JPH0961426A - Dry measuring instrument for blood specimen utilizing hapten - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a small-sized dry measuring instrument capable of simultaneously measuring HbA and HbA1c by providing a structure for catching hemoglobin A1c (HbA1c) and a structure catching hemoglobin A (HbA) on a support side by side. SOLUTION: A structure consisting of a hemolytic layer 11 coated with a polymer of hapten containing a hemolytic agent, a modifier and an epitope of an anti-HbA1c antibody in order to catch HbA1c, a developing layer 10, a coagulation layer 9 coated with fine particles having an anti-HbA1c antibody immobilized thereon, a developing layer 8 and a measuring layer 7 catching flock particles is provided. A dry measuring instrument is obtained by successively arranging a hemolytic layer 4 coated with a hemolytic agent in order to catch HbA, a developing layer 3 catching HbA on a support 1 side by side in a laminated state and providing absorbing layers 12, 5 absorbing residual developing solns. after development so as to surround the measuring layers 7, 2.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to HbA and HbA.
By simultaneously measuring _lc , H for the amount of HbA
The present invention relates to a dry-type measuring tool for simply measuring the ratio of bA _lc amounts.

[0002]

2. Description of the Related Art Glycohemoglobin in which sugar is bound to hemoglobin in the blood reflects the average blood sugar concentration in the past 1-2 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 _lc (HbA _lc ), which is a specific glycohemoglobin in hemoglobin A (HbA), as an index. In general, the ratio of HbA _lc to HbA is usually about 4 to 6%.

As the HbA _lc measuring method, an electrophoresis method,
The boronic acid affinity chromatography method, ion exchange chromatography method, immunoassay method and the like are known, but the ion exchange chromatography method is predominant in terms 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 and acetylated hemoglobin, which are problems in ion exchange chromatography due to the high specificity of the antibody used, and the immunoassay method 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 _lc , the site is not sufficiently exposed, and therefore, when carrying out an immunoassay method, a denaturing operation for exposing the site is often necessary.

The conventional ion-exchange chromatography method and immunoassay method require a relatively large-scale analyzer, 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 test device can be measured by the patient himself, it requires hemolysis as a pretreatment, and in addition to that, HbA
_It does not measure _{glycohemoglobin} such as _lc . 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 capable of measuring glycated hemoglobin such as HbA _1c and capable of measuring 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 practitioners with a small number of specimens, or even patients themselves, can use HbA with a simple operation using simple equipment.
It is an object of the present invention to provide a dry measuring tool capable of directly measuring _HbAlc and HbAlc simultaneously.

[0010]

The above object is a dry test device for measuring the amount of HbA _lc with respect to HbA in blood by using a developing solution as a whole, which is formed of a material capable of transferring a liquid. And on one support, the following i
Coating the structure i.e. i) the hemolytic agent vi, and modifiers, particulates anti HbA _lc hapten comprising the epitope of an antibody polymer (polyhapten) and was applied hemolysis layer ii) anti-HbA _lc antibody is immobilized from Aggregated layer iii) Development layer iv) Measurement layer having a function of capturing agglomerated fine particles v) Development layer vi) Measurement layer for capturing agglomerated fine particles A structure in which layers are laminated in order, and the following v to ix, that is, vii ) Hemolytic layer coated with hemolytic agent viii) Development layer ix) Measurement layer that captures HbA is placed in parallel with a structure in which layers are sequentially stacked, and the absorption layer that absorbs the development residual liquid after development is measured as described above. surrounds around the layers, using a blood HbA _lc measured for dry measurement tool, the amount of HbA and HbA _lc as measured by an optical method after deployed spotted blood sample Sometimes determined by calculating the ratio of _{HbA lc} for HbA from each value, it can be achieved. 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. In addition, 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. The principle of the present invention is the so-called "immunoinhibition method". The details will be described below.

[0012]

DETAILED DESCRIPTION OF THE INVENTION FIG.
FIG. 3 is a cross-sectional view of a measurement tool according to the present invention having a structure for capturing _lc and a structure for capturing HbA. The structure for capturing HbA has a measurement layer 2 having an anti-HbA antibody or an ion-exchange substance immobilized on a support 1, and a development layer made of a material capable of developing a liquid on the measurement layer. 3 and a hemolytic layer 4 coated with a hemolytic agent on the spreading layer. These constituent elements are covered with a cover 6 having holes for supplying a sample and a developing solution, and are laminated and fixed to a support at the same time. Around the measurement layer 2, an absorption layer 5 made of an absorbent that absorbs the development residual liquid is provided.

The structure for trapping HbA _lc has a measurement layer 7 made of a filtration membrane capable of trapping only aggregated fine particles on the support 1, and a liquid is spread on each of the measurement layers 7. It has a spreading layer 8 made of a possible material, has an agglutinating layer 9 coated with fine particles on which the anti- _HbAlc antibody is immobilized, and spreads the liquid on the agglomerating layer 9. It has a spreading layer 10 made of a possible material, and has a hemolysis layer 11 coated with a hapten polymer (polyhapten) containing a hemolytic agent, a denaturant, and an anti- _HbAlc antibody epitope on the spreading layer 10. . These components are covered with a cover 13 having a hole, and are laminated and fixed to a support at the same time. An absorption layer 12 made of an absorbent that absorbs the unfolded residual liquid is provided around the measurement layer.

In use, the hemolyzing layer 4 of the measuring tool is first used.
It is started by depositing the same amount of blood sample on the hemolysis layer 11 and. The blood sample is hemolyzed in the hemolysis layer 4, and then a developing solution is added to the hemolysis layer 4 to develop HbA. The developed HbA is captured by the anti-HbA antibody or ion exchange substance immobilized on the measurement layer 2, and the development residual liquid is absorbed by the absorption layer 5. On the other hand, in the hemolysis layer 11, the blood sample is denatured at the same time as hemolysis. When the developing solution is subsequently added to the hemolytic layer 11, the sample and polyhapten are developed. Since this polyhapten is a polymer of a peptide containing the HbA _lc epitope of the anti-HbA _lc antibody, the anti-H in the aggregation layer 9 is
bA _lc antibody immobilized particles, aggregate reacts with polyhapten.

However, when HbA _lc is present in the sample, polyhapten and H are added to the antibody-bound fine particles.
The bA _lc competes, resulting in quantitatively less aggregation. The more HbA _lc , the less aggregation. At this time, the degree of aggregation in the case where HbA _lc does not exist at all is investigated in advance, and it is confirmed how much HbA _lc exists and how much the degree of aggregation decreases. Since the aggregated fine particles are colored, the concentration associated with the aggregation can be easily determined. The aggregated fine particles are captured by the measurement layer 7 having a capture function, and the development residual liquid containing the non-aggregated fine particles is absorbed by the absorption layer 12. Measurement layer 2
The degree of coloring of HbA captured by and the degree of coloring of the fine particles themselves (corresponding to HbA _lc ) captured in the measurement layer 7 are measured by an optical method. After the HbA and HbA _lc contents were obtained from the results, respectively, HbA _lc relative to HbA was _calculated.
Calculate the ratio of

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, and in the present invention, the lower part of the measurement layer must be light-transmissive in order to perform optical measurement after completion of development 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. In the present invention, a material through which a liquid can pass is referred to as a "developing film", and a material having a constituent layer such as a hemolytic layer or an agglutinating layer provided on the developing film is referred to as a "developing 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 and polyethylene glycol-p-isooctyl ether. Among them, Triton X-100 is particularly preferable as a commercial product from the viewpoint of hemolytic ability. At the same time, the denaturing agent applied to the hemolyzed layer denatures HbA _lc to expose a site (that is, a sugar moiety) having a chemical structure different from that of HbA to expose anti-HbA
_{The lc} antibody has a role of _facilitating immunological recognition. Therefore, it is not particularly necessary for the structure for capturing hemoglobin A. As types, acids such as thiocyanate,
A hydrolase such as a protease is preferable, and it may be simply acidified.

The hapten containing the epitope of the anti- _HbAlc antibody in the hemolyzed layer is obtained by binding one glucose to the valine portion of a polypeptide having valine at one end. A polymer of a hapten containing an epitope of an anti-HbA _lc antibody (polyhapten) binds a carboxyl group in the hapten to a polypeptide containing several lysines and having an amino group on the surface, and the hapten is bound to the surface of the polypeptide. Obtained by introducing. Alternatively, it can be obtained by binding the amino group in the hapten to a polypeptide containing several glutamic acids and having a carboxyl group on the surface, and introducing the hapten to the surface of the polypeptide.

The fine particles for immobilizing the anti- _HbAlc antibody contained in the agglutination layer may be the same as those used in the usual immunoagglutination method, such as commercially available colored latex used in an immunoagglutination reaction, Colored or uncolored metal colloids (gold, silver, copper) and the like can also be used. However, the coloring of these fine particles must be capable of being optically distinguished from the color of hemoglobin (red). As a method of immobilizing the antibody on the fine particles, in addition to mixing the two and physically adsorbing them, an amino group is introduced into the fine particles,
There are a method of reacting with the carboxyl group on the antibody side, a method of binding with a coupling agent, and the like.

The measuring layer having a capturing function is preferably a porous matrix having a pore size capable of capturing agglomerated fine particles by utilizing filtration. Examples of the filtration membrane include polytetrafluoroethylene, polysulone, polypropylene, polyvinylidene difluoride, cellulose mixed ester and the like. Further, in order to observe the coloration of the fine particles captured here, a film having a color different in wavelength from the coloration is preferable. It is preferably white. As a method of capturing, it is generally desirable to filter, 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 residual liquid for development, 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 a developing solution used for 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.

In the structure for capturing HbA, it is not necessary to distinguish between HbA and HbA _lc in the measurement layer, and since there are no aggregated fine particles, the measurement layer has an anti-HbA antibody or an ion exchange resin (for example, , Diethylaminoethyl cellulose, carboxymethyl cellulose) and the like. Anti-HbA antibodies are HbA and HbA
binds to both _lc .

The hemolysis layer, aggregation layer and measurement layer are prepared by impregnating, coating or binding 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.

[0028]

EFFECTS OF THE INVENTION According to the present invention, a large-scale analyzer is not used, hemolysis and / or denaturation treatment operations are not required, and small and medium-sized hospitals or medical practitioners with a small number of specimens, or patients themselves can use small instruments. , HbA and HbA with simple operation
_{and lc} can be measured directly at the same time. Further, it is not necessary to label the antibody with a dye or the like, and 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 measuring tool of the present invention.

[Explanation of Codes] 1: Support 2,7: Measurement layer 5,12: Absorption layer 3,8,10: Development layer 9: Aggregation layer 4,11: Hemolysis layer 6,13 ,: Cover

Claims (4)

[Claims] 1. A hemoglobin A _lc (Hb) relative to hemoglobin A (abbreviated as HbA) in a blood sample by using a developing solution, which is entirely formed of a material capable of transferring a liquid.
abbreviated as “bA _lc” ), which is a dry test device comprising the following components i to vi, i) a hemolytic agent, a denaturing agent, and an anti-HbA _lc antibody on one support. Hemolytic layer coated with hapten polymer (polyhapten) containing the epitope of ii) Aggregation layer coated with fine particles to which anti- _HbAlc antibody is immobilized iii) Development layer iv) Measurement layer having a function of capturing aggregated fine particles v) Development layer vi) Structure in which a measurement layer that captures aggregated fine particles is sequentially stacked, and the following vii to ix configuration, vii) Hemolytic agent-coated hemolysis layer viii) Development layer ix) Measurement to capture HbA A dry measurement device for a blood sample, in which a structure in which layers are sequentially stacked is installed in parallel, and an absorption layer that absorbs a development residual liquid after development surrounds the measurement layer. 2. The measuring tool according to claim 1, wherein the method of capturing the aggregated fine particles in the measuring layer is by a filtering action of a filtering material. 3. The measuring tool according to claim 1 or 2, wherein the fine particles are colored in a color that can be optically distinguished from the color of hemoglobin. 4. For optical measurement in the measuring layer, the support is wholly or partly light-transmitting or the lower part of the measuring layer is perforated. The measurement tool according to any one of item 3.