JPH09113511A - Dry test piece for measuring glyco-albumin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a dry test piece capable of measuring the glyco-albumin value with simple operation without any pre-treatment of serum separation. SOLUTION: A test piece is a dry test piece for measuring glyco-albumin which uses the expansion solution and provided with a blood cell separation layer 3, an reagent layer containing a first pigment to color albumin, a second pigment to color glyco-albumin, and a serum degeneration agent, a measuring layer 5 in which the specific binding substance to albumin and glyco-albumin is solidified to capture them, and a residual solution absorbing layer 6 to absorb the residual expansion solution on the expansion layer fitted on a supporting body 1 in this order. The pigments are used as the marker of the specific substance because the pigment is very stable compared with the enzyme and the radioactive substance, and inexpensive, and two kinds of pigments can sufficiently be discriminated with a photometer having the function of measuring two wavelengths.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a test piece for simultaneously measuring albumin and its glycosylated protein, glycoalbumin, in a clinical test.

[0002]

2. Description of the Related Art It is known that albumin, which is a protein in blood, and hemoglobin in red blood cells, react non-enzymatically with glucose to become glycoalbumin and glycohemoglobin, respectively. This so-called glycated protein concentration reflects the blood sugar concentration during the period in which the protein was present in the living body, and therefore its measurement is very useful for diagnosing diabetes and observing diabetes.

As an index of such a glycated protein, glycated hemoglobin hemoglobin A _1C (HbA
_1C ), glycoalbumin, fructosamine and the like. HbA _1C reflects the blood sugar concentration in the past 1-2 months, whereas glycoalbumin and fructosamine reflect the blood sugar concentration in the past 1-2 weeks, and therefore their concentrations are higher than the HbA _1C concentration. Also, a tendency to move sharply and with a large fluctuation range is observed. That is, the concentration of glycoalbumin or fructosamine is more useful as an index of blood glucose control in a short period than the HbA _1C level. Since the fructosamine measurement method is a colorimetric method based on its reducing property, it is susceptible to reducing substances (bilirubin, ascorbic acid, etc.) coexisting in the reaction system. Further, since the measured value of fructosamine is displayed as an absolute value, there is a problem that it is affected by the amount of protein in the sample. Therefore,
Glycoalbumin, which measures relative values, is more reliable than measuring fructosamine.

It is clinically generalized that the value of glycoalbumin is expressed by using the ratio of the amount of glycoalbumin to the amount of total albumin as an index, and the percentage is usually 11 to 1
6% is a standard for its normal value. That is, in order to know the value of glycoalbumin, it is necessary to simultaneously measure the total albumin amount and the glycoalbumin amount.

Glycated proteins have been conventionally measured by electrophoresis, ion exchange chromatography, high performance liquid chromatography (HPLC) method with boronic acid affinity, etc. The HPLC method has become the mainstream in the field. However, HPL
The method C has high accuracy and separability, but has a drawback that a large number of samples cannot be processed at the same time because the time required for measurement per sample is long.

Recently, a measurement method combining a boronic acid affinity and an immunoassay method has been used. In general, immunoassays are excellent in specificity for glycated proteins and large-volume sample processing.

At present, both the HPLC method and the immunoassay method are used in a test center or a large hospital by using a relatively large-sized device. Therefore, there is a demand for a method that can be used by small and medium-sized hospitals, medical practitioners, or patients themselves to measure a sample in a short time with a simple small device. However, hitherto, no one satisfying the demand has been reported.

As a solution for fulfilling this demand, a dry test piece can be considered. In fact, although not glycoalbumin, the type of hemoglobin in the blood (hemoglobin A,
A disposable type dry inspection tool for determining S and C) has been reported (Screening 3, 67 to 76,
1994).

Since the method for measuring glucoalbumin uses serum or plasma as a sample, it is necessary to perform pretreatment for separating blood cell components from whole blood to obtain serum or plasma.
The serum separation work not only involves complicated operations, but also poses a problem of infection risk (biohazard). This work of separating serum also becomes a factor that hinders the measurement of glycated proteins in dry test strips.

[0010]

Therefore, an object of the present invention is to use a small photometric device instead of a large-sized analyzer, and without requiring pretreatment for serum separation. It is an object of the present invention to provide a dry-type test piece capable of measuring the glycated albumin level at home with a simple operation, that is, to measure the glycated albumin level simultaneously by measuring the total albumin amount and the glycated albumin amount.

[0011]

[Means for Solving the Problems] The above object is a reagent containing a blood cell separation layer, a first dye for coloring albumin and a second dye for coloring glycoalbumin, on a developing membrane mounted on a support. A whole layer was prepared by using a developing solution, in which a layer, a measuring layer on which a substance that specifically binds to both albumin and glycoalbumin is immobilized, and a residual solution absorbing layer that absorbs the developing residual solution are provided in this order. It can be solved by a dry test piece for simultaneously measuring albumin and glycoalbumin in the sample.

At the time of use, albumin and glycoalbumin are colored with different dyes simply by dropping a blood sample and developing with a developing solution, which is optically measured by a photometric device. The ratio of glycoalbumin to albumin is calculated from the amounts of albumin and glycoalbumin thus obtained.

As the blood sample used at this time, not only whole blood but also serum or plasma separated in advance can be used. When using the latter, a blood cell separation layer is not particularly required, but may be provided.

A blood modifying agent may be added to the reagent layer in order to denature albumin and glycoalbumin so that the epitope is exposed as much as possible to facilitate binding to the antibody.

Japanese Unexamined Patent Publication (Kokai) No. 5-87809 discloses a method for measuring glycoalbumin by capturing both albumin and glycoalbumin with a solid phase antibody and labeling only glycoalbumin with a boronic acid derivative. However, this method, which does not label albumin, captures a fixed amount of albumin by applying a sample (ie, albumin containing glycoalbumin) in excess of the known amount of antibody for the solid phase. The capture capacity of the phase antibody has to be constant, which is subject to various effects and causes errors. Therefore, strict conditions are required for the solid phase preparation method and storage, and skill is required. In addition, a large amount of sample is required.

In the case of glycated hemoglobin, since hemoglobin itself has a red color, it suffices to label only glycated hemoglobin, but albumin is colorless,
Both albumin and glycoalbumin must be labeled with different labeling substances. Therefore, in the present invention, the "total amount of albumin" including glycoalbumin is completely grasped by intentionally labeling albumin.

In the present invention, a dye is used as a labeling substance because the dye is very inexpensive and stable as compared with an enzyme or a radioactive substance, and a photometric device having a dual wavelength measuring function is sufficient. This is because it is possible to determine the types of dyes, that is, to measure albumin and glycoalbumin.
Many dyes emit fluorescence, which is also very useful.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the accompanying drawings. 1 to 4 are a plan view and a sectional view showing an embodiment of the present invention.

(Test Piece A) FIG. 1 shows a test piece according to the present invention in which two spreading layers 2 and 2 ′ made of the same material capable of developing a developing solution are mounted on a support 1. FIG. 3 is a plan view and a cross-sectional view of one embodiment (hereinafter referred to as test piece A). On the spreading layer 2, a spreading start point P, a sample spot E, a blood cell separating layer 3 composed of a blood cell separating membrane, a dye a which is a first dye for coloring albumin, and a reagent coated with a denaturant are sequentially applied. A layer 4, a measurement layer 5 on which an anti-albumin antibody or an ion-exchange substance is immobilized, and a residual liquid absorption layer 6 made of an absorbent that absorbs a residual development liquid are provided. In the development layer 2 ', a development start point P', a sample spot E ', a blood cell separation layer 3'consisting of a blood cell separation membrane, a dye b which is a second dye for coloring glycoalbumin, and a modification A reagent layer 4'coated with the agent, a measurement layer 5'on which an anti-albumin antibody or an ion exchange substance is immobilized, and a residual liquid absorption layer 6'made of an absorbent that absorbs the residual development liquid are provided. Both the anti-albumin antibody and the ion exchange substance bind to both albumin and glycoalbumin.

When a blood sample is dropped on the sample spot E of the test piece A and then the developing solution is added to the development start point P, the blood sample is developed and blood cells are removed by the blood cell separation membrane of the blood cell separation layer. . Albumin therein is denatured by the denaturing agent in the reagent layer 4 and colored by the dye a. After that, dye a
Albumin colored with is captured in the measurement layer 5,
Other substances are absorbed by the development residual liquid absorption layer 6. On the other hand, when the same amount of the blood sample is dropped on the sample spot E ′ and then the developing solution is added to the development start point E ′, the blood sample is developed and blood cells are removed by the blood cell separation membrane of the blood cell separation layer. Glycoalbumin therein is denatured by the denaturing agent in the reagent layer 4 ′ and colored by the dye b. Then, the glycoalbumin colored with the dye b is captured in the measurement layer 5 ′,
Other substances are absorbed by the development residual liquid absorption layer 6 '. Then, from the color development in the complex of albumin and dye a captured in the measurement layer 5 and the color development in the complex of glycoalbumin and dye b captured in the measurement layer 5 ', each dye is detected by an optical detection method. The concentration is determined, and the amounts of albumin and glycoalbumin are determined from the results, and then the ratio of glycoalbumin to albumin is calculated.

As shown in FIG. 4, the test piece A may share the unfolded residual liquid absorbing layers (6 and 6 ') of the two unfolded layers (2 and 2'). The residual liquid absorption layer shared is 7 in the figure.

In FIG. 1, the development layers 2 and 2'are arranged close to one end of the support 1, but this is for the measurer to hold one end of the support with a finger. Of course, it can be arranged at any position on the support 1. The same applies to test pieces B and C shown below. Any known material may be used here, but specifically, the following materials are used.

○ Support: polystyrene, polyester,
Film, sheet or plate containing cellulose acetate as a main component The support may be light transmissive or light non-transmissive, but it is transparent if the measurement after completion of development is performed from the lower part of the support,
For example, transparent polyethylene terephthalate (PET) or the like is desirable.

Development membrane: filter paper-like body or porous matrix containing cellulose, cellulose derivative (cellulose acetate, nitrocellulose, etc.), glass fiber, etc. as the main components. The development film processed into a long and narrow tape shape as described above is physically fixed using a double-sided tape, an adhesive, or a hot melt. The width and length when processed into a tape conforms to the chromatography technology.

Blood cell separation membrane: a filtration membrane mainly composed of polytetrafluoroethylene, polysulone, polypropylene, polyvinylidene difluoride, cellulose mixed ester, etc.

Denaturing agent; surfactant, acid, saponin, guanidine and its salt etc. denaturing agent is added in order to denature albumin and glycoalbumin to expose the epitope as much as possible and facilitate binding to antibody. Depending on the antibody used, it is not always necessary, but it may be added.

Absorbent: filter paper-like body or porous matrix mainly composed of cellulose, cellulose derivative (cellulose acetate, nitrocellulose, etc.), glass fiber etc. As a condition of the absorbent for absorbing the residual liquid after development Is
It has a pore volume sufficient to absorb the developing solution after the development.

Developing solution: buffer solution containing a trace amount of protein, salt, chelate compound, surfactant, etc. As a developing solution used for developing a sample specimen, a developing solution such as that used in usual immunoassay method or chromatography is used. , A buffer solution containing a trace amount of protein, salt, chelate compound, surfactant, etc. is desirable, and as an example, 0.1 bovine serum albumin, 0.9% sodium chloride and 0.01
% EDTA and 0.01% Triton X-100.
1M phosphate buffer is mentioned.

Dye: As the dye a, a dye having no specific affinity for other proteins but a specific affinity for albumin can be used. For example, indicators such as bromcresol purple, bromphenol blue, bromcresol green, and cibacron blue F3G-A are preferable. These dyes selectively stain only albumin. Or
A dye that does not have a specific affinity for albumin can also be used by binding it to an anti-albumin antibody. For example, anthraquinone, an azo dye, an azine dye, a triazine dye, a phycoerythrin dye, a coumarin, an acridine, a rhodamine, a fluorescein, or a phthalocyanine bound to an anti-albumin antibody can be used. Incidentally, since some of these dyes emit fluorescence, the fluorescence intensity may be detected. A dye that specifically stains albumin is very inexpensive and stable, and there is no need for an antibody or the like to intervene between the dye and albumin, which is very useful. As the dye b, a dye having a specific affinity for glycoalbumin can be used. It is advisable to use a dye having no specific affinity for glycoalbumin by binding it to a dihydroxyboronic acid derivative or an anti-glycalbumin antibody. For example, those in which anthraquinones, azo dyes, azine dyes, triazine dyes, phycoerythrin dyes, coumarins, acridines, rhodamines, fluorescenes, and phthalocyanines are bound to dihydroxyboronic acid derivatives or anti-glycoalbumin antibodies are preferable. . Preferred examples include xylene cyanol boronic acid and dimethylaminonaphthalenesulfonyl anti-glycalbumin antibody.

The anti-albumin antibody and the anti-glycoalbumin antibody may be a polyclonal antibody or a monoclonal antibody. Only the antibody molecule may be used, but Fab, Fab ′, (Fab ′) ₂ obtained by enzymatic treatment of these may be used.
Antibody active fragments such as can also be used. When the dye is used as a dihydroxyboronic acid derivative, since the antibody has a sugar, the sugar chain is removed by previously treating it with an enzyme such as glucopeptidase or N-glycase, or the Fab, Fab ',
It is desirable to use (Fab ') ₂ fragments.

In order to provide each layer in the spreading layer, various reagents are impregnated / applied or bonded to the spreading film. The method may be a method known to those skilled in the art, and one or more substances to be applied are impregnated. It can be impregnated by dissolving it in a solution and applying it, or spraying with an inkjet printer. In any case, it is finally dried. However, when forming the reagent layer, there is no point unless the reagents are floated from the initial position by the developing solution and chromatographed, so it may be supported by impregnation / coating, but when forming the measurement layer described below. It is necessary that the specific binding substance is chemically bound to the developing membrane and immobilized so that it does not move from its original position (“enzyme immunoassay”, Medical Shoin 1987).

As the dyes a and b, the above-mentioned dyes may be used, but albumin and glycoalbumin must be colored independently of each other. As an example of a suitable combination, the dye a is bromcresol purple and the dye b is xylene cyanol boronic acid. Since the measurement layers of the test piece A are prepared separately for albumin and for glycoalbumin, there is an advantage that even dyes having the same kind or the same absorption wavelength can be distinguished.

(Test Specimen B) FIG. 2 is a plan view of one embodiment of a test specimen according to the present invention in which one developing film 2 capable of developing a developing solution is mounted on a support 1. It is a sectional view (hereinafter referred to as a test piece B). The deployment film 2 is
A development start point P, a sample spot E, a blood cell separation layer 3 composed of a blood cell separation membrane, a dye a that is a first dye that colors albumin, and a dye b that is a second dye that colors glycoalbumin. A reagent layer 4 coated with a denaturant, a measurement layer 5 on which an anti-albumin antibody or an ion exchange substance is immobilized, and a residual liquid absorption layer 6 made of an absorbent that absorbs a residual development liquid are provided.

When the blood sample is dropped on the sample spot E of the test piece B and then the developing solution is added to the development start point P, the blood sample is developed and blood cells are removed by the blood cell separation membrane of the blood cell separation layer. . Next, albumin and glycoalbumin therein are modified by the modifying agent in the reagent layer 4, albumin is colored by the dye a, and glycoalbumin is colored by the dye b. Then, the albumin colored with the dye a and the glycoalbumin colored with the dye b are captured in the measurement layer 5, and the other substances are absorbed in the development residual liquid absorption layer 6. After that, the concentration of each dye is determined by detecting the color of the dye a and the color of the dye b captured in the measurement layer 5 by optically distinguishing them from each other, and from each result, the amount of albumin and glycoalbumin is determined. Then, the ratio of glycoalbumin to albumin is calculated.

Since this test piece B has only one spreading film,
The developed film has a simpler structure than the two test pieces A, the manufacturing cost is low, the spotting of the sample is only required once, and the measuring method is simple.

Here, the material of each constitution is the same as that of the test piece A, but when the dye a is used as a dye, the dye a dyes albumin but also glycoalbumin. In other words, because glycoalbumin is colored with two dyes (a and b), dyes that can be optically distinguished from each other in order to observe a and b in one measurement layer, that is, dyes having different absorption wavelengths from each other. This is different from the test piece A in this respect. Of course, the dye b must label only glycoalbumin. Further, when the dyes a and b are mediated by an antibody and the specific binding substance in the measurement layer contains an antibody, the antibodies must have mutually different epitopes and can simultaneously bind to an antigen.

(Test Specimen C) FIG. 3 is a plan view of one embodiment of a test specimen according to the present invention in which one developing film 2 capable of developing a developing solution is mounted on a support 1. It is a sectional view (hereinafter referred to as a test piece C). The deployment film 2 is
A development start point P, a sample spot E, a blood cell separation layer 3 composed of a blood cell separation membrane, a dye a that is a first dye that colors albumin, and a dye b that is a second dye that colors glycoalbumin. And a reagent layer 4 coated with a denaturant, a measurement layer 5 on which an anti-glycoalbumin antibody or a dihydroxyboronic acid derivative is fixed, a measurement layer 5'on which an anti-albumin antibody or an ion exchange substance is fixed, and a development residual liquid is absorbed. A residual liquid absorption layer 6 made of an absorbent is provided.

When a blood sample is dropped on the sample spot E of the test piece C and then a developing solution is added to the development starting point P, the blood sample is developed and blood cells are removed by the blood cell separation membrane of the blood cell separation layer. . Next, the albumin and glycoalbumin contained therein are modified by the denaturing agent in the reagent layer 4 to convert albumin into the dye a.
And the glycoalbumin is colored with the dye b. Then, the glycoalbumin colored with the dye b is captured in the measurement layer 5, the albumin colored with the dye a is captured in the measurement layer 5 ′, and the other substances are absorbed in the development residual liquid absorption layer 6. Thereafter, the concentration of each dye is determined by optically distinguishing and detecting the color of the dye b and the color of the dye a captured in the measurement layers 5 and 5 ', respectively. After determining the amounts of albumin and glycoalbumin from the results, the ratio of glycoalbumin to albumin is calculated.

This test piece C has the same advantages as the test piece B, but since two measurement layers, one for albumin and one for glycoalbumin, are prepared, dyes each having the same kind or the same absorption wavelength are provided. But they can be distinguished. As with the test piece B, the dye a may be one that specifically colors only albumin or one that colors both albumin and glycoalbumin. However, the dye b must label only glycoalbumin.

Further, the test piece C has an anti-glycalbumin antibody immobilized on one measurement layer and an anti-albumin antibody immobilized on the other measurement layer, and as the pigment, a pigment that colors both albumin and glycoalbumin at once. Good. The type of dye and the absorption wavelength are exactly the same, but there are two measurement layers, one for albumin and one for glycoalbumin, so they can be distinguished from each other.

Although the above has illustrated a part of the embodiments of the present invention, the technical idea of the present invention encompasses various modifications. For example, when the dry test piece of the present invention has no support,
Alternatively, the constitution can be changed according to the purpose of use, such as when a support is used as a part of the spreading film.

[0042]

EFFECTS OF THE INVENTION Since the present invention is a structure having the above-mentioned constitution, it is a novel and highly practical technique of simultaneously measuring glycoalbumin and albumin with a dry test piece, and at the same time, the operation is This test piece is simple and can be used for home use.

[Brief description of the drawings]

1 to 3 are respectively a plan view and a sectional view of one embodiment of a test piece according to the present invention (test pieces A, B,
C). FIG. 4 is a plan view and a cross-sectional view showing another aspect of FIG.

[Explanation of symbols]

 1: Support 2,2 ': Development membrane 3,3': Blood cell separation layer 4,4 ': Reagent layer 5,5': Measurement layer 6,6 ': Residual liquid absorption layer 7: Residual liquid absorption layer (two E, E ': Sample point landing point P, P': Deployment starting point

Claims (10)

[Claims] 1. A dry test piece for simultaneously measuring albumin and glycoalbumin in whole blood using a developing solution, comprising a blood cell separation layer and albumin on a developing film mounted on a support. A reagent layer containing a first dye for coloring and a second dye for coloring glycoalbumin, a measurement layer having a specific binding substance immobilized thereto for capturing albumin and glycoalbumin, and a development residual liquid A dry test piece for measuring glycoalbumin in which each layer of the residual liquid absorbing layer to be absorbed is provided in this order. 2. The dry method for measuring glycoalbumin according to claim 1, wherein the first dye and the second dye have different absorption wavelengths or different fluorescence intensities, respectively. Test pieces. 3. The first dye stains albumin via an antibody specific to albumin, and the second dye colors glycoalbumin via an antibody specific to glycoalbumin or a dihydroxyboronic acid derivative or a combination thereof. The dry test strip for measuring glycoalbumin according to claim 1 or 2. 4. The dry test strip for measuring glycoalbumin according to claim 1 or 2, wherein the first dye is a dye capable of specifically coloring albumin directly. 5. The dye is selected from bromcresol purple, bromphenol blue, bromcresol green, cibacron blue F3G-A,
The dry test strip for measuring glycoalbumin according to claim 4. 6. The layer according to claim 1, wherein each layer is provided on each of two spreading membranes, and albumin and glycoalbumin pass through each reagent layer and are captured by different measurement layers. The dry test strip for measuring glycoalbumin according to 1. 7. The dry test strip for measuring glycoalbumin according to claim 6, wherein the spreading liquids share the same residual liquid absorption layer. 8. The layer according to claim 1, wherein each layer is provided on one spreading film, and both albumin and glycoalbumin pass through the same reagent layer and are captured by the same measurement layer. A dry test piece for measuring glycoalbumin as described above. 9. The method according to claim 1, wherein each layer is provided on one spreading film, and albumin and glycoalbumin both pass through the same reagent layer and are captured by different measurement layers. A dry test piece for measuring glycoalbumin as described above. 10. The dry test strip for measuring glycoalbumin according to claim 1, wherein the reagent layer further contains a blood modifying agent.