogen peroxide generated in the step of reacting the non-test substance saccharide of the present invention with the glycated amino acid oxidase to reduce the influence of the non-test substance saccharide, Catalase in whole blood is decomposed into oxygen and water, and thus catalase can be added as needed.

As the enzyme used in the present invention, any kind of enzyme can be used as long as it is an enzyme that catalyzes a reaction of disproportionation of hydrogen peroxide generated when the non-test substance saccharide is eliminated into oxygen and water. Catalase derived from animals and from microorganisms can be cited. Among them, from the viewpoints of purity, availability, price, and the like, a catalase derived from a microorganism is preferred. As a commercial product, it is suitable to use catalase C3515 (made by SIGMA) from Aspergillus, catalase 02071 (made by SIGMA) of the genus Corynebacterium, and Micrococcus. ) Catalase 60638 (manufactured by SIGMA) and the like.

The concentration of the catalase is not particularly limited, but preferably ^{0.1U / cm 2 ~ 10000U / cm} 2, more preferably ^{1U / cm 2 ~ 1000U / cm} 2. If the catalase concentration is less than 0.1 U/cm ² , it is sometimes impossible to remove the hydrogen peroxide generated when the non-test substance saccharide is removed, resulting in an apparent increase in the measured value. On the other hand, if the catalase concentration is higher than 10000 U/cm ² , it sometimes causes an increase in the background to cause a high price. The pH at which the above-described catalase reaction is carried out may not be adjusted, but is preferably adjusted by a suitable pH adjusting agent such as the following buffer to bring the catalase used to the most suitable pH.

In order to exclude the influence of catalase after the above-described catalase reaction, a catalase inhibitor such as sodium azide may be carried on a layer lower than the catalase. Alternatively, by using catalase and peroxidase to have different affinity to the substrate, by appropriately setting the concentration ratio of catalase and peroxidase, it is also possible to carry the catalase without the lower layer of catalase. The structure of the inhibitor.

(buffering agent)

As the buffer which can be used in the present invention, any kind of buffering agent can be used as long as it has sufficient buffering ability in the target range of pH, and examples thereof include Tris, phosphoric acid, phthalic acid, citric acid, and Malay. Acid, succinic acid, nitric acid, boric acid, tartaric acid, acetic acid, carbonic acid and good buffer (MES, ADA, PIPES, ACES, cholamine, BES, TES, HEPES, acetaminoglycan, flavonoids, gan Amine amide, broad bean glucoside, etc.

Among them, from the viewpoint of having a sufficient buffering capacity in the most suitable pH range of the protease, the glycated amino acid oxidase, and the peroxidase used in the present invention of 6.0 to 8.5 (preferably 6.0 to 7.5), it is preferred. Tris, phosphoric acid, MES, PIPES, TES, HEPES, more preferably MES, PIPES.

The concentration of the buffer is not particularly limited, and is preferably about 50 mM to 100 mM with respect to the reagent at the time of production of the multilayer test piece.

(other reagents)

In the present invention, in addition to the above reagents (surfactant, protease, glycosylated amino acid oxidase, peroxidase, redox-based color reagent, etc.), an oxidizing agent (ferrocyanide, ferrocene) may be added as needed. a chelating agent (ethylenediamine, dipyridyl, ethylenediaminetetraacetic acid, phenanthroline, porphyrin, crown ether, etc.) for capturing ions which hinder the enzymatic reaction, and using a chelating agent (such as ethylenediamine, dipyridylamine, ethylenediaminetetraacetic acid, phenanthroline, porphyrin, crown ether, etc.) Ascorbic acid oxidase, salt (sodium chloride, potassium chloride, calcium chloride, magnesium chloride, aluminum chloride, etc.) and enzyme stabilizer (monosaccharide, low) for removing ascorbic acid as a substance that hinders the quantification of hydrogen peroxide Glycans, polysaccharides, sugar alcohols, glycerol, gluconates, amino acids, albumin, globulins, fibrous proteins, etc.), redox-based color reagent stabilization agents (cyclodextrin) Classes, reducing thiols, reducing sulfates, etc.). These may be used alone or in combination of two or more.

[Examples]

The invention is specifically illustrated by the following examples, which are not intended to limit the invention. In addition, the evaluation methods in the specification are as follows.

[various evaluation methods]

<1. Measurement of protease activity>

The activity of the protease was calculated by the casein Folin method using Folin-Ciocalteu reagent. Here, the casein was hydrolyzed at 37 ° C for 1 minute at the optimum pH of the protease, and the amount of the enzyme which produced a color corresponding to 1.0 μmol of tyrosine was defined as IU.

<2. Measurement of activity of glycosylated amino acid oxidase>

The activity of glycosylated amino acid oxidase is developed by the reaction of hydrogen peroxide and redox system produced by the reaction of saccharyl histidine and glycated amino acid oxidase in the presence of peroxidase. The reagent produces a reaction that is calculated from the change in absorbance. Here, the hydrolysis of the glycated amidoxime histidine at 37 ° C for 1 minute at the most suitable pH (pH = 6.5) of the glycated amino acid oxidase will produce an amount of enzyme equivalent to 1.0 μmol of hydrogen peroxide. Defined as IU.

<3. Measurement of activity of peroxidase>

The peroxidase activity is calculated from the change in absorbance from the produced Purpurogallin by reacting hydrogen peroxide with pyrolyl in the presence of peroxidase. Here, at a most suitable pH (pH=6.0) of peroxidase, the reaction was carried out at 20 ° C for 20 seconds, and the amount of the enzyme which produced a color corresponding to 1.0 mg of Purpurogallin was defined as IU.

4. Measurement of the activity of catalase

The activity of catalase is calculated by the decomposition of hydrogen peroxide into water and oxygen in the presence of peroxidase from the change in absorbance at 240 nm from hydrogen peroxide. Here, the amount of the enzyme which decomposes 1.0 μmol of hydrogen peroxide is defined as IU by reacting at 25 ° C for 1 minute at the most suitable pH of the peroxidase.

<5. Measurement of Hydrophile Lipophile Balance Value (HLB Value)>

The HLB value was calculated by using the Griffin method from the HLB value = 20 × (sum of the formula amount of the hydrophilic moiety / molecular weight). Further, the HLB value of the mixture of surfactants is represented by a weighted average of the HLB values of the respective components.

<6. Storage stability of glycosylated amino acid oxidase>

On the Kiryu filter paper No. 5A (manufactured by Tokyo Glass Instruments Co., Ltd.) of 8 mm, 10 μL of the following reagent 1 was dropped, and dried in a light-shield dryer 3909-04 (manufactured by Tokyo Glass Instruments Co., Ltd.) at 25 ° C for 2 hours to prepare a blank test. sheet.

<Reagent 1>

100 mM PIPES (manufactured by Tongren Chemical Research Co., Ltd.) pH 6.5

500 U/mL glycosylated amino acid oxidase FPO-301 (manufactured by Toyobo Co., Ltd.)

Next, as an acceleration test, the blank test piece was incubated for 5 hours in a programmable cryostat IN604 (manufactured by Yamato Scientific Co., Ltd.) at 37 °C. Then, the above blank test piece and 1000 μL of the following reagent 2 were placed in a microtube, and the mixture was stirred for 1 minute with a vortex mixer (manufactured by MS Instruments Co., Ltd.) to extract saccharification in the blank test piece. Amino acid oxidase. Next, the above extract was centrifuged at 14,000 G in a centrifugal ultrafiltration tube (MICROCON) 3 (manufactured by Millipore) to obtain 200 μL of a concentrated liquid. Next, 50 μL of the above-mentioned concentrate was mixed with 47.5 μL of Laemmli sample buffer (Bio-Rad Laboratiories), 2.5 μL of 2-mercaptoethanol (manufactured by Bio-Rad Laboratiories), and boiled at 95 ° C for 5 minutes. Blank solution.

<Reagent 2>

100 mM PIPES (manufactured by Tongren Chemical Research Co., Ltd.) pH 6.5

100-fold diluted general protease inhibitor cocktail, 100 times concentrated (Nacalai Tesque)

Next, as a speed test, the multilayer test piece of the present invention was incubated for 5 hours in a programmable cryostat IN 604 (manufactured by Yamato Scientific Co., Ltd.) at 37 °C. Then, the multilayer test piece and 1000 μL of the above reagent 2 were placed in a microtube, and the interfacial activity in the multilayer test piece was extracted by stirring with a vortex mixer (manufactured by MS Instruments) for 1 minute. Agent, protease, glycosylated amino acid oxidase, peroxidase, redox system color reagent. Next, the extract was centrifuged at 14000 G in a centrifugal ultrafiltration tube (MICROCON) 3 (manufactured by Millipore) to remove a low molecular weight component (a surfactant, a redox-based color developing reagent, etc.) having a molecular weight of 3,000 or less. 200 μL of concentrate was obtained. Next, 50 μL of the above-mentioned concentrate was mixed with 47.5 μL of Laemmli sample buffer (Bio-Rad Laboratiories), 2.5 μL of 2-mercaptoethanol (manufactured by Bio-Rad Laboratiories), and boiled at 95 ° C for 5 minutes. Sample solution.

The prepared blank solution and the sample solution were subjected to SDS-PAGE under the following conditions 1 using an electrophoresis best package (pre-formed gel (manufactured by Bio-Rad Laboratiories)). In addition, the blank solution and the sample solution flow through different channels of the same gel.

<Condition 1>

Precast gel: pre-made glue J 10%T, 12well

Electrophoresis system: mini vertical tee cell (mini protean Tetra cell)

Power: Basic electrophoresis power supply (power pac Basic)

Standard: high precision double standard (precision Plus dual standard)

Electrophoresis Buffer: Premixed Buffer Tris/Glycine/SDS

Standard quantity: 10μL

Electrophoresis loading: 20μL

Electrophoresis conditions: 100V fixed voltage, 90 minutes

Temperature: 25 ° C

Next, the gel after electrophoresis and 200 mL of distilled water were added to a tight box of No. 3 (manufactured by AS ONE Co., Ltd.), and shaken for 5 minutes using a rocking mixer RM-80 (manufactured by AS ONE). After removing distilled water, three such cleaning operations were carried out. Next, 50 mL of a staining solution of Bio-Safe CBB G-250 stain (manufactured by Bio-Rad Laboratiories Co., Ltd.) was added, and the mixture was shaken by the above-described rocking mixer for 1 hour, and then the staining solution was removed. Finally, 200 mL of distilled water was added, and the mixture was shaken for 1 hour with the above-described rocking mixer to remove distilled water to obtain an electrophoresis pattern.

The band thickness (the size of the band in the direction of migration) and the concentration from the glycated amino acid oxidase in the vicinity of 50 kDa in the obtained electrophoresis pattern were measured by a usual method using a GS-800 Calibrated Densitometer (manufactured by Bio-Rad Laboratiories Co., Ltd.). (Absorbance of the band) is measured. The band retention ratio 1 in the following formula (1) was calculated from the obtained blank and the band thickness of the sample, respectively, and the band retention ratio in the following formula (2) was calculated from the obtained blank and the band concentration of the sample. , band retention rate 1 and band retention rate 2 90% is set to ◎ (excellent), and the band retention ratio 1 and the band retention ratio 2 <90% are set to × (poor), and the storage stability (ie, decomposition resistance) of the glycated amino acid oxidase is performed. Evaluation.

[Math 1]

Band retention rate 1 (%) = (sample band thickness / blank band thickness) × 100 (1)

[Math 2]

Band retention 2 (%) = (sample band concentration / blank band concentration) × 100 (2)

<7. Storage stability of redox-based color reagents>

The multilayer test piece of the present invention was placed in an aluminum sealed bag (Alumi lamizip) AL-9 (manufactured by Tokyo Glass Instruments Co., Ltd.), and after sealing with nitrogen, in a programmable cryostat IN 604 (manufactured by Yamato Scientific Co., Ltd.), Incubate at 0, 24, 48, 72, 96, 120, 144, 168, 336, 504 hours at 4 ° C, or incubate 0, 24, 48, 72, 96, 120, 144, 168, 336 at 25 ° C, 504 hours. Next, the multilayer test piece was sandwiched and fixed by a jig (see Fig. 1) from the upper and lower sides, and the reflectance of the side of the layer carrying the redox-based color developing reagent was measured under the following condition 2.

<Condition 2>

Device name: Spectrophotometer UV-2450 (made by Shimadzu Corporation)

Attachment name: integrating sphere ISR-2200 (made by Shimadzu Corporation)

Standard Whiteboard: Barium Sulfate Standard Whiteboard

Measurement wavelength: 666 nm (λmax of DA67)

Angle of incidence: 0°

Slit width: 2.0nm

Beam size: 3mm × 5mm

Temperature: 25 ° C

The K/S value was calculated from the obtained reflectance by the Kubelka-Munk conversion of the following formula (3), and the K/S value (4 ° C) was obtained for the K/S value after 3 weeks (504 hours). 0.25 and K/S value (25 ° C) 0.50 is set to ◎ (excellent), and 0.25 < K/S value (4 ° C) 0.35 and 0.50<K/S value (25°C) 0.60 is set to ○ (good), and 0.35<K/S value (4 ° C) 0.40 and 0.60<K/S value (25°C) 1.30 is set to △ (acceptable), and 0.40<K/S value (4°C) and 1.30<K/S value (25°C) are set to ×(bad), and the storage stability of the redox-based color developing reagent is That is, it is evaluated by self-coloring resistance. It is known that an increase in the K/S value is self-developing if the redox coloring reagent is self-developing. Further, %R in the formula (3) means the reflectance.

[Math 3]

K/S value = (1-%R) 2 / (2 × % R) (3)

<8. Reactivity of proteases>

In the multilayer test piece of the present invention, 20 μL of an aqueous solution of 100 g/L of human heme H7379 (manufactured by SIGMA) was spotted on the first layer, and in a programmable cryostat IN 604 (manufactured by Yamato Scientific Co., Ltd.), five 37 Incubate for 0, 5, 10, 15, 30, 60 minutes at °C. Next, the multilayer test piece and 1000 μL of the above reagent 2 were placed in a microtube, and the interfacial activity in the multilayer test piece was extracted by stirring with a vortex mixer (manufactured by MS Instruments) for 1 minute. Agent, protease, glycosylated amino acid oxidase, peroxidase, redox system color reagent. Then, the extract was concentrated by centrifugation at 14000 G in a centrifugal ultrafiltration tube (MICROCON) 3 (manufactured by Millipore) to remove a low molecular weight component (a surfactant, a redox-based color developing reagent) having a molecular weight of 3,000 or less. 200 μL of concentrate. Next, 2.5 μL of the above-mentioned concentrate was mixed with 47.5 μL of distilled water, 47.5 μL of Laemmli sample buffer (Bio-Rad Laboratiories), and 2.5 μL of 2-mercaptoethanol (Bio-Rad Laboratiories), and boiled at 95 ° C. In minutes, sample solutions 1 to 6 were prepared.

SDS-PAGE was performed on the prepared sample solutions 1 to 6 under the above condition 1 using an electrophoresis best package (pre-formed gel (manufactured by Bio-Rad Laboratiories)). In addition, sample solutions 1 through 6 flow through different channels of the same gel.

Next, the gel after electrophoresis and 200 mL of distilled water were added to a tight box of No. 3 (manufactured by AS ONE Co., Ltd.), and shaken for 5 minutes using a rocking mixer RM-80 (manufactured by AS ONE). After removing distilled water, three such cleaning operations were carried out. Next, 50 mL of a staining solution of Bio-Safe CBB G-250 stain (manufactured by Bio-Rad Laboratiories Co., Ltd.) was added, and the mixture was shaken by the above-described rocking mixer for 1 hour, and then the staining solution was removed. Finally, 200 mL of distilled water was added, and the mixture was shaken for 1 hour with the above-described rocking mixer to remove distilled water to obtain an electrophoresis pattern.

Using the GS-800 Calibrated Densitometer (manufactured by Bio-Rad Laboratiories Co., Ltd.), the band thickness (the size of the band in the direction of migration) and the concentration (spectrum) from the heme subunit near 16 kDa in the obtained electropherogram according to the usual method. The absorbance of the belt is measured. The band disappearance rate in the following formula (4) was calculated from the band thickness after incubation at 37 ° C for 0 minutes to 60 minutes, respectively, and the band concentration after incubation from the obtained 37 ° C for 0 minutes to 60 minutes. The band disappearance rate 2 in the following formula (5) is calculated, and the band disappearance rate 1 and the band retention rate reach 90% or more (hereinafter, also referred to as band disappearance time), and 0 minute <band Time of disappearance 5 minutes is set to ◎ (excellent), 5 minutes < band disappearing time 15 minutes is set to ○ (good), 15 minutes < band disappearing time The temperature was set to Δ (acceptable) for 30 minutes, and the band disappearance time was set to × (bad) for 30 minutes, and the reactivity of the protease was evaluated.

[Math 4]

Band disappearance rate 1 (%) = {1 (band thickness (0 minutes to 60 minutes) / band thickness (0 minutes))} × 100 (4)

[Math 5]

Band disappearance rate 2 (%) = {1 - (band concentration (0 minutes to 60 minutes) / band concentration (0 minutes))} × 100 (5)

<9. Sensitivity and correlation of redox-based color reagents>

The synthetic fructosyl-valyl-histidine (hereinafter sometimes referred to as F-VH) is dissolved in an aqueous solution of 100 g/L of human heme H7379 (manufactured by SIGMA) to make it Measurement samples of 0 μm, 50 μm, 100 μm, 200 μm, and 500 μM were prepared and prepared into five levels. Next, the multilayer test piece of the present invention was clamped and fixed from above and below by a jig (refer to Fig. 1), and 20 μL of the above-mentioned measurement sample was spotted on the first layer, and after incubation at room temperature for 1 minute, under the following condition 3, The reflectance of the surface opposite to the spot surface of the measurement sample was measured.

<Condition 3>

Measurement sample: human heme H7379 100g/LF-VH 0μM, 50μM, 100μM, 200μM, 500μM catalase

Device name: Spectrophotometer UV-2450 (made by Shimadzu Corporation)

Attachment name: integrating sphere ISR-2200 (made by Shimadzu Corporation)

Standard Whiteboard: Barium Sulfate Standard Whiteboard

Measurement wavelength: 666 nm (DA67), 727 nm (DA64), 555 nm (TOOS), 630 nm (MAOS)

Angle of incidence: 0°

Slit width: 2.0nm

Beam size: 3mm × 5mm

Temperature: 25 ° C

The obtained F-VH was converted by Kubelka-Munk of the above formula (3) at a reflectance of 0 μM to 500 μM to obtain a K/S value of F-VH at 0 μM to 500 μM. The K/S volatility of the following equation (6) was calculated from the obtained K-S value of F-VH at 0 μM and the K/S value of F-VH at 500 μM, and the K/S volatility was obtained. 2.0 was set to ◎ (excellent), and the K/S fluctuation rate < 2.0 was set to × (bad), and the sensitivity of the redox-based color developing reagent was evaluated. Further, the K/S value (0 μM) and the K/S value (500 μM) in the formula (6) mean the K/S value of F-VH at 0 μM and the K/S value of F-VH at 500 μM, respectively.

[Math 6]

K/S fluctuation rate = K/S value (500 μM) / K / S value (0 μM) (6)

In addition, the Pearson correlation coefficient r of the obtained K/S value and F-VH concentration is calculated, and r>0.95 is set to ◎ (excellent), and 0.95 is obtained. r<0.90 is set to ○ (good), will be 0.90 r<0.85 is set to △ (to be), r will be 0.85 was set to × (bad), and the correlation was evaluated.

<10. Production of Heme A1c Value Calibration Curve>

The multi-layer test piece of the present invention was clamped and fixed from the upper and lower sides by a jig (refer to FIG. 1), and the sample of the HbA1c measurement performance evaluation sample QRM HbA1c 2007-1 (manufactured by General Corporate Legal Examination Material Standards Co., Ltd.) was spotted in the first place. One layer was incubated for 15 minutes in a programmable cryostat IN 604 (manufactured by Yamato Scientific Co., Ltd.) at 37 ° C, and the reflectance of the surface opposite to the spot surface of the measurement sample was measured under the following condition 4.

<Condition 4>

Measurement sample: HbA1c measurement performance evaluation sample QRM LEVEL 1, 2, 3, 4, 5

Device name: Spectrophotometer UV-2450 (made by Shimadzu Corporation)

Attachment name: integrating sphere ISR-2200 (made by Shimadzu Corporation)

Standard Whiteboard: Barium Sulfate Standard Whiteboard

Measuring wavelength: 480nm, 666nm

Angle of incidence: 0°

Slit width: 2.0nm

Beam size: 3mm × 5mm

Temperature: 25 ° C

From the resulting reflectance at 480 nm and the reflectance at 666 nm, the K/S value at 480 nm and the K/S value at 666 nm were obtained by Kubelka-Munk conversion of the above formula (3). Next, the K/S ratio of the following formula (7) was calculated from the obtained K/S value at 480 nm and the K/S value at 666 nm. The K/S ratio is proportional to the heme A1c value (see Non-Patent Document 1). Further, the K/S value (480 nm) and the K/S value (666 nm) in the formula are the K/S value at 480 nm and the K/S value at 666 nm, respectively.

K/S ratio = K/S value (666 nm) / K / S value (480 nm) (7)

The HbA1c value (LEVEL 1 = 4.67%, LEVEL 2 = 5.29%, LEVEL 3 = 6.96%, LEVEL 4 = 9.08%, LEVEL 5) specified in the obtained K/S ratio and the HbA1c measurement performance evaluation sample QRM was calculated. 1:1.9%) Pearson correlation coefficient r, set r>0.95 to ◎ (excellent), will be 0.95 r<0.90 is set to ○ (good), will be 0.90 r<0.85 is set to △ (to be), r will be 0.85 was set to × (bad), and the correlation was evaluated.

<11. Measurement of hemoglobin A1c value>

The synthesized fructosyl-valyl-histidine (hereinafter sometimes referred to as F-VH) is dissolved in healthy human blood to form 0 μm, 10 μm, 20 μm, 50 μm, and 100 μM. Modulated into 5 levels of measurement samples.

Next, the multilayer test piece of the present invention was clamped and fixed from above and below by a jig (refer to FIG. 1), and 20 μL of the sample to be tested was spotted on the first layer, and a programmable cryostat IN 604 at 37 ° C (Yamato Science) After the incubation for 15 minutes in the company, the reflectance of the surface opposite to the spot surface of the measurement sample was measured under the following condition 5. In addition, Hitachi Automatic Analyzer 7180 (manufactured by Hitachi High-Technologies Corporation), which is a commercially available automatic analyzer, and Nordia N HbA1c (manufactured by Sekisui Medical Technology Co., Ltd.), which is a commercially available heme A1c measuring reagent, are used as usual. The method was measured and the hemoglobin A1c value was calculated.

<Condition 5>

Measurement sample: healthy human blood F-VH 0μM, 10μM, 20μM, 50μM, 100μM

Device name: Spectrophotometer UV-2450 (made by Shimadzu Corporation)

Attachment name: integrating sphere ISR-2200 (made by Shimadzu Corporation)

Standard Whiteboard: Barium Sulfate Standard Whiteboard

Measuring wavelength: 480nm, 666nm

Angle of incidence: 0°

Slit width: 2.0nm

Beam size: 3mm × 5mm

Temperature: 25 ° C

From the resulting reflectance at 480 nm and the reflectance at 666 nm, the K/S value at 480 nm and the K/S value at 666 nm were obtained by Kubelka-Munk conversion of the above formula (3). Next, the K/S ratio of the above formula (7) was calculated from the obtained K/S value at 480 nm and the K/S value at 666 nm. The hemoglobin A1c value was calculated from the obtained K/S and the calibration curve obtained in the previous item. From the obtained hemoglobin A1c value of healthy human blood to which F-VH of 0 μM was added and the hemoglobin A1c value of healthy human blood to which 100 μM of F-VH was added, the difference in HbA1c value of the following formula (8) was calculated. The difference in HbA1c value 0.5 is set to ◎ (excellent), and 0.5<HbA1c value difference 1.0 is set to ○ (good), will be 1.0 <HbA1c value difference 1.5 is set to △ (acceptable), and 1.5<HbA1c value difference is set to × (bad), for glycosylated amino acids and/or glycated peptides which may be present in the sample to be tested but not by glycated hemoglobin The impact was evaluated. Further, the HbA1c value (0 μM) and the HbA1c value (100 μM) of the formula (8) represent the hemoglobin A1c value of healthy human blood to which F-VH of 0 μM was added, and the blood red of healthy human blood to which 100 μM of F-VH was added, respectively. The meaning of the A1c value.

[Math 8]

HbA1c value difference = HbA1c value (100 μM) - HbA1c value (0 μM) (8).

[Production of test piece]

[Example 1]

10 μL of the following reagent 3 was dropped on the Kiryu filter paper No. 5A (manufactured by Tokyo Glass Instruments Co., Ltd.) of 8 mm ,, and 10 μL of the following reagent 4 was dropped on the other 8 mm 桐Kanayama filter paper No. 5A (manufactured by Tokyo Glass Instruments Co., Ltd.). This was dried for 2 hours in a light-shielding dryer 3909-04 (manufactured by Tokyo Glass Instruments Co., Ltd.) at 25 ° C to prepare two kinds of single-layer test pieces. A multilayer test piece was produced by laminating the obtained two single-layer test pieces. In addition, the concentration of the surfactant carried is 0.1 mg/cm ² , the protease concentration is 10 U/cm ² , the glycated amino acid oxidase concentration is 10 U/cm ² , the peroxidase concentration is 40 U/cm ² , and the redox system The chromogenic reagent concentration was 0.1 mg/cm ² .

The details of the obtained multilayer test piece are shown in Table 1. In addition, NP40, XIV, FPO, PEO, and DA67 in the table are Nonidet (registered trademark) P-40, protease Type-XIV, glycosylated amino acid oxidase FPO-301, peroxidase PEO-302, and DA67, respectively. The meaning of each layer. The same is true below.

<Reagent 3>

100 mM PIPES (manufactured by Tongren Chemical Research Co., Ltd.) pH 6.5

5.0 mg/mL Nonidet (registered trademark) P-40 (manufactured by Nacalai Tesque Co., Ltd.)

500 U/mL glycosylated amino acid oxidase FPO-301 (manufactured by Toyobo Co., Ltd.)

<Reagent 4>

100 mM PIPES (manufactured by Tongren Chemical Research Co., Ltd.) pH 6.5

5.0 mg/mL Nonidet (registered trademark) P-40 (manufactured by Nacalai Tesque Co., Ltd.)

500U/mL protease Type-XIV (made by SIGMA)

2000U/mL peroxidase PEO-302 (manufactured by Toyobo Co., Ltd.)

5.0 mg/mL DA67 (manufactured by Wako Pure Chemical Industries, Ltd.)

[Examples 2 to 4]

A multilayer test piece was produced in the same manner as in Example 1 except that the layer of the surfactant, the protease, the glycated amino acid oxidase, the peroxidase, and the redox-based color developing reagent was different. The obtained multilayer test piece is specifically shown in Table 1.

[Example 5]

10 μL of the following reagent 5 was dropped on the Kiryu filter paper No. 5A (manufactured by Tokyo Glass Instruments Co., Ltd.) of 8 mm, and 10 μL of the following reagent 6 was dropped on the other 8 mm 桐Kanayama filter paper No. 5A (manufactured by Tokyo Glass Instruments Co., Ltd.). 10 μL of the following reagent 7 was dropped on the other 8 mm 桐Kanayama filter paper No. 5A (manufactured by Tokyo Glass Instruments Co., Ltd.), and dried in a light-shield dryer 3909-04 (manufactured by Tokyo Glass Instruments Co., Ltd.) at 25 ° C for 2 hours. Three single layer test pieces were made. A multilayer test piece was produced by laminating the obtained three single-layer test pieces. In addition, the concentration of the surfactant carried is 0.1 mg/cm ² , the protease concentration is 10 U/cm ² , the glycated amino acid oxidase concentration is 10 U/cm ² , the peroxidase concentration is 40 U/cm ² , and the redox system The chromogenic reagent concentration was 0.1 mg/cm ² .

The details of the obtained multilayer test piece are shown in Table 2.

<Reagent 5>

100 mM PIPES (manufactured by Tongren Chemical Research Co., Ltd.) pH 6.5

5.0 mg/mL Nonidet (registered trademark) P-40 (manufactured by Nacalai Tesque Co., Ltd.)

500 U/mL glycosylated amino acid oxidase FPO-301 (manufactured by Toyobo Co., Ltd.)

<Reagent 6>

100 mM PIPES (manufactured by Tongren Chemical Research Co., Ltd.) pH 6.5

5.0 mg/mL Nonidet (registered trademark) P-40 (manufactured by Nacalai Tesque Co., Ltd.)

500U/mL protease Type-XIV (made by SIGMA)

<Reagent 7>

100 mM PIPES (manufactured by Tongren Chemical Research Co., Ltd.) pH 6.5

5.0 mg/mL Nonidet (registered trademark) P-40 (manufactured by Nacalai Tesque Co., Ltd.)

2000U/mL peroxidase PEO-302 (manufactured by Toyobo Co., Ltd.)

5.0 mg/mL DA67 (manufactured by Wako Pure Chemical Industries, Ltd.)

[Examples 6 to 9]

A multilayer test piece was produced in the same manner as in Example 5 except that the layer of the surfactant, the protease, the glycated amino acid oxidase, the peroxidase, and the redox coloring reagent was carried. The obtained multilayer test piece is shown in Table 2.

[Comparative Example 1]

10 μL of the following reagent 8 was dropped on Kiryu filter paper No. 5A (manufactured by Tokyo Glass Instruments Co., Ltd.) of 8 mm, and dried in a light-shield dryer 3909-04 (manufactured by Tokyo Glass Instruments Co., Ltd.) at 25 ° C for 2 hours to prepare a single sheet. Layer test piece. In addition, the concentration of the surfactant carried is 0.1 mg/cm ² , the protease concentration is 10 U/cm ² , the glycated amino acid oxidase concentration is 10 U/cm ² , the peroxidase concentration is 40 U/cm ² , and the redox system The chromogenic reagent concentration was 0.1 mg/cm ² .

The details of the obtained multilayer test piece are shown in Table 3.

<Reagent 8>

100 mM PIPES (manufactured by Tongren Chemical Research Co., Ltd.) pH 6.5

5.0 mg/mL Nonidet (registered trademark) P-40 (manufactured by Nacalai Tesque Co., Ltd.)

500U/mL protease Type-XIV (made by SIGMA)

500 U/mL glycosylated amino acid oxidase FPO-301 (manufactured by Toyobo Co., Ltd.)

2000U/mL peroxidase PEO-302 (manufactured by Toyobo Co., Ltd.)

5.0 mg/mL DA67 (manufactured by Wako Pure Chemical Industries, Ltd.)

[Comparative Examples 2 to 7]

A multilayer test piece was produced in the same manner as in Example 1 except that the layer of the surfactant, the protease, the glycated amino acid oxidase, the peroxidase, and the redox-based color developing reagent was different. The details of the obtained multilayer test piece are shown in Table 4.

[Comparative Examples 8, 9]

A multilayer test piece was produced in the same manner as in Example 5 except that the layer of the surfactant, the protease, the glycated amino acid oxidase, the peroxidase, and the redox coloring reagent was carried. The details of the obtained multilayer test piece are shown in Table 5.

<Storage stability of glycosylated amino acid oxidase and redox chromogenic reagent>

Using the multilayer test pieces of Examples 1 to 9 and Comparative Examples 1 to 9, the storage stability of the glycated amino acid oxidase and the storage stability of the redox-based color developing reagent were evaluated.

The evaluation results obtained are shown in Tables 6 and 7.

According to the evaluation results of the storage stability of the glycated amino acid oxidase, when the protease and the glycated amino acid oxidase are carried in the same layer, the inactivation and decomposition of the glycated amino acid oxidase caused by the protease occurs, resulting in stable storage. Significantly reduced sex. In addition, inactivation and decomposition of the glycated amino acid oxidase sometimes cause measurement failure.

Further, according to the storage stability evaluation result of the redox-based color developing reagent, when the peroxidase and the redox-based color developing reagent are carried in the same layer, the redox-based color developing reagent self-develops color, resulting in remarkable storage stability. reduce. In addition, since the redox coloring reagent self-develops color, it sometimes causes a decrease in sensitivity. According to the above results, the layer structure of the multilayer test piece excellent in stability is required to carry the protease and the glycated amino acid oxidase in different layers, and examples thereof include the multilayer test pieces of Examples 1 to 9. More preferably, the peroxidase and the redox-based color developing reagent are respectively carried in different layers, and examples thereof include multilayer test pieces of Examples 2, 3 and 6 to 9.

[Examples 10 to 13]

In addition, the protease was changed from Type-XIV (manufactured by SIGMA Co., Ltd.) to Toyoteam NEP (manufactured by Toyobo Co., Ltd.), Type-X (manufactured by SIGMA Co., Ltd.), proteinase K (manufactured by Roche), and Type-XIII (manufactured by SIGMA Co., Ltd.). The same procedure as in Example 2 was carried out to prepare a multilayer test piece. The details of the obtained multilayer test piece are shown in Table 8. In addition, NEP, X, K, and XIII in the table mean that each of Toyoteam NEP, Type-X, Proteinase K, and Type-XIII is carried on each layer.

[Comparative Example 10]

A multilayer test piece was produced in the same manner as in Example 2 except that the protease was changed from Type-XIV (manufactured by SIGMA Co., Ltd.) to Type-I (manufactured by SIGMA Co., Ltd.). The details of the obtained multilayer test piece are shown in Table 8. In addition, I in the table means that Type-I is carried in each layer.

<Protease reactivity>

The reactivity of the protease was evaluated using the multilayer test pieces of Examples 2, 10 to 13, and Comparative Example 10.

The evaluation results obtained are shown in Table 9.

According to the evaluation results of the protease reactivity, the protease is preferably selected from a protease derived from Bacillus, a protease derived from Aspergillus, a protease derived from Streptomyces, and a strain from Tritirachium. More than one of the groups consisting of proteases.

[Embodiment 14]

The same procedure as in Example 2 was carried out except that the redox coloring reagent was changed from DA67 (manufactured by Wako Pure Chemical Industries, Ltd.) of λmax = 666 nm to DA64 (manufactured by Wako Pure Chemical Industries, Ltd.) of λmax = 727 nm. Multi-layer test piece.

The details of the obtained multilayer test piece are shown in Table 10. In addition, DA64 in the table means that DA64 is carried in each layer.

[Comparative Examples 11, 12]

In addition, the redox-based coloring reagent was changed from DA67 (manufactured by Wako Pure Chemical Industries, Ltd.) of λmax = 666 nm to 4AA (manufactured by Nacalai Tesque Co., Ltd.) and TOOS (manufactured by Toray Chemical Research Co., Ltd.) of λmax = 555 nm, and λmax = 630 nm. A multilayer test piece was produced in the same manner as in Example 2 except that 4AA (manufactured by Nacalai Tesque Co., Ltd.) and MAOS (manufactured by Tongren Chemical Research Co., Ltd.). Further, 4AA carrier concentration of 0.06 mg / cm ^2, TOOS concentration of 0.09mg / cm ^2, MAOS concentration of 0.09mg / cm ^2.

The details of the obtained multilayer test piece are shown in Table 10. In addition, 4AA, TOOS, and MAOS in the table mean that 4-aminoantipyrine, TOOS, and MAOS are each carried on each layer.

<Sensitivity and correlation of redox-based color reagents>

Using the multilayer test pieces of Examples 2 and 14 and Comparative Examples 11 and 12, the sensitivity and correlation of the redox-based color developing reagent were evaluated.

The evaluation results obtained are shown in Table 11.

According to the evaluation results of the sensitivity and correlation of the redox-based coloring reagent, it is preferable that the redox-based color developing reagent has a high molar absorption coefficient and is less susceptible to the hue of heme, and has a maximum at 650 nm to 800 nm. A colorless pigment that absorbs wavelengths.

[Examples 15 to 17]

In addition to changing the surfactant from Nonidet (registered trademark) P-40 (manufactured by Nacalai Tesque Co., Ltd.) having an HLB value of 17.6 to Triton (registered trademark) X-100 (manufactured by Nacalai Tesque Co., Ltd.) having an HLB value of 13.5, HLB value = A Tween (registered trademark) 20 (manufactured by Wako Pure Chemical Industries, Ltd.) of 16.7 and a Brij (registered trademark) 35 (manufactured by Wako Pure Chemical Industries, Ltd.) having an HLB value of 16.9 were used in the same manner as in Example 10 to produce a plurality of layers. Test piece.

The details of the obtained multilayer test piece are shown in Table 12. In addition, TX100, Tw20, and Br35 in the table mean that Triton (registered trademark) X-100, Tween (registered trademark) 20, and Brij (registered trademark) 35 are carried in each layer.

[Comparative Examples 13, 14]

In addition to the surfactant Nodidet (registered trademark) P-40 (manufactured by Nacalai Tesque Co., Ltd.) having an HLB value of 17.6, DK Ester (registered trademark) F50 (manufactured by Daiichi Kogyo Co., Ltd.) having an HLB value of 6.0, HLB value = A multilayer test piece was produced in the same manner as in Example 10 except that DK Ester (registered trademark) F70 (manufactured by Dai-ichi Kogyo Co., Ltd.) of 8.0 was used.

The details of the obtained multilayer test piece are shown in Table 12. In addition, F50 and F70 in the table mean that DK Ester (registered trademark) F50 (manufactured by Daiichi Kogyo Co., Ltd.) and DK Ester (registered trademark) F70 are carried in each layer.

<Protease reactivity>

The reactivity of the protease was evaluated using the multilayer test pieces of Examples 10, 15 to 17, and Comparative Examples 13 and 14.

The evaluation results obtained are shown in Table 13.

According to the evaluation result of the protease reactivity, the surfactant is preferably a nonionic surfactant having an excellent effect of promoting the protease reaction and having an HLB value of 10 to 20.

<Preparation of Hemoglobin A1c Value Calibration Curve: Sample for HbA1c Measurement Performance Evaluation>

Using the multilayer test pieces of Examples 1 to 4 and Comparative Example 2, a calibration curve of HbA1c value was prepared, and the correlation was evaluated. The evaluation results obtained are shown in Table 14.

<Measurement of heme A1c value>

Using the multilayer test pieces of Examples 1 to 4 and Comparative Example 2, the HbA1c value was calculated using the calibration curve of the HbA1c value obtained in the previous item. The evaluation results obtained are shown in Table 15.

From the above results, the heme A1c value measured using the multilayer test piece of the present invention was very consistent with the heme A1c value measured using the biochemical automatic analyzer. Moreover, since the multilayer test piece of the present invention is not easily affected by the glycated amino acid and/or the glycated peptide which may be present in the sample to be tested but not by glycated hemoglobin, and has excellent storage stability, Using the multilayer test piece of the present invention, the amount of glycated hemoglobin in the test sample can be accurately, simply and quickly subjected to colorimetric quantification.

Industrial availability

By using the multilayer test piece of the present invention, it is possible to perform colorimetric quantification of the amount of glycated hemoglobin in the test sample correctly, simply and quickly at the diagnosis site, and since the multilayer test piece of the present invention is less susceptible to being tested It is not affected by glycated amino acids and/or glycated peptides derived from glycated hemoglobin, and has excellent storage stability. Therefore, it is hoped to be in the field of clinical examination based on preventive medicine, in the field of diagnostic medicine, and in the field of medicine. And the industry in the field of life sciences, which is headed by the field of health care, has made great contributions.

Fig. 1 is a plate-shaped jig used in the present embodiment for holding a test piece up and down to fix it. This fixture is provided with two through holes.

Claims (11)

A multilayer test piece for colorimetric quantification of glycated hemoglobin content, characterized in that the multilayer test piece is laminated with at least the following layers (a) and (b), and the (a) layer and (b) layers are stacked in the order of layers (a) and (b) from the sample to be tested; and the multilayer test piece does not have a blood separation layer; wherein (a) layer: at least carrying a glycosylated amine a polymer substrate of a base acid oxidase; (b) a layer: a polymer substrate carrying at least a protease. A multilayer test piece for colorimetric quantification of glycated hemoglobin content, characterized in that the multilayer test piece is laminated with at least the following layers (a) and (b), and the (a) layer and (b) Layers are stacked in the order of layers (a) and (b) from the sample to be tested; peroxidase and redox are respectively carried on different layers in layers (a) and (b) a color developing reagent; and the multilayer test piece does not have a blood separation layer; wherein (a) layer: a polymer substrate carrying at least a glycated amino acid oxidase; (b) a layer: at least a protease carrying Molecular substrate. The multilayer test piece according to claim 1 or 2, wherein the multilayer test piece further carries a surfactant on at least one of layers (a) and (b). A multilayer test piece for colorimetric quantification of glycated hemoglobin content, characterized in that the multilayer test piece is laminated with at least the following layers (a), (b) and (c), (a) layer, (b) layer and (c) layer from (a) layer and (b) layer and (c) layer, (a) layer and (c) layer and (b) Or a sequential layer of (a) layer and (a) layer and (b) layer; and the multilayer test piece does not have a blood separation layer; wherein (a) layer: at least loaded with glycated amino acid oxidase Polymer substrate; (b) layer: a polymer substrate carrying at least a protease; (c) layer: a polymer substrate carrying at least any reagent other than a protease and a glycosylated amino acid oxidase. A multilayer test piece for colorimetric quantification of glycated hemoglobin content, characterized in that the multilayer test piece is laminated with at least the following layers (a), (b) and (c), (a) layer, (b) layer and (c) layer from (a) layer and (b) layer and (c) layer, (a) layer and (c) layer and (b) Or a sequential layer of (c) and (a) and (b) layers; carrying peroxidase and redox on the different layers in layers (a), (b) and (c) a color developing reagent; and the multilayer test piece does not have a blood separation layer; wherein (a) layer: a polymer substrate carrying at least a glycated amino acid oxidase; (b) a layer: at least a protease carrying Molecular substrate; (c) layer: a polymer substrate carrying at least any reagent other than a protease and a glycosylated amino acid oxidase. The multilayer test piece of claim 4 or 5, wherein the multilayer test piece further carries a surfactant on at least one of the layers (a), (b) and (c). The multilayer test piece according to any one of claims 1 to 6, wherein the protease is selected from the group consisting of at least a protease derived from Bacillus, a protease derived from Aspergillus, and Streptomyces. One or more of the protease and the group consisting of proteases of Tritirachium. The multilayer test piece according to any one of claims 1 to 7, wherein the redox-based color developing reagent is a colorless dye having a maximum absorption wavelength of from 600 nm to 800 nm. The multilayer test piece according to any one of claims 1 to 8, wherein the surfactant is a nonionic surfactant having a Hydrophile Lipophile Balance Value (HLB Value) of 10-20. A measuring method, characterized in that the measuring method uses a multilayer test piece according to any one of claims 1 to 9 to perform colorimetric comparison of glycated hemoglobin content through at least the following steps (i) to (iii); Quantitative; step (i): a step of spotting a sample to be tested on the upper surface of the multilayer test piece; and step (ii): measuring the reflection from the surface opposite to the sample to be tested of the multilayer test piece a step of rate and/or absorbance; and step (iii): calculating, from the obtained reflectance and/or absorbance, a group selected from the group consisting of a heme content, a glycated hemoglobin content, a glycated hemoglobin content, and a heme content. More than one step in the process. For example, the measuring method of claim 10, wherein the sample to be tested is a whole blood sample.