JPH08285850A - Dry test kit for blood specimen - Google Patents

Abstract

PURPOSE: To obtain a highly practical new dry test kit for measuring glucosyl hemoglobin (HbAIC) and hemoglobin A(HbA) simultaneously which can be used easily even in home. CONSTITUTION: One or more than one developing layer 2 held on a support 1 in order to carry a blood specimen on a developing liquid through capillarity is provided with a development starting point E being applied with the developing liquid, and a hemolysis layer 3 coated with a hemolytic agent and/or modifier. The dry test kit for blood specimen is also provided, as essential requirements, solid phase antibody layers 4, 4' (or solid phase adsorbent layer) for capturing specified substances or an equivalent hemolysis layer 3, a labeled substance layer 6, and the solid phase antibody layers 4, 4' (or solidified adsorbent layer) arranged sequentially.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention
Robin A_1C(HbA _1C) (Also known as: glycosylated hemoglobin)
Bin) and hemoglobin A (HbA ) And simple measurement at the same time
The present invention relates to a test tool.

[0002]

2. Description of the Related Art Glycosylated hemoglobin in which sugar is bound to hemoglobin in the blood reflects the average blood sugar concentration in the past 1-3 months. It is widely used as an index suitable for.

Conventionally, the measurement of glycosylated hemoglobin has been carried out by an electrophoresis method, a mini-column method, an HPLC method, etc. Among them, in the field of clinical examination, the HPLC method is required from the viewpoint of required time and separability. Is the mainstream. However, recently, a method of utilizing an immune reaction and optically measuring the result, that is, a so-called immunoassay method has been used.

Since this immunoassay method utilizes the specificity of the antibody for glycosylated hemoglobin, it produces accurate and quick results without measuring the unstable influencing factors that are problematic in HPLC. High processing capacity. Furthermore, this immunoassay method is simplified,
Disposable test tools for visual examination of blood hemoglobin (hemoglobin A, S and C) are also known ("Screening" 3, 67-76).
(1994)). However, this is to determine the difference in type among hemoglobins A, S and C, and not to measure glycosylated hemoglobin in each hemoglobin.

The conventional HPLC method and immunoassay method require a relatively large-scale analyzer, and the measurement is performed in a hospital laboratory or a laboratory. On the other hand, the above-mentioned disposable test devices currently known can be measured by the patient themselves, but require hemolysis as a pretreatment, and hemoglobin can be measured, but glycosylated hemoglobin can be measured. Can not be measured. When the pretreatment is performed by the patient himself / herself, the problem of contamination occurs in addition to the complexity of the operation.

Particularly in the measurement of glycosylated hemoglobin,
Hemoglobin A (HbA ) Specific glycosylated hemoglobin in
Hemoglobin A, which is globin _1C(HbA _1C) Of
It is clinically generalized to use it as an index, and
It is always displayed as a percentage, and about 4 to 6% is a standard for its normal value.
It is said that. These HbA _1CValue, requires preprocessing
A kit has been developed that enables simple and quick measurement without
If so, the merit is extremely large.

[0007]

SUMMARY OF THE INVENTION The present invention is directed to a large analytical instrument.
Patient at home without pretreatment
HbA and HbA with a simple operation _1CAnd can be measured at the same time
The purpose is to provide a dry test device.

However, HbA _1CSpecific antibody against Hb
A The site having a different chemical structure from
And the site is HbA _1CExposed too much in
Because of the lack of immunoassays,
HbA _1CMust be completely exposed by denaturation of
However, the denaturing operation was necessary.

[0009]

SUMMARY OF THE INVENTION The present invention is supported on a support.
And transfer the blood sample by capillary action by the developing solution
The developing solution is applied to one or more developing layers
The starting point of development and the application of hemolytic and / or denaturing agents
Hemolysis layer and solid-phase antibody layer for capturing specific substances (or
Or a solid-phased adsorbent layer), or a hemolysis layer that works in the same way as above
, The labeling substance layer, and the immobilized antibody layer (or the immobilized adsorption layer).
It is essential that the adhesive layer) is installed in this order.
It is a dry test device for blood samples, and various antigens in blood
Which is applicable to the measurement of hemoglobin A
And A _1CA tool suitable for measurement and according to the present invention
The physical aspect will be specifically described with reference to the drawings.

HbA _1CTo measure the
Anti-HbA antibody and anti-HbA _1CThe above is bound to an antibody
After preparing a test device and dropping and deploying a blood sample,
HbA and HbA in the adsorption layer _1CThe amount of
HbA to HbA using the value of _1CCalculate the percentage of
Put out. The blood sample used at this time is whole blood.
Of course, use isolated red blood cells alone
You can also.

[0011]

Embodiments of the present invention will be described below with reference to the accompanying drawings. 1 to 5 are a plan view and a sectional view showing an embodiment of the present invention.

(Test tool A) FIG.
Deployment made of a pair of identical deployment membranes (materials that can deploy liquid)
Layer 2, the same hemolytic agent and / or the
Is the same hemolytic layer 3 coated with denaturant, anti-HbA
Antibodies and anti-HbA _1CSolid-phase immobilization of antibodies
Absorb the antibody layers 4, 4 ', and the development residual liquid after development
Test tool provided with the same development residual liquid absorption layer 5 made of an absorbent
It is the top view (a) and sectional drawing (b) of A.

A blood sample S is dropped on the hemolyzed layer 3 of the test device A.
Then, it is degenerated at the same time as hemolysis. Next, develop and open the developing solution.
When added to the starting point E, the denatured hemoglobin is unfolded.
HbA in the developing solution And HbA _1CSolid phase
Anti-HbA in antibody layer 4 In the antibody and the immobilized antibody layer 4 '
Anti-HbA _1CIt is captured by the antibody, and other substances are absorbed by the development residual liquid.
It is absorbed by the collecting layer 5. Hemoglobin is colored
The hemoglobin in the immobilized antibody layers 4 and 4 '
It is measured by the method (reflected light, transmitted light). Hb respectively
A And HbA _1CThen, the value is used for HbA
To HbA _1CCalculate the percentage of.

In the figure, the development layer 2, the hemolysis layer 3, the solid-phased antibody layers 4, 4'and the development residual liquid absorption layer 5 are arranged close to one end of the support 1, but this is different from the measurer. The end can be held by a finger, and can of course be arranged at any position on the support 1. The following test tools B, C, D,
The same applies to E.

Here, the materials are all known materials.
However, it is specifically as follows. Support: polystyrene, polyester, cellulose acetate
Films, sheets or plate-like bodies mainly made of etc., Deployable films: cellulose, cellulose derivatives (cellulosic acetate
Filter, mainly made of glass, etc.)
Paper or continuous porous membrane, absorbent: cellulose, cellulose derivative, polyacrylic
Granules composed mainly of acid salts and polyvinyl alcohol, hemolytic agents: saponins, surfactants, etc. Denaturing agents: thiocyanates, proteases, etc., anti-HbA Antibodies and anti-HbA _1CAntibody: polyclonal anti
Either body or monoclonal antibody. Developing solution: Buffer containing trace amounts of proteins, salts, and surfactants
liquid

As a method of arranging the spreading film on the support, a spreading film processed into an elongated tape shape as shown in each figure is used.
Physically fix with double-sided tape, adhesive or hot melt. The width and length when processed into a tape conforms to the chromatography technology.

The support used may be light transmissive or light non-transmissive, but if the measurement after completion of development is carried out from the lower part of the support, transparent polyethylene terephthalate (PE) is used.
T) and the like are desirable.

The condition of the absorbent for absorbing the residual liquid after the development is that it has a pore volume sufficient to absorb the developing liquid after the development.

Saponin is effective as a hemolytic agent, and examples of the surfactant used as a hemolytic agent include polysorbate, polyethylene glycol-p-isooctylphenyl ether and the like. Among them, Triton X-100 is particularly preferable as a commercial product from the viewpoint of hemolytic ability.

The denaturing agent contained in the hemolytic layer, HbA _IC a site at which the difference in chemical structure between HbA denature the HbA _IC
It has the role of exposing it to the outside and making the anti-HbA _IC antibody easier to recognize immunologically. Acids such as thiocyanate and degrading enzymes such as proteases are preferred. The pH may simply be in the acidic range.

The developing solution used for developing the sample specimen is preferably a buffer solution containing a trace amount of proteins, salts and surfactants as used in chromatography. For example, 0.1% bovine A 0.1 M phosphate buffer containing serum albumin, 0.9% sodium chloride and 0.01% Triton X-100 can be mentioned.

In order to form each layer in the spreading layer, various reagents are impregnated / coated or bonded to the spreading film. The method may be a method known to those skilled in the art, and the substance to be applied may be one or more kinds. It can be impregnated by dissolving it in an impregnating solution and applying it, or spraying with an inkjet printer. In any case, it is finally dried. However, when forming the hemolyzed layer in the example and the labeling substance layer described below, it is meaningless if the reagents do not rise from the starting position in the developing solution, so it is sufficient to impregnate / apply, but the adsorption layer described below. Since it must not move from its original position, it must be chemically bound and immobilized (“enzyme immunoassay”, Medical Shoin 1987).

(Test tool B) FIG.
A development layer 2 made of a pair of identical development films, which is arranged on each of the development layers in order.
Then apply the same hemolytic and / or denaturing agents, respectively.
The same hemolyzed layer 3 coated, labeled anti-HbA Antibodies and labeled anti
HbA _1CLabeled substance layer (hereinafter, labeled
6,6 ', which is a solid phase of an adsorbent.
Absorbs one solid-phased adsorbent layer 7 and development residual liquid after development
The same development residual liquid absorption layer 5 composed of the absorbent to be provided was provided.
It is a top view (C) and a sectional view (D) of a test tool.

A blood sample S is dropped on the hemolyzed layer 3 of the test device B.
Then, it is degenerated at the same time as hemolysis. Next, develop and open the developing solution.
When added to the starting point E, the denatured hemoglobin is unfolded.
HbA in the developing solution And HbA _1CIs the label
In the labeled anti-HbA antibody in the body layer 6 and in the labeled antibody layer 6 '
Labeled anti-HbA _1CBinds to antibodies and their immune complex
Each body is captured by the solid-phased adsorbent layer 7,
The quality is absorbed by the development residual liquid absorption layer 5. Its solidified adsorption layer
Labeled substance (probe) captured by 7 is the same as test tool A
It measures by the method of. HbA respectively And HbA _1C
HbA with respect to HbA after obtaining _1Cof
Calculate the percentage. This test tool B is captured hemo
A machine rather than Test Tool A, which visually measures the coloration of globin.
The measurement accuracy is high for
It has the advantage of being expensive.

Here, the support, spreading film, hemolytic agent, denaturing agent, antibody, absorbent and developing solution are the same as those in Test Tool A, and the following materials can be used as other materials. Labeled antibody probe (labeling means): metal colloid (gold,
Silver, copper), fine particles (latex, etc.) and their colorants, dyes (hematoxylin, malachitegrin, rhodamine B, galocyanine, neal blue A, azure C, xylene cyanol, etc.), fluorescent dyes (fluorescein, rhodamine, etc.) adsorption Agents: Ion-exchange substances (diethylaminoethyl cellulose, carboxymethylated polyvinyl alcohol, etc.), antibodies and dihydroxyboryl derivatives immobilized on filter paper or powder containing cellulose or polyvinyl alcohol as a main component

Examples of the adsorbent in the solid-phased adsorption layer include ion exchange resins such as diethylaminoethyl (DEAE) cellulose and carboxymethyl (CM) -modified polyvinyl alcohol. These ion exchange resins cannot separately capture HbA and HbA _IC , which are measurement target substances. Since the antibody as an adsorbent utilizes the specificity for HbA and HbA _IC based on the antigen-antibody reaction,
It is possible to separately capture HbA and HbA _IC .

Dihydroxyboryl derivatives, such as aminophenylboric acid (Aminophenylboric)
acid) is a substance that chemically bonds with two cis-type hydroxyl groups in the sugar chain, and can be used as an adsorbent after being immobilized.
It can also be used as a labeling substance by binding to a dye or the like. HbA and HbA _IC for specific binding to sugar chains
It is also possible to distinguish between. If this substance is used as a labeling substance and hemoglobin is captured by an ion exchange resin, there is an advantage that an antigen-antibody reaction is not involved and an expensive antibody is not used.

(Test tool C) FIG.
A spreading layer 2 made of two spreading films, and the spreading layer 2 is sequentially melted.
Hemolytic layer 3 coated with blood and / or denaturant, anti-Hb
A Antibodies and anti-HbA _1CSolid phase with each antibody immobilized
Absorb the derivatized antibody layers 4, 4 ', and the unfolded residual liquid after unfolding
A flat surface of a test tool C provided with a development residual liquid absorption layer 5 made of an absorbent.
It is a front view (e) and a sectional view (f).

When the blood sample S is dropped on the hemolyzing layer 3, hemolysis occurs.
Simultaneously denatured, then add the developing solution to the starting point E
Denatured hemoglobin is expanded and HbA in it is expanded.
And HbA _1CIs the anti-HbA in the immobilized antibody layer 4, respectively.
Antibodies and anti-HbA in immobilized antibody layer 4 ' _1CCaptured by antibody
Other substances are absorbed by the development residual liquid absorption layer 5.
The hemoglobin in the solid-phased antibody layers 4, 4'is tested by the test tool A.
HbA measured by the same method as And Hb
A _1CAsk for. Here, support, spreading membrane, hemolytic agent, denaturation
Agents, antibodies, absorbents, adsorbents and developing solutions are the test tools described above.
The same as A.

(Test tool D) FIG.
Development layer 2 made of two development membranes, and on the development layer 2 sequentially,
Hemolytic layer 3 coated with hemolytic agent and / or denaturant, label
Anti-HbA Labeled substance layer coated with antibody (hereinafter, labeled antibody layer
6.) anti-HbA antibody and anti-HbA_1CAntibody
Solid phase immobilized antibody layers 4 and 4 ', respectively, and
Development residual liquid consisting of an absorbent that absorbs the development residual liquid after development
A plan view (g) and a sectional view of the test device D provided with the absorption layer 5.
(H). However, the anti-body in the labeled antibody layer and the immobilized antibody layer
HbA antibodies have different epitopes to anti-HbA antibodies.
However, both can bind anti-HbA, and the former is Hb
A_1CCan also be combined with.

When the blood sample S is dropped on the hemolyzed layer 3 of the test device D, it is denatured at the same time as hemolysis. Next, when a developing solution is added to the starting point E, the denatured hemoglobin is developed, and HbA and HbA _1C therein are bound to the labeled anti-HbA antibody (labeled antibody layer 6), respectively, and the labeled anti-HbA antibody-HbA complex is added. And the labeled anti-HbA antibody-HbA _1C complex with the body. Furthermore, each of these complexes is anti-HbA.
The antibody (immobilized antibody layer 4) and the anti-HbA _IC antibody (immobilized antibody layer 4 ′) are immunologically captured, and other substances are absorbed by the residual liquid absorption layer 5. The respective labels (probes) captured by the solid-phased antibody layers 4, 4'are measured by the same method as in the test tool B, and HbA and HbA _1C are respectively measured.
Ask for. Here, the support, the spreading film, the hemolytic agent, the denaturing agent,
The labeled antibody, the adsorbent, the absorbent and the developing solution are the same as in the test device B.

(Test tool E) FIG.
Development layer 2 made of two development films, and on the development layer 2 sequentially
A hemolytic layer 3 coated with a hemolytic agent and / or a denaturing agent,
Dyes bound to dihydroxyboryl derivatives
Labeling substance layer 8 coated with anti-HbA Antibody solid phase
Absorption that absorbs the derivatized antibody layer 4 and the development residual liquid after development
Plan view of a test tool E provided with a development residual liquid absorption layer 5 made of a chemical
It is (i) and sectional drawing (nu). However, in the immobilized antibody layer
The anti-HbA antibody has an epitope on the hemoglobin side and
bA and HbA_1CCan also be combined with.

When the blood sample S is dropped on the hemolyzed layer 3 of the test device E, it is denatured at the same time as hemolysis. Then the addition of expanded fluid to the mapping start point E, denatured hemoglobin is developed, among HbA and HbA _1C therein, HbA _1C
A dihydroxyboryl derivative having a dye as a label is bound to the exposed sugar moiety of the. Then, HbA that does not bind anything and HbA that the dihydroxyboryl derivative has bound
_1C occurs. These are each immunologically captured by the anti-HbA antibody (immobilized antibody layer 4). Other substances are absorbed by the residual liquid absorption layer 5.

No Hb is bound to the immobilized antibody layer 4.
A and HbA _1C bound to the dihydroxyboryl derivative are captured, but the amount of HbA is measured from the coloring of itself, and HbA _1C is obtained from the dye bound to the dihydroxyboryl derivative. In order to distinguish it from the coloring of HbA, it is desirable that the dye bound to the dihydroxyboryl derivative is on the long wavelength side. Here, the support, the spreading film, the hemolytic agent,
The modifier, the adsorbent, the absorbent and the developing solution are the same as those of the test tool B.

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 device of the present invention has no support,
Alternatively, it can be appropriately changed depending on the purpose of use such as partial use.

[0036]

EFFECTS OF THE INVENTION Since the present invention is a structure having the above structure, it is novel and practical to simultaneously measure glycosylated hemoglobin (HbA _IC ) and hemoglobin A (HbA) with a dry test tool. It is a high-tech test device that is easy to operate and can be used for household purposes.

[Brief description of drawings]

FIG. 1 is a plan view (a) and a sectional view (b) of a test device A. Since the cross-sectional views have the same structure except for 4 and 4 ',
Shown in one cross-section.

FIG. 2 is a plan view (C) and a cross-sectional view (D) of the test tool B. Since the cross-sectional views have the same structure except 6 and 6 ',
Shown in one cross-section.

FIG. 3 is a plan view (e) and a cross-sectional view (f) of the test tool C.

FIG. 4 is a plan view (G) and a cross-sectional view (H) of the test device D.

5A and 5B are a plan view and a cross-sectional view of the test tool E, respectively.

[Explanation of symbols]

 1 Support 2 Development Layer 3 Hemolysis Layer 4 Immobilized Antibody Layer (Antibody is anti-HbA In the case of antibody) 4'immobilized antibody layer (antibody is anti-HbA _1CIn the case of antibody 5 Absorption layer of residual liquid 6 Labeled substance layer (antibody used is anti-HbA In case of antibody 6 ′ labeling substance layer (antibody used is anti-HbA _1CPlace of antibody
7) Solid phase adsorption layer 8 Labeled substance layer (in the case of dihydroxyboryl derivative) S Blood sample E Starting point of development

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[Procedure amendment]

[Submission date] August 9, 1995

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 5

[Correction method] Change

[Correction content]

[Figure 5]

Claims (7)

[Claims] 1. A development starting point at which a development solution is applied to one or more development layers, which are held on a support and can transfer a blood sample by a capillary action by the development solution, and a hemolytic agent and / or denaturation. A hemolytic layer coated with an agent and a solid-phased antibody layer (or solid-phased adsorbent layer) for capturing a specific substance,
Alternatively, a dry test for a blood sample, which requires that a hemolysis layer that works in the same manner as above, a labeling substance layer, and a solid-phased antibody layer (or a solid-phased adsorbent layer) are installed in this order Ingredient 2. The dry test device according to claim 1, wherein the labeling substance layer contains a labeled antibody or a labeled boric acid derivative. 3. A hemolytic layer and an immobilized antibody layer are provided.
In the test tool of No. 1, the same hemolysis layer and
And, as the antibody immobilized on the immobilized adsorbent layer,
Anti-hemoglobin A (HbA) antibody layer and anti-hemoglobin respectively
Globin A _1C(HbA _1C) Using the antibody layer,
A hemoglobin with the same developed residual liquid absorption layer for each
Bins A and A_{I c}Dry test device for blood samples for simultaneous measurement. 4. A hemolytic layer, a labeled antibody layer and a solid-phased adsorbent
The test device according to claim 2, wherein layers are provided, and a pair of development layers are provided.
To the same hemolytic layer and labeled antibody layer, respectively,
Labeled anti-HbA antibody layer and labeled anti-HbA _1CAntibody layer and same
Solid-phase adsorbents of
Hemoglobin A and A, which are provided with a residual liquid absorption layer_{I c}
Dry test device for blood samples for simultaneous measurement. 5. A hemolytic layer and a solid phase antibody layer are provided.
In the test device of No. 1, one development layer and a solid-phased antibody layer
Then, immobilized anti-HbA _1CAntibody layer and immobilized anti-HbA Anti
The body layer is used, and one development residual liquid absorption layer is provided.
Hemoglobin A and A_{I c}Blood sample for simultaneous measurement
Dry test tool. 6. A hemolytic layer, a labeled antibody layer, and a solid-phased antibody layer
The test device according to claim 2, wherein one development layer is provided.
And a labeled anti-HbA antibody layer as a labeled antibody layer,
Immobilized anti-HbA as body layer _1CAntibody layer and immobilized anti-Hb
A antibody layer is used, and one development residual liquid absorption layer is further provided.
Hemoglobin A and A_{I c}Blood for simultaneous measurement
Dry test device for specimens. 7. The solid-phased adsorbent layer according to claim 1, wherein the method of capturing the specific substance or the labeled specific substance utilizes an ion exchange reaction or a boric acid derivative reaction. The dry test device for blood samples according to any one of 6 above.