CN202837169U - Multi-layer test piece for glycated hemoglobin measurement - Google Patents

Multi-layer test piece for glycated hemoglobin measurement Download PDF

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Publication number
CN202837169U
CN202837169U CN 201220111588 CN201220111588U CN202837169U CN 202837169 U CN202837169 U CN 202837169U CN 201220111588 CN201220111588 CN 201220111588 CN 201220111588 U CN201220111588 U CN 201220111588U CN 202837169 U CN202837169 U CN 202837169U
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China
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layer
proteinase
amino acid
testing sheet
peroxidase
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Expired - Fee Related
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CN 201220111588
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冈本淳
塚本晓
中森雅彦
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Toyobo Co Ltd
Toyo Textile Co Ltd
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Toyo Textile Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • G01N33/723—Glycosylated haemoglobin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • G01N33/726—Devices

Abstract

At least a layer a and a layer b stack to form a multi-layer test piece for the glycated hemoglobin measurement, wherein the layer a and the layer b stack according to the orders of the layer a and the layer b from a sample point and a sample surface under test, the different layers of the layer a and the layer b bear peroxidase and redox color reagents, a blood separation layer is not provided, the layer a bears at least polymer base materials of protease, and the layer b bears at least polymer base materials of glycated amino acid oxidase. The multi-layer test piece for the glycated hemoglobin measurement has the advantages of being capable of performing the colorimetric analysis on the glycated hemoglobin levels under test right, simply and rapidly at the scene of the diagnosis and being excellent in saving stability.

Description

Glycosylated hemoglobin is measured and is used the tier testing sheet
Technical field
The utility model relates to a kind of for on-the-spot correct, easy and promptly the saccharification hemoglobin content of sample to be tested is carried out the tier testing sheet of colorimetric assay in diagnosis.
Background technology
It is extremely important aspect the diagnosis of carrying out diabetes, prevention and treatment to measure glycated proteins.Non-enzymatic reaction occurs and generates in the amino (mainly be the alpha-amido of end amino acid, the gamma-amino of lysine residue) of glycated proteins by protein in vivo glucose and the blood.Since the degree of protein glycation directly and in the blood concentration of glucose (hereinafter referred to as blood glucose value) proportional, therefore, for example, in the situation of glycosylated hemoglobin, can grasp glycemic control state in about 2～3 months by measuring glycated proteins, in the situation of glycosylated albumin, can grasp interior glycemic control state of about 2～3 weeks by measuring glycated proteins.
Especially be considered to most important as a kind of glycated hemoglobin of glycosylated hemoglobin when the diagnosis of diabetes.
As the measuring method of above-mentioned glycated proteins, known have high performance liquid chromatography (HPLC) method, immunization and an enzyme process etc.In the prior art, high performance liquid chromatography (HPLC) method and immunization occupy major part, but in recent years since enzyme process can be rapidly, in a large number, correctly and at an easy rate great amount of samples is measured, therefore also begin frequently to be adopted.
Relevant enzyme process, for example known have a technology (for example, with reference to patent documentation 1～4) of glycated proteins being carried out colorimetric assay by following operation.
(1) make erythrocyte hemolysis in the sample to be tested to extract the operation of glycosylated hemoglobin.
(2) thus make above-mentioned glycosylated hemoglobin and mmp reaction cut out the operation of glycated amino acid and/or glycated peptide from the β chain N end of the saccharification of glycosylated hemoglobin.
(3) thus make the operation of above-mentioned glycated amino acid and/or glycated peptide and glycated amino acid oxydase reaction Hydrogen Peroxide.
(4) in the presence of peroxidase, make the reaction of above-mentioned hydrogen peroxide and redox class chromogenic reagent and the operation of colour developing.
(5) saccharification hemoglobin content in the sample to be tested is carried out the operation of colorimetric assay according to above-mentioned colour developing.
But, related prior art is the dilution sample to be tested, add the reagent of liquid condition, measure the method for the absorbance of reactant liquor after making it to react in reaction vessel, with the method for using the biological chemistry automatic analysing apparatus as main flow, but described biological chemistry automatic analysing apparatus exist be large-scale plant and expensive, need skilled detection technician operate, from Mining blood long these problems of time till go out to report the result, be difficult to be applicable to POCT(point of care testing(namely, clinical scene checks immediately)).
On the other hand, for addressing the above problem, having invented glycated proteins measures with test film (for example patent documentation 5), this test film has utilized so-called dry chemical method, in the method, by with drying regime reagent being carried on the base material, adding again the sample to be tested of liquid condition to it, thereby reaction is carried out the moisture in the sample to be tested.This invention improvement point is as follows: installs as small-sized and cheap, and easy and simple to handle, can finish the short time till going out to report the result from Mining blood.But, there is the problem of the storage stability can not get standing actual uses, namely the problem that oneself develops the color occurs in glycated amino acid oxidase generation inactivation, decomposition, redox class chromogenic reagent.In addition, from the viewpoint of POCT, the blood sample of sample to be tested must carry out the pre-treatment this point such as haemolysis and also have problems.
The invention (for example with reference to patent documentation 6) of the reagent test sheet that is made of multilayer that has utilized dry chemical method is also disclosed in addition.This invention also has following improvement point: install as small-sized and cheap, easy and simple to handle, can finish the short time till going out to report the result from Mining blood, still, have the problem of the storage stability can not get standing actual uses, i.e. the problem that oneself develops the color occurs in redox class chromogenic reagent.In addition, as from the blood separating layer being configured in ground floor also can know, this invention is only imagined the glycosylated albumin as plasma proteins in the glycated proteins is measured, and does not disclose any relevant object lesson of measuring glycosylated hemoglobin.
The prior art document
Patent documentation
Patent documentation 1: international disclosing 2002-006519 number
Patent documentation 2: JP 2004-222570 communique
Patent documentation 3: JP 2007-181466 communique
Patent documentation 4: JP 2010-148515 communique
Patent documentation 5: JP 2004-333452 communique
Patent documentation 6: JP 2004-004071 communique
Non-patent literature
Non-patent literature 1:Clinical Chemistry43:122390-2396(1997)
The utility model content
Utility model technical matters to be solved
The utility model is to carry out take the technical matters of relevant prior art as background.That is, the purpose of this utility model is for providing a kind of for on-the-spot correct, easy and promptly the saccharification hemoglobin content of sample to be tested is carried out tier testing sheet and the measuring method of colorimetric assay in diagnosis.More specifically, the purpose of this utility model is to provide tier testing sheet and measuring method a kind of excellent storage stability, that be used for saccharification hemoglobin content is carried out colorimetric assay.
The means of technical solution problem
The present application people finds, when proteinase and glycated amino acid oxidase are carried on identical layer, and generation is caused by proteinase in preservation glycated amino acid inactivating oxidase and/or decomposition, thus cause storage stability significantly to reduce.
Also find in addition, when peroxidase and redox class chromogenic reagent are carried on identical layer, the certainly colour developing of the redox class chromogenic reagent that generation is caused by peroxidase in preservation, thus cause storage stability significantly to reduce.Because compare with concentration of glucose etc., the glycosylated hemoglobin concentration in the sample to be tested is generally low concentration, therefore when measuring glycosylated hemoglobin, preferably use highly sensitive colourless pigment, but colourless pigment is unstable especially, occur from colour developing easily.
Thereby the present application people is carried on proteinase and glycated amino acid oxidase on the different layers, and proteinase is carried on the layer than the more close sample to be tested point sample of glycated amino acid oxidase face.Find that thus the oxidasic storage stability of glycated amino acid significantly improves, and the reactivity of proteinase improves also, measuring accuracy also improves, thereby has finished the utility model.In addition, by peroxidase and redox class chromogenic reagent are carried on respectively on the different layers, the storage stability that improves redox class chromogenic reagent are succeeded simultaneously, thereby finished the utility model.
That is, the utlity model has following inscape.
1, a kind of tier testing sheet, be used for saccharification hemoglobin content is carried out colorimetric assay, it is characterized in that: this tier testing sheet is laminated with following a layer and b layer at least, described a layer and b layer are pressed the sequential cascade of a layer, b layer from sample to be tested point sample face, carry respectively peroxidase and redox class chromogenic reagent on the different layer in a layer and b layer, and described tier testing sheet does not have the blood separating layer, wherein, a layer: carry at least the polymer base material of proteinase, the b layer: carry at least the oxidasic polymer base material of glycated amino acid.
2, according to 1 described tier testing sheet, it is characterized in that: also carry surfactant on described tier testing sheet at least one deck in a layer and b.
3, a kind of tier testing sheet, be used for saccharification hemoglobin content is carried out colorimetric assay, it is characterized in that: described tier testing sheet is laminated with following a layer at least, b layer and c layer, described a layer, b layer and c layer are pressed a layer from sample to be tested point sample face, b layer and c layer, the a layer, c layer and b layer or c layer, the sequential cascade of a layer and b layer, at a layer, carry respectively peroxidase and redox class chromogenic reagent on the different layers in b layer and the c layer, and described tier testing sheet does not have the blood separating layer, wherein, a layer: the polymer base material that carries at least proteinase, b layer: carry at least the oxidasic polymer base material of glycated amino acid, the c layer: the polymer base material that carries at least any agent except proteinase and glycated amino acid oxidase.
4, according to 3 described tier testing sheets, it is characterized in that: also carry surfactant on described tier testing sheet at least one deck in a layer, b layer and c layer.
5, according to each described tier testing sheet in 1 to 4, it is characterized in that: proteinase is to be selected from least by from the proteinase of rod bacterium (Bacillus), from the proteinase of Aspergillus (Aspergillus), the group that forms from the proteinase of streptomycete (Streptomyces) and from the proteinase of reading coccus (Tritirachium) more than one.
6, according to each described tier testing sheet in 1 to 5, it is characterized in that: redox class chromogenic reagent is that maximum absorption wavelength is the colourless pigment of 600nm～800nm.
7, according to each described tier testing sheet in 1 to 6, it is characterized in that: surfactant is that hydrophilic lipophilic balance (Hydrophile Lipophile Balance Value:HLB Value) is 10～20 nonionic surfactant.
The utility model technique effect
According to the utility model, a kind of tier testing sheet for saccharification hemoglobin content being carried out colorimetric assay can be provided, this tier testing sheet has suppressed the oxidasic inactivation of glycated amino acid or decomposition, and has suppressed the certainly colour developing of redox class chromogenic reagent, and excellent storage stability.
Description of drawings
Fig. 1 is the tabular anchor clamps that the clamping test film is fixed about being used for of using in the present embodiment.These anchor clamps are provided with two through holes.
Embodiment
Below, the utility model is elaborated.
(measuring object thing)
Measuring object thing of the present utility model is glycosylated hemoglobin, and from the viewpoint that diabetes diagnosis is used, the β chain N end of preferred haemoglobin is by the glycated hemoglobin of saccharification (the following HbA1c that sometimes is also referred to as).
(sample to be tested)
As sample to be tested of the present utility model, can list organism sample (sample of namely taking from biosome) or the foodstuffs such as drinking water, condiment such as whole blood, haemocyte, blood plasma, serum, marrow liquid, sweat, urine, tear, saliva, skin, mucous membrane, hair.In addition, organism sample is not limited only to from human body, also is object from the organism samples of the mammals such as dog, cat, ox.Wherein, from the viewpoint that diabetes diagnosis is used, preferred whole blood or haemocyte from the viewpoint of POCT, more preferably do not carry out the whole blood that haemocyte/blood plasma or serum separate pre-treatment.
The amount of sample to be tested is not particularly limited in the utility model, for example is preferably 1 μ L～50 μ L, more preferably 1 μ L～40 μ L, more preferably 1 μ L～30 μ L.If the sample to be tested amount is less than 1 μ L, then probably can't be deployed into orlop from the superiors of tier testing sheet.On the other hand, if the sample to be tested amount is when then for example sample to be tested is blood, because patient's burden becomes large, therefore not preferred more than 50 μ L.
(measuring principle)
The measuring principle of glycosylated hemoglobin is to measure by so-called enzymic colorimetric in the utility model, in the method, carry out enzymatic reaction (so-called dry chemical method) by the moisture in the sample to be tested, thereby make the colour developing of tier testing sheet, then measure its colour developing degree by measuring reflected light.By using above-mentioned measuring principle, small-sized, light weight, the low price of device become possibility.In addition, correct, easy and rapidly measurement becomes possibility.
Below, the reaction during to the saccharification hemoglobin content in the measurement red blood cell describes, but this does not consist of any restriction of the present utility model.
(1) make red blood cell and surfactant in the sample to be tested react, make it haemolysis to extract the operation of glycosylated hemoglobin.
(2) thus make above-mentioned glycosylated hemoglobin and mmp reaction cut out the operation of glycated amino acid and/or glycated peptide from the β chain N end of the saccharification of glycosylated hemoglobin.
(3) thus make the operation of above-mentioned glycated amino acid and/or glycated peptide and glycated amino acid oxydase reaction Hydrogen Peroxide.
(4) above-mentioned hydrogen peroxide and redox class chromogenic reagent are reacted and the operation that develops the color.
(5) saccharification hemoglobin content in the sample to be tested is carried out the operation of colorimetric assay according to above-mentioned colour developing
(tier testing sheet)
Tier testing sheet of the present utility model is made of a plurality of polymer base materials (the following layer that sometimes is also referred to as).The number of plies of above-mentioned tier testing sheet can change according to embodiment, is preferably 2～7 layers, and more preferably 2～6 layers, more preferably 2～5 layers.In addition, carry the polymer base material of reagent (surfactant, proteinase, glycated amino acid oxidase, peroxidase, redox class chromogenic reagent etc.) except the utility model is needed, also comprise in the number of plies for the developer layer that launches blood or clear support layer etc.When the number of plies is one deck, proteinase and glycated amino acid oxidase and peroxidase and redox class chromogenic reagent etc. have to be carried on same layer, probably can produce the certainly colour developing of the oxidasic inactivation of the glycated amino acid that is caused by proteinase, decomposition or redox class chromogenic reagent.That is, the storage stability of tier testing sheet probably can significantly reduce.On the other hand, the number of plies is during more than 7 layers, and is because it is many to launch required sample to be tested quantitative change from the superiors to the orlop, therefore not preferred.
Above-mentioned tier testing sheet can adopt arbitrarily, and operation is made.Typically, can adopt after being immersed in respectively a plurality of layer in more than one the reagent solution drier habitual operation to make a plurality of layers, then be assembled into final test film.
Being characterized as of above-mentioned tier testing sheet do not contain the blood separating layer.In addition, the blood separating layer refer to for by obtain from the Whole Blood Filtration haemocyte blood plasma or serum the layer.Measuring object thing of the present utility model is not glycosylated albumin contained in blood plasma or the serum, but contained glycosylated hemoglobin in the haemocyte, therefore, then can't measure after haemocyte filtered.
(layer structure)
The layer structure of above-mentioned tier testing sheet need to be carried on proteinase on the layer (below, sometimes be also referred to as the upper strata) than the more close sample to be tested point sample of glycated amino acid oxidase face.When proteinase being carried on when upper further from the layer (below, sometimes be also referred to as lower floor) of sample to be tested point sample face than glycated amino acid oxidase, probably can cause proteinase fully not react, thus desensitization.
In addition, the layer structure of above-mentioned tier testing sheet need to be carried on proteinase and glycated amino acid oxidase respectively on the different layers, further preferably peroxidase and redox class chromogenic reagent is carried on the different layers respectively.When proteinase and glycated amino acid oxidase are carried on identical layer, the oxidasic inactivation of glycated amino acid, the decomposition that are caused by proteinase probably can occur, when peroxidase and redox class chromogenic reagent were carried on identical layer, the certainly colour developing of redox class chromogenic reagent probably can occur.That is, the storage stability of tier testing sheet probably can significantly reduce.The object lesson of layer structure below is shown, but this does not consist of to any restriction of the present utility model.
When the ground floor side of establishing above-mentioned tier testing sheet is sample to be tested point sample face, when second layer side is the reflected light measurement face, can lists proteinase and be carried on than the glycated amino acid oxidase following structure on upper strata more.
1, ground floor: proteinase, peroxidase; And the second layer: glycated amino acid oxidase, redox class chromogenic reagent.
2, ground floor: proteinase, redox class chromogenic reagent; And the second layer: glycated amino acid oxidase, peroxidase.
3, ground floor: proteinase; And the second layer: glycated amino acid oxidase, peroxidase, redox class chromogenic reagent.
4, ground floor: proteinase, peroxidase, redox class chromogenic reagent; And the second layer: glycated amino acid oxidase.
Wherein, more preferably peroxidase and redox class chromogenic reagent are carried on respectively the following structure of different layers.
1, ground floor: proteinase, peroxidase; And the second layer: glycated amino acid oxidase, redox class chromogenic reagent.
2, ground floor: proteinase, redox class chromogenic reagent; And the second layer: glycated amino acid oxidase, peroxidase.
Further preferably redox class chromogenic reagent is carried on following structure on the reflected light measurement face (second layer), that can expect to carry out highly sensitive measurement.
1, ground floor: proteinase, peroxidase; And the second layer: glycated amino acid oxidase, redox class chromogenic reagent.
When the ground floor side of establishing above-mentioned tier testing sheet is sample to be tested point sample face, when the 3rd layer of side is the reflected light measurement face, can lists proteinase and be carried on than the glycated amino acid oxidase following structure on upper strata more.
1, ground floor: proteinase; The second layer: glycated amino acid oxidase; And the 3rd layer: peroxidase, redox class chromogenic reagent.
2, ground floor: proteinase; The second layer: peroxidase; And the 3rd layer: glycated amino acid oxidase, redox class chromogenic reagent.
3, ground floor: proteinase; The second layer: redox class chromogenic reagent; And the 3rd layer: glycated amino acid oxidase, peroxidase.
4, ground floor: peroxidase; The second layer: proteinase; And the 3rd layer: glycated amino acid oxidase, redox class chromogenic reagent.
5, ground floor: redox class chromogenic reagent; The second layer: proteinase; And the 3rd layer: glycated amino acid oxidase, peroxidase.
6, ground floor: proteinase; The second layer: glycated amino acid oxidase, peroxidase; And the 3rd layer: redox class chromogenic reagent.
7, ground floor: proteinase; The second layer: glycated amino acid oxidase, redox class chromogenic reagent; And the 3rd layer: peroxidase.
8, ground floor: proteinase; The second layer: peroxidase, redox class chromogenic reagent; And the 3rd layer: the glycated amino acid oxidase.
9, ground floor: peroxidase; The second layer: proteinase, redox class chromogenic reagent; And the 3rd layer: the glycated amino acid oxidase.
10, ground floor: redox class chromogenic reagent; The second layer: proteinase, peroxidase; And the 3rd layer: the glycated amino acid oxidase.
11, ground floor: proteinase, peroxidase; The second layer: glycated amino acid oxidase; And the 3rd layer: redox class chromogenic reagent.
12, ground floor: proteinase, redox class chromogenic reagent; The second layer: glycated amino acid oxidase; And the 3rd layer: peroxidase.
13, ground floor: peroxidase, redox class chromogenic reagent; The second layer: proteinase; And the 3rd layer: the glycated amino acid oxidase.
14, ground floor: proteinase, peroxidase; The second layer: redox class chromogenic reagent; And the 3rd layer: the glycated amino acid oxidase.
15, ground floor: proteinase, redox class chromogenic reagent; The second layer: peroxidase; And the 3rd layer: the glycated amino acid oxidase.
Wherein, more preferably peroxidase and redox class chromogenic reagent are carried on respectively following structure on the different layers.
2, ground floor: proteinase; The second layer: peroxidase; And the 3rd layer: glycated amino acid oxidase, redox class chromogenic reagent.
3, ground floor: proteinase; The second layer: redox class chromogenic reagent; And the 3rd layer: glycated amino acid oxidase, peroxidase.
4, ground floor: peroxidase; The second layer: proteinase; And the 3rd layer: glycated amino acid oxidase, redox class chromogenic reagent.
5, ground floor: redox class chromogenic reagent; The second layer: proteinase; And the 3rd layer: glycated amino acid oxidase, peroxidase.
6, ground floor: proteinase; The second layer: glycated amino acid oxidase, peroxidase; And the 3rd layer: redox class chromogenic reagent.
7, ground floor: proteinase; The second layer: glycated amino acid oxidase, redox class chromogenic reagent; And the 3rd layer: peroxidase.
9, ground floor: peroxidase; The second layer: proteinase, redox class chromogenic reagent; And the 3rd layer: the glycated amino acid oxidase.
10, ground floor: redox class chromogenic reagent; The second layer: proteinase, peroxidase; And the 3rd layer: the glycated amino acid oxidase.
11, ground floor: proteinase, peroxidase; The second layer: glycated amino acid oxidase; And the 3rd layer: redox class chromogenic reagent.
12, ground floor: proteinase, redox class chromogenic reagent; The second layer: glycated amino acid oxidase; And the 3rd layer: peroxidase.
14, ground floor: proteinase, peroxidase; The second layer: redox class chromogenic reagent; And the 3rd layer: the glycated amino acid oxidase.
15, ground floor: proteinase, redox class chromogenic reagent; The second layer: peroxidase; And the 3rd layer: the glycated amino acid oxidase.
Further preferably redox class chromogenic reagent is carried on following structure on the reflected light measurement face (the 3rd layer), that can expect to carry out highly sensitive measurement.
2, ground floor: proteinase; The second layer: peroxidase; And the 3rd layer: glycated amino acid oxidase, redox class chromogenic reagent.
4, ground floor: peroxidase; The second layer: proteinase; And the 3rd layer: glycated amino acid oxidase, redox class chromogenic reagent.
6, ground floor: proteinase; The second layer: glycated amino acid oxidase, peroxidase; And the 3rd layer: redox class chromogenic reagent.
11, ground floor: proteinase, peroxidase; The second layer: glycated amino acid oxidase; And the 3rd layer: redox class chromogenic reagent.
At least one deck in preferred above-mentioned each layer (ground floor, the second layer, the 3rd layer) further carries surfactant.In addition, as long as proteinase and glycated amino acid oxidase and peroxidase and redox class chromogenic reagent are not present in same layer, then mentioned reagent (surfactant, proteinase, glycated amino acid oxidase, peroxidase, redox class chromogenic reagent etc.) can repeat to be carried on each layer (ground floor, the second layer, the 3rd layer).In addition, each layer can carry buffering agent as required.
(polymer base material)
As the polymer base material that consists of tier testing sheet of the present utility model, as long as can carry the mentioned reagent (surfactant, proteinase, glycated amino acid oxidase, peroxidase, redox class chromogenic reagent etc.) of aequum, and sample to be tested can be in the horizontal direction and vertical direction suitably launch, then can use the material of any form, composition.The form of polymer base material, the object lesson of composition below are shown, but this does not consist of to any restriction of the present utility model.
(form)
As the form of above-mentioned polymer base material, can list the material that material that filter paper, fiber construct, multiple aperture plasma membrane (film filter), film etc. can self-supporting or high-molecular gel etc. can not self-supportings.In addition, when using the material that high-molecular gel etc. can not self-supporting, material that filter paper, fiber construct, multiple aperture plasma membrane (film filter), film etc. can self-supporting preferably is set as supporter.
(composition)
As the composition of above-mentioned polymer base material, can list: as polyethylene terephthalate (PET), polybutylene terephthalate (PBT), PEN (PEN) and the PBN (PBN) etc. of vibrin; Tygon (PE), polypropylene (PP) and polybutylene etc. as olefin resin; Polyvinylchloride (PVC), Vingon and polyvinyl acetate etc. as vinylite; Acryl resin; Acrylate resin; As the polytetrafluoroethylene (PTFE) of fluororesin and Vingon (PVDF) etc.; Polycarbonate resin; Polyoxymethylene (POM), polyphenylene oxide (PPO), polyetherketone (PEK), polyetheretherketone (PEEK), polyphenylene sulfide (PPS), polysulfones (PSU), polyethersulfone (PES) and polyetherimide (PEI) etc. as polyether resin; As the nylon of polyamide and fragrant acid amides etc.; And as cellulose acetate, nitrocellulose and the regenerated cellulose etc. of cellulose family.
The shape of above-mentioned polymer base material is not particularly limited, and square, rectangle, circle, ellipse etc. are lamellar such as listing.
From small-sized, the light weight of device, the viewpoint of low price, the area of above-mentioned polymer base material is the smaller the better, but for example is preferably 1mm 2～1000mm 2, 2mm more preferably 2～500mm 2, 5mm more preferably 2～200mm 2
Extensibility and the reactive viewpoint of enzyme (proteinase, glycated amino acid oxidase, peroxidase) from sample to be tested, (one deck) thickness of above-mentioned polymer base material for example is preferably 10 μ m～2000 μ m, more preferably 20 μ m～1000 μ m, more preferably 50 μ m～500 μ m.
(detection)
When reaction was detected, the easiest was to the tier testing sheet irradiation light (incident light) that has developed the color, and then detects its reflected light, but also can use method in addition.Be not particularly limited as light source, such as listing UV lamp, xenon lamp, krypton lamp, mercury vapor lamp, deuterium lamp, tungsten lamp, Halogen lamp LED, light emitting diode (LED), laser etc.Wherein, from small-sized, the light weight of the easiness of optical wavelength control and device, the viewpoint of low price, preferred light emitting diode (LED).The angle of above-mentioned incident light (incident angle) is not particularly limited, and can adopt arbitrarily angle.On the other hand, catoptrical detection is not particularly limited, is preferably perpendicular to detection faces.In addition, if use photodiode or integrating sphere then can easily detect.
(proteinase)
As the employed proteinase of the utility model, as long as can be with the β chain N end effect of the saccharification of glycosylated hemoglobin and cut out glycated amino acid and/or glycated peptide, then can use the proteinase of any kind, such as listing proteinase from animal, plant, microorganism etc.The object lesson of proteinase below is shown, but this does not consist of to any restriction of the present utility model.
(from the proteinase of animal)
As the proteinase from animal, can list factor Xa(factor Xa), plasmin (plasmin), fibrin ferment (thrombin), pepsin (pepsin), leucine amino peptidase (leucinaminopeptidase), pancreatin (pancreatin), pancreas peptase (elastase), trypsase (trypsin), chymotrypsin A(chtmotrypsin A), Aminopeptidase M (aminopeptidase M), Carboxypeptidase A (carboxypeptidase A), protaminase (carboxypeptidase B), calpain (calpain), cathepsin B (cathepsin B), cathepsin C (cathepsin C), cathepsin D (cathepsin D), endo protease Arg-C(endoprotinaseArg-C) etc.
(from the proteinase of plant)
As the proteinase from plant, can list carboxypeptidase W(carboxypeptidase W), kassinin kinin releasing agent (kallikrein), ficin (ficin), papain (papain), chymopapain (chimopapain), bromelain (bromelain) etc.
(from the proteinase of microorganism)
As the proteinase from microorganism, can list: the proteinase from rod bacterium (Bacillus) take subtilopeptidase A (subtilisin), thermolysin (thermolysin), dispase (dispase), protease N (proteinase N) etc. as representative; The proteinase from Aspergillus (Aspergillus) take IP enzyme etc. as representative; The proteinase from streptomycete (Streptomyces) take pronase (pronase) etc. as representative; Take Proteinase K (proteinaseK) etc. as representative from the proteinase of reading coccus (Tritirachium); Take peptase R(peptidase R) etc. be the proteinase from head mold (Rhizopus) of representative; Take carboxypeptidase P(carboxypeptidase P), PD enzyme etc. is the proteinase from Penicillium notatum (Penicillium) of representative; Take endo protease Glu-C(endoprotinase Glu-C) etc. be the proteinase from staphylococcus (Staphylococcus) of representative; The proteinase from clostridium (Clostridium) take clostripain (clostripain) etc. as representative; Take endo protease Lys-C(endoprotinase Lys-C) etc. be the proteinase that comes self-dissolving bacillus (Lysobacter) of representative; The proteinase from umbellate pore furgus (Grifola) take metalloproteinases (metalloendopeputidase) etc. as representative; The proteinase from yeast (Yeast) take carboxypeptidase y (carboxypeptidase Y), protease A (proteinase A) etc. as representative; Take aminopeptidase T(aminopeptidase T) etc. be the proteinase from hot bacterium (Thermus) of dwelling of representative; Take endo protease Asp-N(endoprotinase Asp-N) etc. be the proteinase from false pseudomonas bacillus (Pseudomonus) of representative; And take mass spectrum level lysine peptide chain restriction endonuclease (lysylendopeputidase), colour killing exopeptidase (achromopeputidase) etc. as representative from proteinase of achromobacter (Achromobacter) etc.
Wherein, from reasons such as stability, reactive (cut-off velocity of haemoglobin), the easiness that obtains, prices, preferably from the proteinase of microorganism, more preferably be selected from by from the proteinase of rod bacterium (Bacillus), from the proteinase of Aspergillus (Aspergillus), the group that forms from the proteinase of streptomycete (Streptomyces) and from the proteinase of reading coccus (Tritirachium) more than one.As commercially available product, as the Toyoteam NEP(ト ヨ チ ー system NEP from the proteinase of rod bacterium (Bacillus), Japan's textile company system), Type-X(SIGMA company system), Type-XXIV(SIGMA company system), thermolysin (large and change into company's system), high temperature protease P C10(is large and change into company's system), as the Type-XIII(SIGMA company system from the proteinase of Aspergillus (Aspergillus)), Type-XXIII(SIGMA company system), as from the Type-XIV of the proteinase of streptomycete (Streptomyces) and as being suitable for using from the Proteinase K (Roche company system) of the proteinase of reading coccus (Tritirachium) etc.
Wherein, from measuring the viewpoint of glycated hemoglobin, as the Toyoteam NEP(Japan textile company system from the proteinase of rod bacterium (Bacillus)), Type-X(SIGMA company system), Type-XXIV(SIGMA company system), thermolysin (large and change into company's system), high temperature protease P C10(is large and change into company's system), be more suitable for using as the Type-XIV from the proteinase of streptomycete (Streptomyces).
As long as reach targeted activity, then above-mentioned proteinase can be highly finished product, also can be thick highly finished product.In addition, also can be the material of making by genetic manipulation, and no matter have or not chemical modification.And above-mentioned proteinase can use separately, also can be used in combination.
The concentration of above-mentioned proteinase is not particularly limited, and is preferably 0.1U/cm 2～10000U/cm 2, 1U/cm more preferably 2～1000U/cm 2Protease concentration is lower than 0.1U/cm 2The time, owing to reactive the reduction prolongs Measuring Time, therefore not preferred.On the other hand, protease concentration is higher than 10000U/cm 2The time, cause sometimes the background rising or cause high price to be formatted.
PH when carrying out above-mentioned mmp reaction can not adjust, but preferred by suitable pH adjusting agent, for example following buffering agent is adjusted, so that employed proteinase reaches only pH.
(glycated amino acid oxidase)
The employed glycated amino acid oxidase of the utility model (the following Fructoamino-acid-oxidase FAOD that sometimes is also referred to as) is called as following various title in known document: Fructosylamine oxidase, Fructoamino-acid-oxidase, fructosyl peptide oxidase, Fructosylamine oxidase, Fructoamino-acid-oxidase, fructosyl peptide oxidase, the saccharified amine oxidase, the glycated amino acid oxidase, saccharification peptide oxidase, the saccharified amine oxidase, the glycated amino acid oxidase, saccharification peptide oxidase, Amadoriase (amadoriase), Ketoamine oxidase, Ketoamine oxidase etc.
As the employed glycated amino acid oxidase of the utility model, so long as produce specific effect and the enzyme of Hydrogen Peroxide with glycated amino acid and/or glycated peptide, then can use the enzyme of any kind.The oxidasic object lesson of glycated amino acid below is shown, but this does not consist of to any restriction of the present utility model.
As the glycated amino acid oxidase, can list: from the enzyme of gibberella (Gibberella), enzyme from Aspergillus (Aspergillus), enzyme from Penicillium notatum (Penicillium), enzyme from sickle-like bacteria (Fusarium), enzyme from corynebacteria (Corynebacterium), enzyme from shell of ascus bacterium (Coniochaeta), enzyme from penicillium bacterium (Eupenicillium), enzyme from Achaetomiella, enzyme from cupreum (Chaetomium), enzyme from coliform, enzyme from De Balishi enzyme (Debaryomyces), enzyme from Curvularia lunata (Curvularia), the make a fresh start enzyme of red shell bacterium (Neocosmospora), enzyme from cryptococcus (Cryptococcus), enzyme from dark ball chamber bacterium (phaeosphaeria), from the enzyme of candida albicans (Candida) and from enzyme of acrocarp mould (Acremonium) etc.
Wherein, from stability, reactive (oxidation rate of glycated amino acid and/or glycated peptide), the easiness that obtains, the reasons such as price are set out, be preferably the enzyme that is selected from by from shell of ascus bacterium (Coniochaeta), enzyme from penicillium bacterium (Eupenicillium), enzyme from Curvularia lunata (Curvularia), the make a fresh start enzyme of red shell bacterium (Neocosmospora), enzyme from Aspergillus (Aspergillus), in the group that forms from the enzyme of cryptococcus (Cryptococcus) with from the enzyme of dark ball chamber bacterium (phaeosphaeria) more than one more preferably are selected from by the enzyme from shell of ascus bacterium (Coniochaeta), enzyme from Aspergillus (Aspergillus), in the group that forms from the enzyme of cryptococcus (Cryptococcus) with from the enzyme of dark ball chamber bacterium (phaeosphaeria) more than one.
Wherein, be selected from by in the group that forms from the enzyme of shell of ascus bacterium (Coniochaeta) with from the enzyme of dark ball chamber bacterium (phaeosphaeria) more than one from measuring the viewpoint of glycated hemoglobin, being preferably.
As long as reach targeted activity, then above-mentioned glycated amino acid oxidase can be highly finished product, also can be thick highly finished product.In addition, also can be by the made material of genetic manipulation, and no matter have or not chemical modification.And above-mentioned glycated amino acid oxidase can use separately, also can be used in combination.
The oxidasic concentration of above-mentioned glycated amino acid is not particularly limited, and is preferably 0.01U/cm 2～1000U/cm 2, 0.1U/cm more preferably 2～100U/cm 2Glycated amino acid oxidase concentration is lower than 0.01U/cm 2The time, owing to reactive the reduction prolongs Measuring Time, therefore not preferred.On the other hand, glycated amino acid oxidase concentration is higher than 1000U/cm 2The time, cause sometimes the background rising or cause high price to be formatted.
PH when carrying out above-mentioned glycated amino acid oxydase reaction can not adjust, but preferred by suitable pH adjusting agent, for example following buffering agent is adjusted, so that employed glycated amino acid oxidase reaches only pH.
(peroxidase)
As the employed peroxidase of the utility model, so long as can catalyzing hydrogen peroxide and the enzyme of redox class chromogenic reagent reaction, then can use the enzyme of any kind, for example can list from plant, from bacterium, from the peroxidase of basidiomycetes.Wherein, from reasons such as the easiness of purity, acquisition, prices, be preferably the peroxidase from Western mustard, rice, soybean, more preferably from the peroxidase of Western mustard.PEO-301(Japan textile company system), PEO-302(Japan textile company system) etc. as the commercially available prod, PEO-131(Japan textile company system), be suitable for using.
The concentration of above-mentioned peroxidase is not particularly limited, and is preferably 0.01U/cm 2～1000U/cm 2, 0.1U/cm more preferably 2～100U/cm 2Peroxidase concn is lower than 0.01U/cm 2The time, owing to reactive the reduction prolongs Measuring Time, therefore not preferred.On the other hand, peroxidase concn is higher than 1000U/cm 2The time, cause sometimes the background rising or cause high price to be formatted.
PH when carrying out above-mentioned peroxidase reaction can not adjust, but preferred by suitable pH adjusting agent, for example following buffering agent is adjusted, so that employed peroxidase reaches only pH.
(redox class chromogenic reagent)
As the employed redox class of the utility model chromogenic reagent, if can be with hydroperoxidation colour generation, then can use the pigment of any kind, can list such as hydrogen donor and coupling agent, leuco compound etc.In addition, using the representation example of hydrogen donor and coupling agent is to make hydrogen donor and coupling agent carry out the Trinder method that oxidative condensation forms pigment by hydrogen peroxide in the presence of peroxidase.
The object lesson of redox class chromogenic reagent below is shown, but this does not consist of to any restriction of the present utility model.
(hydrogen donor)
As hydrogen donor, can list phenol, amphyl, anil, naphthols, naphthol derivative, naphthylamines, naphthylamine derivative etc.Particularly, can list: N-ethyl-N-(3-sulfopropyl) aniline (ALPS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline (TOPS), N-ethyl-N-(3-sulfopropyl)-3-aminoanisole (ADPS), N-ethyl-N-(3-sulfopropyl)-3, the 5-xylidin, N-ethyl-N-(3-sulfopropyl)-3, the 5-dimethoxyaniline, N-ethyl-N-(2-hydroxyl-3-sulfopropyl) aniline (ALOS), N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-aminoanisole (TOOS), N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-xylidin (MAOS), N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-ethyl-N-(3-aminomethyl phenyl)-N '-acetyl ethylenediamine, N-ethyl-N-(3-aminomethyl phenyl)-N '-succinyl ethylenediamine, N-(2-hydroxyl-3-sulfopropyl)-2, the 5-xylidin, N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N-sulfopropyl aniline, N-sulfopropyl-3,5-dimethoxyaniline etc.
(coupling agent)
As coupling agent, can list the amino antipyrine (4AA) of 4-, amino antipyrine derivant, vanillic aldehyde diamines sulfonic acid (vanillin diamine sulfonic acid), 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH:methybenzthiazoline hydrazone), sulfonation 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (SMBTH:sulphonated methybenzthiazoline hydrazone) etc.
(leuco compound)
As leuco compound, can list triphenyl methane derivant, phenothiazine derivative, diphenylamine derivatives etc.Particularly, can list: 4, two (the N of 4 '-benzal, accelerine), 4,4 '-two (N-ethyl-N-(the 3-sulfopropyl is amino)-2, the 6-xylidin) methane, 1-(ethylamino thiocarbonyl)-2-(3,5-dimethoxy-4 '-hydroxy phenyl)-4, two (the 4-lignocaine phenyl) imidazoles of 5-, 4,4 '-two (dimethylamino) diphenylamine, N-(carboxymethylamino carbonyl)-4,4 '-two (dimethylamino) diphenylamine sodium (DA64), 10-(carboxymethylamino carbonyl)-3, two (dimethylamino) phenothiazine sodium salts (DA67) of 7-etc.
Wherein, from reasons such as molar absorptivity, maximum absorption wavelengths, be preferably leuco compound, N-(carboxymethylamino carbonyl)-4 more preferably, 4 '-two (dimethylamino) diphenylamine sodium (DA64), 10-(carboxymethylamino carbonyl)-3, two (dimethylamino) phenothiazine sodium salts (DA67) of 7-.
The maximum absorption wavelength of above-mentioned redox class chromogenic reagent is preferably 600nm～800nm, more preferably 650nm～750nm.From measuring the viewpoint of glycated hemoglobin, maximum absorption wavelength because the colour developing spectrum of redox class chromogenic reagent overlaps with the spectrum of haemoglobin, be I'm afraid and understood desensitization less than low wavelength one side of 600nm the time.On the other hand, maximum absorption wavelength is greater than high wavelength one side of 800nm the time, and checkout equipment probably can maximize.
The concentration of above-mentioned redox class chromogenic reagent is not particularly limited, and is preferably 0.0001mg/cm 2～10mg/cm 2, 0.001mg/cm more preferably 2～1mg/cm 2Redox class chromogenic reagent concentration is lower than 0.0001mg/cm 2The time, probably can desensitization.On the other hand, redox class chromogenic reagent concentration is higher than 10mg/cm 2The time, cause sometimes the background rising or cause high price to be formatted.
(surfactant)
As the employed surfactant of the utility model, needed only the effect of hemolytic agent and/or protease reaction promoter, then can use the surfactant of any kind, preferably play the surfactant of hemolytic agent and protease reaction promoter.
As above-mentioned surfactant, can list: polyoxyethylene alkyl phenyl ether (Triton(registered trademark) is surfactant etc.), polyoxyethylene alkyl ether (Brij(registered trademark) is surfactant etc.), polyoxyethylene sorbitan fatty acid ester (Tween(registered trademark) is surfactant etc.), the nonionic surfactant such as polyoxyethylene fatty acid ester, sorbitan fatty acid ester, alkyl glucoside, fatty acid cane sugar ester.Wherein, from as the reactivity (haemolysis speed) of hemolytic agent, as reasons such as the functionality of protease reaction promoter, prices, preferred polyoxyethylene alkyl phenyl ether (Triton(registered trademark) is surfactant etc.).As the commercial goods, TritonX(registered trademark)-100(Nacalai Tesque company system), the TritonX(registered trademark)-114(NacalaiTesque company system), the Nonidet(registered trademark) P-40(Nacalai Tesque company system) etc. be suitable for using.In addition, above-mentioned surfactant can use separately, also can be used in combination.
The hydrophilic lipophilic balance of above-mentioned surfactant (Hydrophile Lipophile Balance Value:HLB Value) is preferably 10～20, and more preferably 12～20, more preferably 14～20.The HLB value probably can not get the effect of enough haemolysis effects and promotion mmp reaction less than 10 o'clock.On the other hand, the HLB value does not exist in the definition of HLB greater than 20 surfactant.
The concentration of above-mentioned surfactant is not particularly limited, and is preferably 0.0001mg/cm 2～10mg/cm 2, 0.001mg/cm more preferably 2～1mg/cm 2Surfactant concentration is lower than 0.0001mg/cm 2The time, probably can not get enough haemolysis effects and mmp reaction facilitation effect.On the other hand, surfactant concentration is higher than 10mg/cm 2The time, do not find that effect improves.
(buffering agent)
As can be used for buffering agent of the present utility model, as long as in the target zone of pH, have sufficient surge capability, then can use the buffering agent of any kind, such as listing: Tris, phosphoric acid, phthalic acid, citric acid, maleic acid, succinic acid, nitric acid, boric acid, tartrate, acetic acid, carbonic acid and Good ' s buffer(MES, ADA, PIPES, ACES, hydrochloric acid cholamine (cholamine), BES, TES, HEPES, acetoglycocoll, triein, glycine amide, vicine) etc.
Wherein, be preferably 6.0～7.5 from the optimum pH scope 6.0～8.5(at the employed proteinase of the utility model, glycated amino acid oxidase and peroxidase) in have the reasons such as sufficient surge capability, be preferably Tris, phosphoric acid, MES,, PIPES, TES, HEPES, more preferably MES, PIPES.
The concentration of above-mentioned buffering agent is not particularly limited, and the reagent when making with respect to the tier testing sheet is preferably about 50mM～100mM.
(other reagent)
In the utility model except mentioned reagent (surfactant, proteinase, the glycated amino acid oxidase, peroxidase, redox class chromogenic reagent etc.) outside, can also add as required the oxygenant (ferrocyanide of haemoglobin, azide, nitrite, nitrate etc.), catch the chelating reagent (ethylenediamine of the ion that hinders enzyme reaction, bipyridine, ethylene dinitrilotetra-acetic acid, phenanthroline, porphyrin, crown ether etc.), be used for removing the ascorbic acid oxidase as the ascorbic acid of the quantitative material that hinders hydrogen peroxide, salt (sodium chloride, potassium chloride, lime chloride, magnesium chloride, aluminum chloride etc.), enzyme stabilization agent (monosaccharide, oligosaccharides, polysaccharide, sugar alcohol, glycerine, gluconate, amino acids, the albumin class, Globulins, stringiness protein etc.), redox class chromogenic reagent stabilization agent (cyclodextrin (cyclodextrin) class, the reducible sulfur alcohols, reducible sulfur Barbiturates etc.).These can use separately, also can be used in combination.
Embodiment
By the following examples the utility model is specifically described, but this does not consist of any restriction of the present utility model.In addition, the evaluation method in the instructions is as described below.
[various evaluation method]
The activity measurement of proteinase＜1, 〉
The activity of proteinase calculates by the casein Folin method of having used Folin-Ciocalteu reagent.At this, under the optimal pH of proteinase, at 37 ℃ casein was added water decomposition 1 minute, the enzyme amount of colour generation that generation is equivalent to the tyrosine of 1.0 μ mol is defined as IU.
＜2, the oxidasic activity measurement of glycated amino acid 〉
The oxidasic activity of glycated amino acid is by in the presence of peroxidase, make the hydrogen peroxide and the redox class chromogenic reagent that are produced by saccharification valyl histidine (valyl histidine) and glycated amino acid oxydase reaction produce reaction, calculate from the variation of its absorbance.At this, at the oxidasic optimum pH(pH=6.5 of glycated amino acid) under, at 37 ℃ saccharification valyl histidine being added water decomposition 1 minute, the enzyme amount that generation is equivalent to the hydrogen peroxide of 1.0 μ mol is defined as IU.
The activity measurement of peroxidase＜3, 〉
The activity of peroxidase reacts hydrogen peroxide and pyrogallol (Pyrogallol) by in the presence of peroxidase, the variation of the absorbance of the purpurogallin of always self-generating (Purpurogallin) and calculating.At this, at the optimum pH(pH=6.0 of peroxidase) under, 20 ℃ of reactions 20 seconds, the enzyme amount of colour generation that generation is equivalent to the purpurogallin (Purpurogallin) of 1.0mg was defined as IU.
The measurement of hydrophilic lipophilic balance ((Hydrophile Lipophile Balance Value:HLB Value))＜4, 〉
The HLB value is by adopting the Griffin method, being calculated by HLB value=20 * (summation/molecular weight of the formula weight of hydrophilic segment).In addition, the HLB value of the potpourri of surfactant represents with the weighted mean of the HLB value of each composition.
＜5, the oxidasic storage stability of glycated amino acid 〉
In filter paper NO.5A(Tokyo, the paulownia mountain of 8mm φ nitre apparatus company system) on, drip the following reagent 1 of 10 μ L, 25 ℃ shading exsiccator 3909-04(Tokyo nitre apparatus company system) in drying 2 hours, be made into the blank test sheet.
＜reagent 1 〉
100mM colleague PIPES(chemistry institute company system) pH6.5
500U/mL glycated amino acid oxidase FPO-301(Japan textile company system)
Then, as accelerated test, with the able to programme cryostat IN604(Yamato scientific company system of blank test sheet at 37 ℃) in cultivated 5 hours.Then, above-mentioned blank test sheet and the following reagent 2 of 1000 μ L are added in the centrifuge tube (microtube), by stirring 1 minute with vortex stirrer (vortex mixer) (MS instrument company system), thereby extract glycated amino acid oxidase in the blank test sheet.Then, with said extracted liquid in centrifugal ultrafiltration pipe (MICROCON) 3(Millipore company system) in carry out centrifugal concentrating with 14000G, thereby obtain 200 μ L concentrates.Then, above-mentioned concentrate 50 μ L are mixed with Laemmli sample buffer (Bio-Rad Laboratiories company system) 47.5 μ L, 2 mercapto ethanol (Bio-Rad Laboratiories company system) 2.5 μ L, 95 ℃ of lower boilings 5 minutes, thereby make blank solution.
＜reagent 2 〉
100mM colleague PIPES(chemistry institute company system) pH6.5
The protease inhibitor cocktail of the general usefulness of 100 times of dilutions, 100 times concentrated (Nacalai Tesque company system)
Then, as accelerated test, with the able to programme cryostat IN604(Yamato scientific company system of tier testing sheet of the present utility model at 37 ℃) in cultivated 5 hours.Then, above-mentioned tier testing sheet and 1000 μ L mentioned reagent 2 are added in the centrifuge tube (microtube), by stirring 1 minute with eddy mixer (vortex mixer) (MS instrument company system), thereby extract surfactant, proteinase, glycated amino acid oxidase, peroxidase, redox class chromogenic reagent in the tier testing sheet.Secondly, with said extracted liquid in centrifugal ultrafiltration pipe (MICROCON) 3(Millipore company system) in the 14000G centrifugal concentrating, remove the low molecular weight compositions (surfactant, redox class chromogenic reagent etc.) of molecular weight below 3000, thereby make 200 μ L concentrates.Then, above-mentioned concentrate 50 μ L are mixed with Laemmli sample buffer (Bio-RadLaboratiories company system) 47.5 μ L, 2 mercapto ethanol (Bio-Rad Laboratiories company system) 2.5 μ L, 95 ℃ of lower boilings 5 minutes, thereby make sample solution.
Use electrophoresis best of breed external member (best package) (pre-prepared colloid with (Bio-Rad Laboratiories company system)), following condition 1 time, prepared blank solution and sample solution are implemented SDS-PAGE.In addition, blank solution and sample solution flow through the different ducts of identical gel.
＜condition 1 〉
Precast gel: pre-prepared colloid J10%T, 12well
Electrophoresis system: small-sized Vertial electrophorestic tank (mini protean Tetra cell)
Power supply: basic electrophoresis apparatus power supply (power pac Basic)
Standard: the two marks of high precision (precision Plus dual standard)
Electrophoretic buffer: premixed damping fluid Tris/ glycocoll/SDS
Standard volume: 10 μ L
Electrophoresis applied sample amount: 20 μ L
Deposition condition: 100V fixed voltage, 90 minutes
Temperature: 25 ℃
Then, in closed box (tight box) NO.3(AS ONE company system) in add gel and 200mL distilled water behind the electrophoresis, with shaker mixer (rocking mixer) RM-80(AS ONE company system) vibration removes distilled water after 5 minutes, carries out such clean operation three times.Then, add the dyeing liquor of 50mL Bio-Safe CBB G-250 coloring agent (Bio-Rad Laboratiories company system), remove above-mentioned dyeing liquor with above-mentioned shaker mixer vibration after 1 hour.At last, add 200mL distilled water, remove distilled water with above-mentioned shaker mixer vibration after 1 hour, can obtain electrophoresis pattern.
Use GS-800Calibrated Densitometer(Bio-Rad Laboratiories company system) according to usual way near measuring from the oxidasic bands of a spectrum thickness of glycated amino acid (sizes of the bands of a spectrum of swimming direction) and concentration (absorbances of the bands of a spectrum) 50kDa in the resulting electrophoresis pattern.Calculate bands of a spectrum conservation rate 1 following formula (1) from the bands of a spectrum thickness of the blank of gained and sample respectively, calculate bands of a spectrum conservation rate 2 following formula (2) from the bands of a spectrum densimeter of the blank of gained and sample, bands of a spectrum conservation rate 1 and bands of a spectrum conservation rate 2 〉=90% are set as ◎ (excellent), bands of a spectrum conservation rate 1 and bands of a spectrum conservation rate 2＜90% are set as * (bad), the oxidasic storage stability of glycated amino acid (that is, anti-decomposability) is estimated.
[mathematical expression 1]
Bands of a spectrum conservation rate 1(%)=(solute band thickness/blank bands of a spectrum thickness) * 100 formulas (1)
[mathematical expression 2]
Bands of a spectrum conservation rate 2(%)=(solute band concentration/blank bands of a spectrum concentration) * 100 formulas (2)
The storage stability of redox class chromogenic reagent＜6, 〉
Tier testing sheet of the present utility model is put into aluminum sealed bag (Alumi lamizip) AL-9(Tokyo nitre apparatus company system), after enclosing nitrogen, in cryostat IN604(Yamato scientific company system able to programme) in, 4 ℃ of lower cultivations 0,24,48,72,96,120,144,168,336,504 hour, or 25 ℃ of lower cultivations 0,24,48,72,96,120,144,168,336,504 hour.Then, with anchor clamps (with reference to Fig. 1) that above-mentioned tier testing sheet is fixing from clamping up and down, following condition 2 times, the reflectivity of a side of the layer that carries redox class chromogenic reagent is measured.
＜condition 2 〉
Device name: company of spectrophotometer UV-2450(Shimadzu Seisakusho Ltd. system)
Auxiliary equipment title: company of integrating sphere I SR-2200(Shimadzu Seisakusho Ltd. system)
Standard white plate: barium sulphate standard white plate
Measure the λ max of wavelength: 666nm(DA67)
Incident angle: 0 °
Slit (slit) width: 2.0nm
Beam sizes: 3mm * 5mm
Temperature: 25 ℃
Calculate the K/S value by the Kubelka-Munk conversion by following formula (3) of the reflectivity of gained, for 3 weeks (504 hours) K/S value afterwards, K/S value (4 ℃)≤0.25 and K/S value (25 ℃)≤0.50 are set as ◎ (excellent), 0.25＜K/S value (4 ℃)≤0.35 and 0.50＜K/S value (25 ℃)≤0.60 are set as zero (very), 0.35＜K/S value (4 ℃)≤0.40 and 0.60＜K/S value (25 ℃)≤1.30 are set as △ (still can), 0.40＜K/S value (4 ℃) and 1.30＜K/S value (25 ℃) are set as * (bad), storage stability (that is, anti-from colour rendering) to redox class chromogenic reagent is estimated.The rising of K/S value is known if redox class chromogenic reagent develops the color certainly.In addition, the %R in the formula (3) is the meaning of reflectivity.
[mathematical expression 3]
The formula (3) of the 2/(2 of K/S value=(1-%R) * %R)
The reactivity of proteinase＜7, 〉
On tier testing sheet of the present utility model, human Hb H 7379(SIGMA company system with 100g/L) aqueous solution 20 μ L point samples are at ground floor, in cryostat IN604(Yamato scientific company system able to programme) in, 37 ℃ of lower cultivations 0,5,10,15,30,60 minute.Then, above-mentioned tier testing sheet and 1000 μ L mentioned reagent 2 are added in the centrifuge tube (microtube), by stirring 1 minute with eddy mixer (vortex mixer) (MS instrument company system), thereby extract surfactant, proteinase, glycated amino acid oxidase, peroxidase, redox class chromogenic reagent in the tier testing sheet.Then, with said extracted liquid in centrifugal ultrafiltration pipe (MICROCON) 3(Millipore company system) in the 14000G centrifugal concentrating, remove the low molecular weight compositions (surfactant, redox class chromogenic reagent) of molecular weight below 3000, thereby make 200 μ L concentrates.Then, above-mentioned concentrate 2.5 μ L are mixed with distilled water 47.5 μ L, Laemmli sample buffer (Bio-Rad Laboratiories company system) 47.5 μ L, 2 mercapto ethanol (Bio-Rad Laboratiories company system) 2.5 μ L, 95 ℃ of lower boilings 5 minutes, thereby make sample solution 1～6.
Use electrophoresis best of breed external member (best package) (pre-prepared colloid with (Bio-Rad Laboratiories company system)), above-mentioned condition 1 time, prepared sample solution 1～6 is implemented SDS-PAGE.In addition, sample solution 1～6 flows through the different ducts of identical gel.
Then, in closed box (tight box) NO.3(AS ONE company system) in add gel and 200mL distilled water behind the electrophoresis, with shaker mixer (rocking mixer) RM-80(AS ONE company system) vibration removes distilled water after 5 minutes, carries out such clean operation three times.Then, add the dyeing liquor of 50mL Bio-Safe CBB G-250 coloring agent (Bio-Rad Laboratiories company system), remove above-mentioned dyeing liquor with above-mentioned shaker mixer vibration after 1 hour.At last, add 200mL distilled water, remove distilled water with above-mentioned shaker mixer vibration after 1 hour, can obtain electrophoresis pattern.
Use GS-800Calibrated Densitometer(Bio-Rad Laboratiories company system) according to usual way near the bands of a spectrum thickness from the haemoglobin subunit the 16kDa in the resulting electrophoresis pattern (sizes of the bands of a spectrum of swimming direction) and concentration (absorbances of bands of a spectrum) are measured.Cultivate 0 minute bands of a spectrum thickness after 60 minutes and calculate bands of a spectrum disappearance rate 1 following formula (4) down from 37 ℃ of gained respectively, cultivate 0 minute bands of a spectrum densimeter after 60 minutes and calculate bands of a spectrum disappearance rate 2 following formula (5) down from 37 ℃ of gained, for bands of a spectrum disappearance rate 1 and bands of a spectrum conservation rate reach time more than 90% (below, be also referred to as the bands of a spectrum die-out time), 0 minute＜bands of a spectrum die-out time≤5 minutes is set as ◎ (excellent), 5 minutes＜bands of a spectrum die-out time≤15 minutes is set as zero (very), 15 minutes＜bands of a spectrum die-out time≤30 minutes is set as △ (still can), 30 minutes＜bands of a spectrum die-out time is set as * (bad), the reactivity of proteinase is estimated.
[mathematical expression 4]
Bands of a spectrum disappearance rate 1(%)=1-(bands of a spectrum thickness (0 minute to 60 minutes)/bands of a spectrum thickness (0 minute)) } * 100 formulas (4)
[mathematical expression 5]
Bands of a spectrum disappearance rate 2(%)=1-(bands of a spectrum concentration (0 minute to 60 minutes)/bands of a spectrum concentration (0 minute)) } * 100 formulas (5)
Sensitivity, the correlativity of redox class chromogenic reagent＜8, 〉
Make synthetic fVH (fructosyl-valyl-histidine) (below, sometimes be also referred to as F-VH) be dissolved in the human Hb H 7379(SIGMA company system of 100g/L) in the aqueous solution, make it form 0 μ m, 50 μ m, 100 μ m, 200 μ m, 500 μ M, be modulated into the measurement sample of 5 levels.Then, with anchor clamps (with reference to Fig. 1) that tier testing sheet of the present utility model is fixing from clamping up and down, with the above-mentioned measurement sample of 20 μ L point sample at ground floor, after at room temperature cultivating 1 minute, following condition 3 times, the reflectivity of the face opposite with measuring sample point sample face is measured.
＜condition 3 〉
Measure sample: human Hb H 7379100g/L
F-VH0μM、50μM、100μM、200μM、500μM
Device name: company of spectrophotometer UV-2450(Shimadzu Seisakusho Ltd. system)
Auxiliary equipment title: company of integrating sphere I SR-2200(Shimadzu Seisakusho Ltd. system)
Standard white plate: barium sulphate standard white plate
Measure wavelength: 666nm(DA67), 727nm(DA64), 555nm(TOOS), 630nm(MAOS)
Incident angle: 0 °
Slit (slit) width: 2.0nm
Beam sizes: 3mm * 5mm
Temperature: 25 ℃
Obtain F-VH in the K/S value of 0 μ M～500 μ M by the F-VH of gained in the Kubelka-Munk conversion by following formula (3) of the reflectivity of 0 μ M～500 μ M.The K/S stability bandwidth that calculates following formula (6) in the K/S value of 500 μ M at K/S value and the F-VH of 0 μ M by resulting F-VH, the K/S rate of change 〉=2.0 are set as ◎ (excellent), the K/S rate of change＜2.0 are set as * (bad), the sensitivity of redox class chromogenic reagent is estimated.In addition, the K/S value in the formula (6) (0 μ M), K/S value (500 μ M) are respectively F-VH in the K/S value of 0 μ M, the F-VH meaning in the K/S value of 500 μ M.
[mathematical expression 6]
The K/S rate of change=K/S value (500 μ M)/K/S value (0 μ M) formula (6)
In addition, calculate the Pearson correlation coefficient r of resulting K/S value and F-VH concentration, r＞0.95 is set as ◎ (excellent), 0.95≤r＜0.90 is set as zero (very), 0.90≤r＜0.85 is set as △ (still can), r≤0.85 is set as * (bad), correlativity is estimated.
The making of glycated hemoglobin value calibration curve＜9, 〉
With anchor clamps (with reference to Fig. 1) that tier testing sheet of the present utility model is fixing from clamping up and down, the evaluation of HbA1c measurement performance is checked company of medical science standard substance mechanism system with the general civic organization of sample QRM HbA1c2007-1() 20 μ L point samples are at ground floor, cryostat IN604(Yamato scientific company system able to programme at 37 ℃) after cultivating 15 minutes in, following condition 4 times, the reflectivity of the face opposite with measuring sample point sample face is measured.
＜condition 4 〉
Measure sample: HbA1c measurement performance evaluation sample QRM
LEVEL1、2、3、4、5
Device name: company of spectrophotometer UV-2450(Shimadzu Seisakusho Ltd. system)
Auxiliary equipment title: company of integrating sphere I SR-2200(Shimadzu Seisakusho Ltd. system)
Standard white plate: barium sulphate standard white plate
Measure wavelength: 480nm, 666nm
Incident angle: 0 °
Slit (slit) width: 2.0nm
Beam sizes: 3mm * 5mm
Temperature: 25 ℃
By obtaining in the K/S at 480nm place value and in the K/S at 666nm place value at the reflectivity at 480nm place and in the Kubelka-Munk conversion by following formula (3) of the reflectivity at 666nm place of gained.Then, by resulting at the K/S at 480nm place value and the K/S ratio that calculates following formula (7) in the K/S at 666nm place value.The K/S ratio is known with glycated hemoglobin value proportional (with reference to non-patent literature 1).In addition, the K/S value (480nm) in the formula, K/S value (666nm) are respectively in the K/S at 480nm place value, in the meaning of the K/S at 666nm place value.
K/S ratio=K/S value (666nm)/K/S value (480nm) formula (7)
Calculate resulting K/S ratio and the Pearson correlation coefficient r of HbA1c measurement performance evaluation with the HbA1c value (LEVEL1=4.67%, LEVEL2=5.29%, LEVEL3=6.96%, LEVEL4=9.08%, LEVEL5=10.79%) of defined among the sample QRM, r＞0.95 is set as ◎ (excellent), 0.95≤r＜0.90 is set as zero (very), 0.90≤r＜0.85 is set as △ (still can), r≤0.85 is set as * (bad), correlativity is estimated.
The measurement of glycated hemoglobin value＜10, 〉
With anchor clamps (with reference to Fig. 1) that tier testing sheet of the present utility model is fixing from clamping up and down, with the whole blood of 20 μ L Healthy Peoples or diabetic's whole blood point sample at ground floor, cryostat IN604(Yamato scientific company system able to programme at 37 ℃) after cultivating 15 minutes in, following condition 5 times, the reflectivity of the face opposite with measuring sample point sample face is measured.In addition, to the blood of Healthy People and diabetic's blood, use is as Gao Ke company of automatic analyzer 7180(Hitachi of the Hitachi system of commercially available automatic analyzer) and the Nordia N HbA1c(ponding medical science and technology company system of measuring reagent as commercially available glycated hemoglobin) measure according to usual way, calculate the glycated hemoglobin value.
＜condition 5 〉
Measure sample: 5 levels of the blood of Healthy People; 5 levels of diabetic's blood
Device name: company of spectrophotometer UV-2450(Shimadzu Seisakusho Ltd. system)
Auxiliary equipment title: company of integrating sphere I SR-2200(Shimadzu Seisakusho Ltd. system)
Standard white plate: barium sulphate standard white plate
Measure wavelength: 480nm, 666nm
Incident angle: 0 °
Slit (slit) width: 2.0nm
Beam sizes: 3mm * 5mm
Temperature: 25 ℃
By obtaining in the K/S at 480nm place value and in the K/S at 666nm place value at the reflectivity at 480nm place and in the Kubelka-Munk conversion by following formula (3) of the reflectivity at 666nm place of gained.Then, by resulting at the K/S at 480nm place value and the K/S ratio that calculates following formula (7) in the K/S at 666nm place value.Calculate the glycated hemoglobin value by resulting K/S and the resulting calibration curve of the next item up.Calculate resulting glycated hemoglobin value at the CV at n=10 place, 0%≤CV＜5% is set as ◎ (excellent), 5%≤CV＜10% is set as zero (very), 10%≤CV＜15% is set as △ (still can), 15%≤CV is set as * (bad), difference is estimated.
[making of test film]
[embodiment 1]
In filter paper NO.5A(Tokyo, the paulownia mountain of 8mm φ nitre apparatus company system) a following reagent 3 of 10 μ L, filter paper NO.5A(Tokyo, the paulownia mountain nitre apparatus company system of the 8mm φ that follows at other) drips the following reagent 4 of 10 μ L, respectively 25 ℃ shading exsiccator 3909-04(Tokyo nitre apparatus company system) in to its dry 2 hours, be made into two kinds of monolayer assay sheets.Stacked by resulting two kinds of monolayer assay sheets are carried out, and be made into the tier testing sheet.In addition, the surfactant concentration that carries is 0.1mg/cm 2, protease concentration is 10U/cm 2, glycated amino acid oxidase concentration is 10U/cm 2, peroxidase concn is 40U/cm 2, redox class chromogenic reagent concentration is 0.1mg/cm 2
The details of resulting tier testing sheet is shown in table 1.In addition, the NP40 in the table, XIV, FPO, PEO, DA67 are the Nonidet(registered trademarks) P-40, proteinase Type-XIV, glycated amino acid oxidase FPO-301, peroxidase PEO-302, DA67 be carried on respectively the meaning of each layer.Below identical.
＜reagent 3 〉
100mM colleague PIPES(chemistry institute company system) pH6.5
5.0mg/mL the Nonidet(registered trademark) P-40(Nacalai Tesque company system)
500U/mL proteinase Type-XIV(SIGMA company system)
＜reagent 4 〉
100mM colleague PIPES(chemistry institute company system) pH6.5
5.0mg/mL the Nonidet(registered trademark) P-40(Nacalai Tesque company system)
500U/mL glycated amino acid oxidase FPO-301(Japan textile company system)
2000U/mL peroxidase PEO-302(Japan textile company system)
5.0mg/mL the pure pharmaceutical worker's industry of DA67(and light company system)
[embodiment 2～4]
Except the layer difference of surfactant, proteinase, glycated amino acid oxidase, peroxidase, redox class chromogenic reagent carried, all the other operate similarly to Example 1, are made into the tier testing sheet.Resulting tier testing sheet specifically is shown in table 1.
Table 1
Figure DEST_PATH_GDA00002708015700311
[embodiment 5]
In filter paper NO.5A(Tokyo, the paulownia mountain of 8mm φ nitre apparatus company system) a following reagent 5 of 10 μ L, filter paper NO.5A(Tokyo, the paulownia mountain nitre apparatus company system of the 8mm φ that follows at other) drips the following reagent 6 of 10 μ L, again in other filter paper NO.5A(Tokyo, the paulownia mountain nitre apparatus company system of 8mm φ) drip the following reagent 7 of 10 μ L, respectively 25 ℃ shading exsiccator 3909-04(Tokyo nitre apparatus company system) in to its dry 2 hours, be made into three kinds of monolayer assay sheets.Stacked by resulting three kinds of monolayer assay sheets are carried out, and be made into the tier testing sheet.In addition, the surfactant concentration that carries is 0.1mg/cm 2, protease concentration is 10U/cm 2, glycated amino acid oxidase concentration is 10U/cm 2, peroxidase concn is 40U/cm 2, redox class chromogenic reagent concentration is 0.1mg/cm 2
The details of resulting tier testing sheet is shown in table 2.
＜reagent 5 〉
100mM colleague PIPES(chemistry institute company system) pH6.5
5.0mg/mL the Nonidet(registered trademark) P-40(Nacalai Tesque company system)
500U/mL proteinase Type-XIV(SIGMA company system)
＜reagent 6 〉
100mM colleague PIPES(chemistry institute company system) pH6.5
5.0mg/mL the Nonidet(registered trademark) P-40(Nacalai Tesque company system)
500U/mL glycated amino acid oxidase FPO-301(Japan textile company system)
＜reagent 7 〉
100mM colleague PIPES(chemistry institute company system) pH6.5
5.0mg/mL the Nonidet(registered trademark) P-40(Nacalai Tesque company system)
2000U/mL peroxidase PEO-302(Japan textile company system)
5.0mg/mL the pure pharmaceutical worker's industry of DA67(and light company system)
[embodiment 6～9]
Except the layer difference of surfactant, proteinase, glycated amino acid oxidase, peroxidase, redox class chromogenic reagent carried, all the other operate similarly to Example 5, are made into the tier testing sheet.Resulting tier testing sheet is shown in table 2.
Table 2
Figure DEST_PATH_GDA00002708015700331
[comparative example 1]
In filter paper NO.5A(Tokyo, the paulownia mountain of 8mm φ nitre apparatus company system) drip the following reagent 8 of 10 μ L, 25 ℃ shading exsiccator 3909-04(Tokyo nitre apparatus company system) in to its dry 2 hours, be made into the monolayer assay sheet.In addition, the surfactant concentration that carries is 0.1mg/cm 2, protease concentration is 10U/cm 2, glycated amino acid oxidase concentration is 10U/cm 2, peroxidase concn is 40U/cm 2, redox class chromogenic reagent concentration is 0.1mg/cm 2
The details of resulting tier testing sheet is shown in table 3.
Table 3
Figure DEST_PATH_GDA00002708015700332
＜reagent 8 〉
100mM colleague PIPES(chemistry institute company system) pH6.5
5.0mg/mL the Nonidet(registered trademark) P-40(Nacalai Tesque company system)
500U/mL proteinase Type-XIV(SIGMA company system)
500U/mL glycated amino acid oxidase FPO-301(Japan textile company system)
2000U/mL peroxidase PEO-302(Japan textile company system)
5.0mg/mL the pure pharmaceutical worker's industry of DA67(and light company system)
[comparative example 2～7]
Except the layer difference of surfactant, proteinase, glycated amino acid oxidase, peroxidase, redox class chromogenic reagent carried, all the other operate similarly to Example 1, are made into the tier testing sheet.The details of resulting tier testing sheet is shown in table 4.
Table 4
Figure DEST_PATH_GDA00002708015700341
[comparative example 8,9]
Except the layer difference of surfactant, proteinase, glycated amino acid oxidase, peroxidase, redox class chromogenic reagent carried, all the other operate similarly to Example 5, are made into the tier testing sheet.The details of resulting tier testing sheet is shown in table 5.
Table 5
＜storage stability of glycated amino acid oxidase, redox class chromogenic reagent 〉
Use the tier testing sheet of embodiment 1～9, comparative example 1～9, the storage stability of the oxidasic storage stability of glycated amino acid, redox class chromogenic reagent is estimated.
Resulting evaluation result is shown in table 6,7.
Figure DEST_PATH_GDA00002708015700361
Figure DEST_PATH_GDA00002708015700371
According to the oxidasic storage stability evaluation result of glycated amino acid, when proteinase and glycated amino acid oxidase are carried on same layer, the oxidasic inactivation of glycated amino acid, the decomposition that are caused by proteinase occur, and cause storage stability significantly to reduce.In addition, owing to glycated amino acid oxidase generation inactivation, decomposition, cause sometimes and can't measure.
In addition, according to the storage stability evaluation result of redox class chromogenic reagent, when superoxide enzyme and redox class chromogenic reagent were carried on same layer, redox class chromogenic reagent occured from colour developing, and causes storage stability significantly to reduce.In addition, because redox class chromogenic reagent causes sensitivity sometimes from colour developing.According to above result, as the layer structure of the tier testing sheet of preserving the stability excellence, proteinase and glycated amino acid oxidase need to be carried on respectively different layers, for example can list the tier testing sheet of embodiment 1～9.More preferably peroxidase and redox class chromogenic reagent are carried on respectively different layers, for example can list embodiment 2,3,6～9 tier testing sheet.
[embodiment 10～13]
Except with proteinase by Type-XIV(SIGMA company system) change to Toyoteam NEP(Japan textile company system), Type-X(SIGMA company system), Proteinase K (Roche company system), Type-XIII(SIGMA company system), all the other operate similarly to Example 2, are made into the tier testing sheet.The details of resulting tier testing sheet is shown in table 8.In addition, the NEP in the table, X, K, XIII represent that Toyoteam NEP, Type-X, Proteinase K, Type-XIII are carried on respectively the meaning of each layer.
[comparative example 10]
Except with proteinase by Type-XIV(SIGMA company system) change to Type-I(SIGMA company system), all the other operate similarly to Example 2, are made into the tier testing sheet.The details of resulting tier testing sheet is shown in table 8.In addition, the I in the table is the meaning that expression Type-I is carried on each layer.
Table 8
Figure DEST_PATH_GDA00002708015700391
＜reactivity of proteinase 〉
Use the tier testing sheet of embodiment 2,10～13, comparative example 10, the reactivity of proteinase is estimated.
Resulting evaluation result is shown in table 9.
Table 9
Figure DEST_PATH_GDA00002708015700392
According to the evaluation result of mmp reaction, be preferably selected from by from the proteinase of rod bacterium (Bacillus), from the proteinase of Aspergillus (Aspergillus), the group that forms from the proteinase of streptomycete (Streptomyces) and from the proteinase of reading coccus (Tritirachium) more than one as proteinase.
[embodiment 14]
Except with redox class chromogenic reagent by the DA67(of λ max=666nm and the pure pharmaceutical worker's industry of light company system) change to DA64(and the pure pharmaceutical worker's industry of the light company system of λ max=727nm), all the other operate similarly to Example 2, are made into the tier testing sheet.
The details of resulting tier testing sheet is shown in table 10.In addition, the DA64 in the table is the meaning that expression DA64 is carried on each layer.
[comparative example 11,12]
Except with redox class chromogenic reagent by the DA67(of λ max=666nm and the pure pharmaceutical worker's industry of light company system) change to the 4AA(Nacalai Tesque company system of λ max=555nm) and colleague's TOOS(chemistry institute company system) and the 4AA(Nacalai Tesque company system of λ max=630nm) and colleague's MAOS(chemistry institute company system), all the other operate similarly to Example 2, are made into the tier testing sheet.The 4AA concentration of carrying in addition, is 0.06mg/cm 2, TOOS concentration is 0.09mg/cm 2, MAOS concentration is 0.09mg/cm 2
The details of resulting tier testing sheet is shown in table 10.In addition, the 4AA in the table, TOOS, MAOS are the meanings that the amino antipyrine of expression 4-, TOOS, MAOS are carried on respectively each layer.
Table 10
Figure DEST_PATH_GDA00002708015700401
＜sensitivity, the correlativity of redox class chromogenic reagent 〉
Use embodiment 2,14, comparative example 11,12 tier testing sheet, sensitivity, the correlativity of redox class chromogenic reagent are estimated.
Resulting evaluation result is shown in table 11.
Table 11
Figure DEST_PATH_GDA00002708015700411
According to the sensitivity of redox class chromogenic reagent, the evaluation result of correlativity, high and be not subject to colourless pigment impact, have maximum absorption wavelength at 650nm～800nm place of the tone of haemoglobin as the preferred molar absorptivity of redox class chromogenic reagent.
[embodiment 15～17]
Except with the Nonidet(registered trademark of surfactant by HLB value=17.6) P-40(Nacalai Tesque company system) change to the Triton(registered trademark of HLB value=13.5) X-100(Nacalai Tesque company system), the Tween(registered trademark of HLB value=16.7) 20(and the pure pharmaceutical worker's industry of light company system), the Brij(registered trademark of HLB value=16.9) 35(and the pure pharmaceutical worker of light company's system already), all the other operate similarly to Example 10, are made into the tier testing sheet.
The details of resulting tier testing sheet is shown in table 12.In addition, the TX100 in the table, Tw20, Br35 are expression Triton(registered trademarks) X-100, Tween(registered trademark) 20, the Brij(registered trademark) 35 be carried on respectively the meaning of each layer.
[comparative example 13,14]
Except the surfactant Nodidet(registered trademark with HLB value=17.6) P-40(NacalaiTesque company system) change to the DK Ester(DK エ ス テ Le of HLB value=6.0, registered trademark) F50(the first industrial drugmaker system), the DK Ester(registered trademark of HLB value=8.0) F70(the first industrial drugmaker system), all the other operate similarly to Example 10, are made into the tier testing sheet.
The details of resulting tier testing sheet is shown in table 12.In addition, the F50 in the table, F70 are expression DK Ester(registered trademarks) F50(the first industrial drugmaker system), DK Ester(registered trademark) F70 is carried on respectively the meaning of each layer.
Table 12
Figure DEST_PATH_GDA00002708015700421
＜reactivity of proteinase 〉
Use embodiment 10,15～17, comparative example 13,14 tier testing sheet, the reactivity of proteinase is estimated.
Resulting evaluation result is shown in table 13.
Table 13
Figure DEST_PATH_GDA00002708015700431
According to the evaluation result of mmp reaction, promote preferably that as surfactant effect excellence, the HLB value of mmp reaction are 10～20 nonionic surfactant.
＜making of glycated hemoglobin value calibration curve: HbA1c measurement performance evaluation sample 〉
Use the tier testing sheet of embodiment 1～4, make the calibration curve of HbA1c value, correlativity is estimated.Resulting evaluation result is shown in table 14.
Table 14
Figure DEST_PATH_GDA00002708015700432
＜measurement of glycated hemoglobin value 〉
Use the tier testing sheet of embodiment 1～4, use the calibration curve of the resulting HbA1c value of the next item up, calculate the HbA1c value.Resulting evaluation result is shown in table 15.
Table 15
Figure DEST_PATH_GDA00002708015700441
According to above result, the glycated hemoglobin value of using tier testing sheet of the present utility model to measure is very consistent with the glycated hemoglobin value of using the biological chemistry automatic analysing apparatus to measure.And, because tier testing sheet of the present utility model has excellent storage stability, therefore by using tier testing sheet of the present utility model, can correct, easy and promptly carry out colorimetric assay to the glycosylated hemoglobin amount of measuring in the sample.
Industrial utilizability
By using tier testing sheet of the present utility model, can be on-the-spot correct, easy and promptly the glycosylated hemoglobin amount of measuring in the sample is carried out colorimetric assay in diagnosis, and then owing to also have excellent storage stability, therefore wish to make larger contribution based on clinical examination field, the diagnostic medical field of preventive medicine, the industrial community of controlling the life science headed by medicine field and the physical-fitness medicine field.

Claims (6)

1. a glycosylated hemoglobin is measured and is used the tier testing sheet, it is characterized in that:
Described tier testing sheet is laminated with following a layer and b layer at least, and described a layer and b layer are pressed the sequential cascade of a layer, b layer from sample to be tested point sample face,
Carry respectively peroxidase and redox class chromogenic reagent on the different layer in a layer and b layer, and
Described tier testing sheet does not have the blood separating layer, wherein,
The a layer: carry at least the polymer base material of proteinase,
B layer: carry at least the oxidasic polymer base material of glycated amino acid.
2. glycosylated hemoglobin according to claim 1 is measured and is used the tier testing sheet, it is characterized in that:
Also carry surfactant on described tier testing sheet at least one deck in a layer and b.
3. a glycosylated hemoglobin is measured and is used the tier testing sheet, it is characterized in that:
Described tier testing sheet is laminated with following a layer, b layer and c layer at least, and described a layer, b layer and c layer are pressed the sequential cascade of a layer, b layer and c layer, a layer, c layer and b layer or c layer, a layer and b layer from sample to be tested point sample face,
Carry respectively peroxidase and redox class chromogenic reagent on the different layers in a layer, b layer and c layer, and
Described tier testing sheet does not have the blood separating layer, wherein,
The a layer: carry at least the polymer base material of proteinase,
The b layer: carry at least the oxidasic polymer base material of glycated amino acid,
C layer: the polymer base material that carries at least any agent except proteinase and glycated amino acid oxidase.
4. glycosylated hemoglobin according to claim 3 is measured and is used the tier testing sheet, it is characterized in that:
Also carry surfactant on described tier testing sheet at least one deck in a layer, b layer and c layer.
According to claim 1 in 4 each described glycosylated hemoglobin measure and use the tier testing sheet, it is characterized in that:
Redox class chromogenic reagent is that maximum absorption wavelength is the colourless pigment of 600nm～800nm.
According to claim 2 or 4 described glycosylated hemoglobins measure and use the tier testing sheet, it is characterized in that:
Surfactant is that hydrophilic lipophilic balance is 10～20 nonionic surfactant.
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