JPH0954087A - Dry testing tool for measuring hemoglobin a1c - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure hemoglobin(Hb) A and A1 C simultaneously by laying a hemolyzing layer, a developing layer, an agglutinating layer, another developing layer, and a measuring layer sequentially on a support, and providing an absorbing layer around the measuring layer. SOLUTION: A measuring layer 2 for trapping agglutinated fine particles, a developing layer 3 capable of developing, an agglutinating layer 4 containing anti-HbA1 C antibody, another developing layer 5 capable of developing, and a hemolyzing layer 6 containing a hemolyzer, a modifier and fine particles, are provided on a support 1. An absorbing layer 8 for absorbing remaining fluid is provided around the layer 2, and the entire layers are covered with a perforated cover 7. When a blood specimen is dropped onto the layer 6, it is hemolyzed and modified, and HbA containing HbA1 o is adsorbed to the surfaces of the fine particles. When a developing solution is added to the layer 6, the fine particles are reacted with the anti-HbA1 C antibody in the layer 4, agglutinated, and trapped in the layer 2. The degrees of coloring of HbA and HbA1 C in the layers 2, 8 and the degree of coloring of the fine particles trapped in the layer 2 are measured for determination of the values of the HbA and HbA1 C, and the ratio between the values is calculated.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to hemoglobin A.
(Abbreviated as HbA) and hemoglobin A _1c (HbA _1c
The present invention relates to a dry-type test device for simply measuring the ratio of the amount of HbA _{1c to the} amount of HbA by simultaneously measuring

[0002]

2. Description of the Related Art Glycohemoglobin in which sugar is bound to hemoglobin in blood reflects the average blood sugar concentration in the past 1 to 2 months, and is therefore useful for diagnosing diabetes or observing diabetes. Widely used as a suitable index.

When measuring glycated hemoglobin,
It is indicated by the ratio of the amount of glycated hemoglobin when the total amount of hemoglobin is used as a control. In this case, of the types of hemoglobin (hemoglobin A, S, C, and E), it is clinical to use the ratio of hemoglobin A _1c (HbA _1c ) that is a specific glycohemoglobin in hemoglobin A (HbA) as an index. In general, the ratio of HbA _1c to HbA is usually about 4 to 6%.

As a method for measuring HbA _1c , an electrophoresis method,
The boronic acid affinity chromatography method, the ion exchange chromatography method, the immunoassay method and the like are known, but at present, the ion exchange chromatography method is predominant in view of required time and separability. However, recently, it has also come to be measured by an immunoassay method using a highly specific antibody.

This immunoassay method is not affected by unstable glycohemoglobin, carbamylated hemoglobin, acetaldehyde hemoglobin, acetylated hemoglobin, etc., which is a problem in ion exchange chromatography due to the high specificity of the antibody used, and is rapid. It also has the advantage of high sample processing capacity.

[0006] However, a specific antibody against glycated hemoglobin has an epitope at a site having a chemical structure different from that of hemoglobin (that is, a portion to which sugar is bound), and further H
In the case of bA _1c , the site is not sufficiently exposed, and therefore, when performing an immunoassay method, a denaturing operation for exposing the site is often necessary.

[0007] The ion exchange chromatography method and the conventional immunoassay method require a relatively large analytical device, and the measurement is performed only in a large hospital laboratory or laboratory.

[0008] On the other hand, an application of the immunoassay method, which is a disposable type dry inspection tool (Scre) capable of visually determining the types of hemoglobins A, S, C and E in blood.
Ening 3, 67-76, 1994) is also known. Although this dry test instrument can be measured by the patient himself, it requires a hemolysis operation as a pretreatment, and HbA _1c
It does not measure glycohemoglobin.
When the pretreatment is performed by the patient himself, contamination is a problem in addition to the complicated operation.

[0009]

If a dry test device that can measure glycohemoglobin such as HbA _1c and that can be measured without pretreatment of a sample is developed, its merit will be extremely great. Therefore, the present invention does not require a large-scale analyzer, and does not require hemolysis and / or denaturation treatment operations, and small and medium-sized hospitals or medical practitioners, or even patients themselves, use a small instrument to perform HbA with a simple operation.
It is an object of the present invention to provide a dry test tool capable of directly measuring HbA _1c and HbA _1c simultaneously.

[0010]

The above object is a dry test tool for measuring the amount of HbA _1c relative to HbA in blood by using a developing solution, which is formed entirely of a material capable of transferring a liquid. And on one support, the following i
From v) i) Hemolytic layer coated with hemolytic agent, denaturant and fine particles ii) Development layer iii) Aggregation layer containing anti-HbA _1c antibody iv) Development layer v) Measurement layer for capturing aggregated particles Blood HbA in which an absorption layer that is laminated and that absorbs a development residual liquid after development is arranged around the measurement layer
HbA and HbA were measured by an optical method after a blood sample was spotted and developed using a dry test device for _1c measurement.
_This can be achieved by simultaneously _obtaining the amount of _1c and calculating the ratio of HbA _1c to HbA from the respective values. The blood sample used at this time may be not only whole blood but also separated red blood cells.

Since the commercially available colored beads are used in the immunoaggregation reaction, it is not necessary to label the antibody with a dye or the like. Since the target substance is not labeled, there is no risk of denaturing the antibody due to labeling. Furthermore, the whole blood, which is the sample, spreads from top to bottom together with the developing solution, so that the solution is uniformly spread.

Next to the above structure, the following v
The accuracy of measurement can be improved by providing a structure in which the structure of i to viii, that is, vi) a hemolytic layer coated with a hemolytic agent, vii) a development layer, and viii) a measurement layer that captures HbA are sequentially stacked. Details will be described later. The details will be described below.

[0013]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows that a support 1 is provided with a measurement layer 2 capable of capturing only agglomerated fine particles, and a development layer 3 made of a developable material is provided on the measurement layer 2. The aggregation layer 4 coated with the anti-HbA _1c antibody is provided on the layer 3, the development layer 5 made of a developable material is provided on the aggregation layer 4, and the hemolytic agent, the fine particles and the denaturant are provided on the development layer 5. A longitudinal cross-sectional view of a test device in which a coated hemolysis layer 6 is provided, an absorption layer 8 for absorbing a development residual liquid after development is provided around the measurement layer 2, and these are laminated with a cover 7 having a hole. Is. This is designated as a test tool A. When a blood sample is spotted on the hemolysis layer 6 of this test tool, hemolysis and denaturation are performed, and Hb is deposited on the surface of the fine particles.
HbA containing A _1c is adsorbed. When the developing solution is subsequently added to the hemolyzing layer 6, the fine particles immunoreact with the anti-HbA _1c antibody in the aggregating layer 4 and agglutinate, the agglomerated fine particles are captured by the measurement layer 2, and the developing residual liquid containing the agglutinated fine particles is It is absorbed by the absorption layer 8. Then, Hb in the measurement layer 2 and the absorption layer 8
The coloring degree of A and HbA _1c and the coloring degree of the fine particles themselves (corresponding to the amount of HbA _1c ) captured in the measurement layer 2 were measured by an optical method, respectively, and from the results, Hb
The values of A and HbA _1c are calculated, and HbA relative to HbA is calculated.
Calculate the ratio of _1c .

Here, the function and material of each component are shown below. Basically, known materials may be used.

The support is preferably a light-transmissive substance. In the present invention, the lower part of the measurement layer must be light-transmissive in order to carry out optical measurement after the development is completed from the lower part of the support. In addition, the support does not have to be a single plate, and for example, the entire support may be impermeable to light and may have a through hole in the lower portion of the measurement layer. The material has a certain level of strength, and examples thereof include glass, polystyrene, polyester, polyethylene terephthalate, and cellulose acetate.

The spreading film forming the spreading layer is used as a place for carrying out an adsorption reaction or an immuno-aggregation reaction and for controlling the speed of the reaction. Further, a hemolytic agent or the like is applied to a part of the spread layer to form a hemolytic layer, and an antibody is applied to form an aggregating layer. As the material, it is necessary that the liquid and fine particles easily pass through such as cellulose acetate, nitrocellulose, cellulose and glass fiber.

As a hemolytic agent contained in the hemolytic layer, saponin is effective, but a surfactant can also be used. Examples of the surfactant include polysorbate, polyethylene glycol-p-isooctyl ether and the like. Among them, Triton X-100 is particularly preferable as a commercial product from the viewpoint of hemolytic ability. At the same time, the denaturant applied to the hemolyzed layer denatures HbA _1c to expose a site (that is, a sugar moiety) having a chemical structure different from that of HbA to expose anti-HbA.
_{The 1c} antibody has a role to facilitate immunological recognition. As the kind, an acid such as thiocyanate or a hydrolase such as protease is preferable, and it may be simply acidified. Further, the hemolytic agent layer of the structure for capturing hemoglobin A _1c contains fine particles for adsorbing the antigen. It may be a commercially available colored bead (made of latex) used in the immunoaggregation reaction, or a colored or uncolored metal colloid (gold, silver, copper) can be used.
However, the coloring of these fine particles must be capable of being optically distinguished from the color of hemoglobin (red).

As the anti-HbA _1c antibody contained in the aggregation layer, both a polyclonal antibody and a monoclonal antibody can be used.

The measuring layer is preferably a porous matrix which is a filtering agent having a pore size capable of capturing aggregated fine particles. Examples of the filtering agent include polytetrafluoroethylene, polysulone, polypropylene, polyvinylidene difluoride, cellulose mixed ester and the like. Also,
In order to observe the coloration of the fine particles captured in this layer, a film having a color different in wavelength from the coloration is preferable. It is preferably white. As a method of capturing, it is preferable to use a filtering agent for filtration, and it is general, but there is also a method of using an antibody or an ion exchange resin.

The condition of the absorbent is a porous matrix having a pore volume sufficient to absorb the development residual liquid, or
A dry gel having a sufficient liquid absorption capacity can be mentioned. Examples include cellulose, cellulose acetate, polyacrylic acid,
For example, polyvinyl alcohol.

As the developing solution used for the developing, a small amount of protein, salt, etc., as used in a usual immunoassay method,
A buffer containing a surfactant is desirable, and as an example,
0.1% bovine serum albumin and 0.01% Triton X
-100 and 0.1 M phosphate buffer containing 0.9% sodium chloride.

The hemolytic layer, the agglutinating layer and the measuring layer are prepared by impregnating, coating or bonding various reagents to the spreading film.
The method may be a method known to those skilled in the art, and the substance to be applied is dissolved in one or more kinds of impregnating solutions and impregnated by spreading, sprayed with an inkjet printer, or chemically bound (“enzyme”). Immunoassay "Medical School,
(1987). In any case, it is finally dried.

As a method for disposing each layer on the support,
The circularly processed films of the respective layers are superposed, and the cover and the support are physically fixed (for example, with an adhesive). Of course, it need not be circular. In that case, examples of the material of the cover include polyethylene, polypropylene, polystyrene, methacrylic resin, polycarbonate and the like.

FIG. 2 shows that HbA _1c is formed on the support 11.
FIG. 3 is a cross-sectional view of a test device having a structure for capturing HbA and a new structure for capturing HbA next to the structure. This test tool is called test tool B. The structure for capturing HbA _1c is the same as that of the test device A in FIG. 1. The structure for capturing HbA has a measurement layer 22 in which an anti-HbA antibody or an ion exchange substance is immobilized on the support 11.
Development layer 2 made of a material capable of developing a liquid on the measurement layer
Hemolyzing layer 2 having 3 and a hemolytic agent applied on the spreading layer
6. These components are covered with a cover 27 having holes for supplying a sample and a developing solution,
It is fixed to the support at the same time as it is laminated. Measurement layer 2
Around the circumference of 2, an absorption layer 28 made of an absorbent that absorbs the development residual liquid is provided. The structure for capturing HbA _1c has a measurement layer 12 made of a filter agent capable of capturing only aggregated fine particles on a support 11, and is made of a material capable of spreading a liquid on the measurement layer 12. Aggregation layer 1 having a spreading layer 13 formed by applying an anti-HbA _1c antibody on the spreading layer 13.
4 and a spreading layer 15 made of a material capable of spreading a liquid on the aggregation layer 14, and a hemolytic agent on the spreading layer 15.
It has a hemolytic layer 16 coated with a modifier and fine particles. These components are covered with a cover 17 having holes, and are laminated and fixed to a support at the same time. An absorption layer 18 made of an absorbent that absorbs the unfolded residual liquid is provided around the measurement layer.

When a blood sample is spotted on the hemolyzed layer 26 of the test device B, it is hemolyzed, and when a developing solution is added to the hemolyzed layer 26,
HbA is the anti-Hb that has been expanded and immobilized on the measurement layer 22.
It is captured by the A antibody or the ion exchange substance. The development residual liquid at that time is absorbed by the absorption layer 28. On the other hand, when a blood sample is spotted on the hemolyzed layer 16, the sample is hemolyzed and denatured, and H
HbA containing bA _1c is adsorbed on the fine particles. Subsequently, when the developing solution was added to the hemolytic layer 16, HbA containing HbA _1c was added.
The fine particles adsorbed with react with the anti-HbA _1c antibody in the aggregation layer 14 and aggregate. The aggregated fine particles are captured by the measurement layer 12, and the development residual liquid containing the non-aggregated fine particles is absorbed by the absorption layer 18. Next, the coloring degree of HbA in the measurement layer 22 and the fine particles themselves (HbA
(Corresponding to the amount of _1c ) and the degree of coloring are optically measured,
After the values of HbA and HbA _1c are obtained from the results, respectively, the ratio of HbA _1c to HbA is calculated.

In the test tool B, the locations where HbA and HbA _1c are captured are different, and the measurement accuracy is improved as compared with the test tool A in order to reliably capture HbA and HbA _1c by different means.

[0027] In hemolysis layer structure in order to capture the _{HbA 1c,} to expose the site (i.e. sugar moiety) having different chemical structure of the HbA denature the _{HbA 1c,} anti HbA
_It is necessary for the _1c antibody to be easily recognized immunologically.
In the hemolyzed layer in the structure for capturing A, HbA _1c
No denaturing agent is specifically required to capture the entire HbA containing H. In addition, since there are no fine particles that agglomerate, the anti-HbA antibody, an ion exchange resin (diethylaminoethyl cellulose, carboxymethyl cellulose as an example of the ion exchange resin) or the like may be immobilized and included in the measurement layer. The anti-HbA antibody recognizes both HbA and HbA _1c , and as the type, both a polyclonal antibody and a monoclonal antibody can be used.

[0028]

EFFECTS OF THE INVENTION According to the present invention, a large-scale analyzer is not used, pretreatment such as hemolysis and / or denaturation treatment is not required, and small and medium-sized hospitals or practitioners with a small number of samples can be used.
Or the patient himself can use a small device and perform H
bA and HbA _1c can be directly measured simultaneously. Moreover,
Since the commercially available colored beads are used in the immunoaggregation reaction which is the principle of the present invention, it is not necessary to label the antibody with a dye or the like. Since the target substance is not labeled, there is no risk of denaturing the antibody due to labeling. Furthermore, since the sample spreads from top to bottom together with the developing solution, the solution is uniformly spread.

[0029]

[Brief description of drawings]

FIG. 1 is a vertical sectional view of a test device A of the present invention.

FIG. 2 is a vertical sectional view of a test device B of the present invention.

[Explanation of symbols]

 1,11: Support 2,12,22: Measurement layer 3,5,13,15,23: Development layer 4,14 ,: Aggregation layer 6,16,26: Hemolysis layer 7,17,27: Cover 8, 18, 28: Absorption layer

Claims (5)

[Claims] 1. A hemoglobin A relative to the amount of hemoglobin A (abbreviated as HbA) in whole blood by using a developing solution.
_1c (abbreviated as HbA _1c ) A dry test tool for measuring the amount of _1c , in which the following constitutions i to v, i) a hemolytic agent, a denaturing agent, and fine particles were applied onto one support. Hemolysis layer ii) Development layer iii) Anti-HbA _1c antibody-containing aggregation layer iv) Development layer v) Measurement layer that captures aggregated fine particles are laminated in this order, and the absorption layer that absorbs the development residual liquid after development is the above. HbA _1c arranged around the measurement layer
Dry test device for measurement. 2. Next to the structure according to claim 1, further comprising the following vi to viii, vi) a hemolytic layer coated with a hemolytic agent vii) a developing layer viii) an anti-HbA antibody or an ion The structure in which the measurement layers on which the exchange resin is immobilized is laminated in order is installed in parallel, and the absorption layer that absorbs the residual liquid after the development is placed around the measurement layer. Dry test tool for HbA _1c measurement 3. The test device according to claim 1 or 2, wherein the method for capturing the aggregated fine particles in the measurement layer is a filtering action by a filtering agent. 4. The test device according to claim 1, wherein the fine particles are colored in a color that can be optically distinguished from the color of hemoglobin for measurement. 5. For optical measurement in the measuring layer, the support is wholly or partly light-transmissive or the measuring layer is perforated at the bottom. The test device according to any one of 4 above.