JP2003262637A - Inspection piece and inspection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inspection piece capable of detecting a substance to be inspected with high accuracy, and to provide an inspection method. <P>SOLUTION: The inspection piece contains a water-absorbing substrate 1 which is situated so as to be separated and to which a substance capable of being specifically bonded to the substance to be inspected is immobilized; a water-absorbing substrate 3 to be impregnated with a buffer for development; and a water-absorbing substrate 2 which contains a labeled substance, capable of being specifically bonded to the substance to be inspected and which is used to add a solution to be inspected. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a test piece and a test method capable of detecting a test substance simply, quickly and highly accurately.

[0002]

2. Description of the Related Art An immunochromatographic method is known as a method for rapidly and simply conducting an inspection of an in-vivo sample or the like. This method is generally carried out by adding a labeling substance for obtaining a detection signal on a test piece on which an antibody capable of specifically binding to a test substance is immobilized on an arbitrary region of the surface of a water-absorbing substrate, The test liquid containing the test substance is brought into contact with an antibody capable of specifically binding to, and the test substance in the test liquid is detected by the immune complex formed on the test piece. For example, the following steps are taken. A test solution and a labeled antibody capable of binding to the test substance in the test liquid are added dropwise to one end of a test piece having a solid phase portion on which an antibody capable of binding to the test substance is immobilized on a water-absorbent substrate. Absorb and develop the mixture. Then, the labeled antibody-test substance complex formed by mixing binds to the immobilized antibody in the solid phase portion. By measuring the labeled antibody bound in the solid phase part, the test substance in the test liquid can be measured.

Further, the immunochromatographic method is not limited to qualitative measurement for determining the presence or absence of a test substance, but it is also possible to measure the amount of signal such as coloration and luminescence obtained from the labeling substance, which appears in the solid phase. Quantitative measurement of the test substance contained in the test liquid is also possible.

However, in the above method, in the step of dropping the test solution onto the test piece made of the water-absorbent substrate and developing the mixture with the labeled antibody, the test solution is dropped using a pipette or a dropper. The reactivity of the solid phase antibody is not constant due to variations in the dropping amount, or variations in the mixing state and concentration distribution of the labeled antibody and the test solution, and as a result, it is contained. It becomes difficult to obtain a signal amount according to the amount of the test substance.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a test piece and a test method capable of detecting a test substance with high accuracy.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that in a test piece used for immunochromatography or the like, a substance capable of specifically binding to a test substance ( Hereinafter, also referred to as “first binding substance”) and the water-absorbent substrate 1 on which the immobilization buffer solution is impregnated and the water-absorbent substrate 3 impregnated with the developing buffer are positioned separately to absorb the test liquid. ,
A water-absorbing substrate 2 containing a labeled substance capable of specifically binding to a test substance (hereinafter also referred to as “labeled second binding substance”) is provided between the water-absorbing substrate 1 and the water-absorbing substrate 3. It has been found that a detection signal corresponding to the amount of the test substance can be obtained by contact crosslinking. That is, when the test liquid is dropped onto the water-absorbent substrate 2 containing the labeled second binding substance to be absorbed and saturated, a fixed amount of the test liquid is contained, and the test liquid and the labeled first The two-binding substance is in a state of being uniformly mixed. When the water-absorbing substrate 2 on which the first binding substance is immobilized and the water-absorbing substrate 3 impregnated with the developing buffer solution are contact-crosslinked by the water-absorbing substrate 2, liquid transfer starts at that point. Then, the test liquid and the labeled second binding substance pass through the region (solid phase portion) of the water-absorbent substrate 1 on which the first binding substance is immobilized in a constant mixed state or concentration distribution to allow a reaction for complex formation. As a result, a signal corresponding to the amount of the test substance is obtained with high accuracy. That is, the present invention is as follows.

(1) Separately positioned water-absorbent substrate 1 on which a substance capable of specifically binding to a test substance is immobilized, water-absorbent substrate 3 for impregnating a developing buffer solution, and test substance A test piece containing a water-absorbent substrate 2 for adding a test liquid containing a labeled substance capable of specifically binding with. (2) The test piece according to (1), wherein the labeling substance of the labeled substance capable of specifically binding to the test substance is a dye-colored water-dispersible polymer or gold colloid. (3) The test piece according to (1) or (2) above, wherein a labeled substance capable of specifically binding to a test substance is dry-immobilized on the water-absorbent substrate 2. (4) The test piece according to any one of (1) to (3) above, wherein a blood cell separation membrane is installed on the water-absorbent substrate 2. (5) The substance capable of specifically binding to the test substance is an immunochemical component, and the labeled substance capable of specifically binding to the test substance is a labeled immunochemical component, (1) ) ~
The inspection piece according to any one of (4). (6) The test piece according to any one of (1) to (5) above, wherein the water-absorbent substrate 2 is made to absorb a test liquid and then impregnated with a developing buffer solution. A method for inspecting a test substance in a test liquid, which comprises contact-crosslinking the water-absorbent substrate 1 with the water-absorbent substrate 2.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail below. The test substance that can be detected by the test strip or the test method of the present invention is the first based on biological affinity such as immunochemical reaction (antigen-antibody reaction) and ligand-receptor binding.
There is no particular limitation as long as it can form a complex by binding to the binding substance and the labeled second binding substance, for example, an antigen,
Examples thereof include antibodies, hormones, hormone receptors, lectins, lectin-binding carbohydrates, drugs or metabolites thereof, drug receptors, nucleic acids and fragments thereof, but those capable of forming an immune complex by an antigen-antibody reaction are preferable. It can be widely applied to test substances that are measured by sandwich immunoassay, competitive immunoassay, and the like. For example, microorganisms such as bacteria (especially Escherichia coli O157, pathogenic bacteria such as Salmonella, Listeria, etc.), actinomycetes, yeasts, fungi, viruses (especially HIV, HBV, HCV, etc.) or antibodies thereto, or tumor marker antigen, phase reaction And substances in biological samples for the purpose of clinical diagnosis, such as active substances and inflammatory marker antigens (especially C-reactive protein).

The test liquid containing the test substance is, for example, a solution extracted from food, its culture supernatant, fecal suspension, plasma,
Examples include liquid samples such as serum and urine, and diluents prepared by diluting these with an appropriate buffer solution.

The test piece of the present invention comprises a water-absorbing substrate 1 on which a first binding substance is immobilized, a water-absorbing substrate 2 containing a labeled second binding substance for adding a test liquid, and a developing buffer solution. It is essential to include the water absorbent base material 3 for impregnation. In addition, in the test piece of the present invention, it is essential that the water-absorbent substrate 1 and the water-absorbent substrate 3 are separately located at an interval such that they can be contact-crosslinked by the water-absorbent substrate 2. In the present specification, the water-absorbent substrate 1 refers to a water-absorbent substrate for immobilizing the first binding substance, and the water-absorbent substrate 2 is for adding a test liquid containing a labeled second binding substance. This refers to a water-absorbent substrate, and the water-absorbent substrate 3 is
A water-absorbent substrate for impregnating with a developing buffer solution.

The first binding substance immobilized on the water-absorbing substrate 1 and the labeled second binding substance contained on the water-absorbing substrate 2 are both biologically compatible with antigen-antibody reaction, ligand-receptor binding and the like. There is no particular limitation as long as it is a substance that can specifically bind to the test substance based on its sex, but a substance that can specifically bind to the test substance by an antigen-antibody reaction (herein, “immunochemical component”) Is preferred (hereinafter, the first binding substance based on the antigen-antibody reaction is the "first immunochemical component" and the labeled second binding substance is the "labeled second immunochemical component".
Also called. ). For example, when the test substance is an antigen (eg, protein, peptide, hapten, etc.), the first immunochemical component and the labeled second immunochemical component are antibodies that can specifically bind to the antigen. The antibody may be a polyclonal antibody or a monoclonal antibody.
The antibody in the present invention means an antibody fragment having a specific affinity with a test substance, such as H chain, L chain, Fa.
b, F (ab ′) ₂ , V _H , V _{L and the} like are also included.
On the other hand, when the test substance is an antibody, the first immunochemical component and the labeled second immunochemical component bind to the antigen that can specifically bind to the antibody or specifically bind to the antibody as the antigen. Secondary antibody. In addition, the antigen in the present invention is meant to include an antigen fragment having an epitope portion that can be recognized and bound by a test antibody.

The water-absorbent substrate (water-absorbent substrate 1, water-absorbent substrate 2, water-absorbent substrate 3) can absorb the test liquid containing the test substance and the liquid containing the labeled second binding substance. There is no particular limitation as long as it is one. In the present invention, the water-absorbent substrate capable of ensuring a sufficient time for the test substance in the test liquid to sufficiently react with the labeled second binding substance and the first binding substance immobilized on the solid phase part. Is preferably used.

If the water-absorbent substrate is poor in water-absorbing property, it takes a long time for the test substance to pass through the solid phase portion, and as a result, rapid measurement cannot be performed. On the other hand, when the water absorbency of the water-absorbent substrate is too high, the time required for the test substance in the test liquid to sufficiently react with the labeled second binding substance and the first binding substance of the solid phase part It is difficult to make accurate measurements due to lack of

Therefore, the preferable degree of water absorption of the water-absorbent substrate is, for example, that one end of the water-absorbent substrate cut into a strip shape having a width of 5 mm is immersed in water, and the water-absorption distance after one minute elapses from 0. It is about 5 to 5 cm.

Preferred specific examples of the water-absorbent substrate include, for example, non-woven fabric, filter paper, glass fiber cloth, glass filter, nitrocellulose filter (nitrocellulose membrane), and porous materials such as nylon. The pore size of the porous material is, for example, 0.1 to 15 μm, preferably 1 to 12 μm. The thickness of the water-absorbent substrate is, for example, 0.05 to 2 mm, preferably 0.1 to 1 mm.

Further, in order to adjust the water absorption of these water-absorbent substrates, the surface of the substrate may be coated with or impregnated with a hydrophilic polymer or a surfactant. Further, as the water-absorbent substrate 1, the water-absorbent substrate 2, and the water-absorbent substrate 3, substrates made of the same material may be used, or substrates made of different materials may be selected and used.

The method for immobilizing the first binding substance on the water-absorbent substrate 1 is not particularly limited, but it is preferable to use the conventionally known physical adsorption method or covalent binding method. When the water-absorbent substrate 1 does not have a functional group for the covalent bonding method, for example, a polymer having an appropriate functional group may be used to attach the substrate to such an extent that the water-absorbing property of the substrate is not impaired. Alternatively, the solid phase part can be prepared by applying a solution containing the first binding substance and the hydrophilic polymer to the water-absorbent substrate 1 and then immersing the solution in a coagulating solvent for coagulating the hydrophilic polymer. As the hydrophilic polymer, for example, hydroxypropylmethyl cellulose, polyvinyl alcohol, hydroxyethyl cellulose or the like can be used. Further, as the coagulation solvent, for example, acetone, ethanol, methanol, ether or the like can be used.

The amount of the first binding substance immobilized on the water-absorbent substrate 1 depends on the type of the substance and the like, but is usually the water-absorbent substrate 1.
It is 1 to 250 μg / cm ² .

The labeled second binding substance is obtained by binding the labeling substance to a substance capable of specifically binding to the test substance. The labeling substance used here may be any labeling substance conventionally used in a chemical assay method, for example, an enzyme (alkaline phosphatase, peroxidase, etc.), a fluorescent substance (FITC (fluorescein isothiocyanate), rhodamine, etc.). ), Phosphors, dyes, etc. are used. In addition, a colloidal metal, or a water-dispersible polymer having an enzyme, a fluorescent substance, a phosphorescent substance, a dye or the like bound or contained therein is used.

In the present invention, colored particles such as colloidal metal or water-dispersible polymer colored with a dye such as a dye or a pigment are preferably used as the labeling substance for the purpose of rapid detection. As the colloidal metal, for example,
Colloids composed of metals such as gold, silver and copper are mentioned, but gold colloids are preferable. Examples of dyes such as dyes and pigments for coloring the water-dispersible polymer include Sudan blue, oil blue, Sudan red IV, Sudan III, oil orange, and quinizarin green. Examples of the water-dispersible polymer include polystyrene latex particles and polymethacrylic acid particles.

The average particle size of the colloidal metal and the colored particles is particularly limited as long as the color development is good at the time of detection and the mobility in the water-absorbent substrate is not reduced. However, in view of storage stability and easy preparation, the range of 0.01 to 3 μm is preferable, and the range of 0.05 to 0.5 μm is more preferable. If the particle size is too small, the degree of coloring per particle is low, so that the degree of color development is low even when bound to the solid phase portion, and the visual confirmation property is poor. On the other hand, if the particle size is too large, the water-absorbent substrate may be clogged with a slight amount of colloidal metal or colored particles to reduce the water absorption, or non-specific coloring may occur. . In the present specification, the average particle size was measured by a laser diffraction / scattering type particle size distribution measuring device.

As a method for labeling the second binding substance with such a labeling substance, a conventionally known method, for example, a covalent binding method, a physical adsorption method, an ionic binding method or the like can be used. The covalent bond method is more preferably used because it is stable without desorption of the labeling substance from the second binding substance such as.

The method of incorporating the labeled second binding substance into the water-absorbent substrate 2 is not particularly limited, and a conventionally known method can be used. For example, there is a method in which a solution of the labeled second binding substance is applied to the water-absorbent substrate 2 and then dried and immobilized under appropriate conditions. As a uniform state of drying, after applying to a water-absorbent substrate, it is dried by a wind of 20 to 60 ° C.,
Alternatively, it can be lyophilized. As another method, there can be mentioned a method in which the labeled second binding substance is dispersed in a solution of a water-soluble polymer or sucrose, and the dispersion is applied to the water-absorbent substrate 2 and then dried.

The labeled second binding substance is preferably contained in the water-absorbent substrate 2 after being dried and immobilized. The labeled second binding substance dried and immobilized on the water-absorbent substrate 2 can be detached from the water-absorbent substrate by contacting the test liquid dropped on the water-absorbent substrate.

The content of the labeled second binding substance in the water-absorbent substrate 2 depends on the type of the substance and the like, but is usually 0.01 to 2.5 μg with respect to the water-absorbent substrate 2.

The shape of the test piece is not particularly limited as long as the test liquid or the like can be spread in the state where the water-absorbent substrate 3 and the water-absorbent substrate 1 are contact-crosslinked with the water-absorbent substrate 2, and for example, Examples include strips.

The test piece includes a water-absorbent substrate 1, a water-absorbent substrate 2,
Other members may be provided in addition to the water absorbent substrate 3. When the test liquid contains insoluble substances such as blood and stool suspension,
The water-absorbent substrate 2 is preferably provided with insoluble matter separating means such as a blood cell separation membrane. The blood cell separation membrane is not particularly limited, but a commercially available one (for example, manufactured by Spectral)
SO904G / 99D, Hemarsep V manufactured by Nippon Pole, etc.) can also be used. The water-absorbent substrate provided with such a blood cell separation membrane can be obtained, for example, by bonding the water-absorbent substrate and the blood cell separation membrane by any means.

The test method using the test piece of the present invention is a water-absorbent substrate in which the test liquid is absorbed in the water-absorbent substrate 2 containing the labeled second binding substance and then impregnated with the developing buffer solution. This is performed by contact-crosslinking the water-absorbent substrate 1 on which 3 and the first binding substance are immobilized with the water-absorbent substrate 2. By the contact crosslinking, the mixed solution of the test liquid and the labeled second binding substance is developed on the test piece and passes through the solid phase portion on which the first binding substance is immobilized to form a complex. Detect the signal originating from the substance. Among such inspection methods, the immunochromatography method in which the first binding substance is the first immunochemical component and the labeled second binding substance is the labeled second immunochemical component is preferable.

Examples of the developing buffer include phosphate buffer, borate buffer, Tris-HCl buffer and the like. The amount of the developing buffer solution impregnated into the water-absorbent substrate 3 may be any amount capable of developing the test substance and the like, and is usually 30 to 200 μl, and preferably 50 to 150 μl with respect to the water absorbent substrate 3. .

The amount of the test liquid added to the water-absorbent substrate 2 is an amount capable of saturating the water-absorbent substrate 2 and depends on the material, size, etc. of the water-absorbent substrate 2. It is 5 to 50 μl, and preferably 10 to 30 μl with respect to the permeable substrate 2.

For the detection of the labeling substance in the solid phase portion, when the labeling substance is an enzyme, a fluorescent substance, or a phosphorescent substance, the detection means conventionally used in EIA or the fluorescent antibody method (FIA) is appropriately selected. When the labeling substance is a colloidal metal or colored particles, a means such as a spectrophotometer (for example, a chromatoscanner (manufactured by Shimadzu Corporation, model number CS-900)) or an image analysis using a CCD camera may be appropriately selected.

[0032]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

Example C Detection of C-reactive protein (CRP) 1) Preparation of test piece with immobilized first antibody (first binding substance) Nitrocellulose membrane (pore size 8 μm, 6 mm × 50 mm)
An anti-human CRP antibody (DAKO, rabbit IgG, 1 mg / ml, 0.1 M-phosphate buffer solution) is placed 25 mm from one end of (water-absorbent substrate 1).
pH7.4) was applied in a line shape using 1.5 μl of a dispenser. This membrane was immersed in an aqueous solution consisting of bovine serum albumin (1% by weight) and polyoxyethylene (10) octylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd., 0.1% by weight) for 10 minutes and then at 40 ° C for 2 hours. Dried. Then, a polyester film (thickness of 100 μm) was attached to the back side of this membrane (the side opposite to the antibody-coated surface) using spray glue.

2) Preparation of labeled second antibody (labeled second binding substance) Blue colored carboxylated polystyrene latex particle dispersion (solid content concentration 5% by weight, average particle diameter 0.1 μm, 0.01 M-
Water-soluble carbodiimide (1mg / in 3ml of borate buffer pH8)
ml, 0.01 M-borate buffer pH 8) 1 ml and anti-human CRP antibody (DAKO, rabbit IgG, 1 mg / ml, 0.01 M-borate buffer p
H8) 1 ml was added and reacted at 10 ° C for 3 hours, followed by centrifugation and washing using a borate buffer (pH 8) as a washing solution,
A blue-colored latex particle-labeled anti-human CRP antibody was prepared. Next, the latex particle-labeled antibody was suspended in 0.01 M-borate buffer (pH 8) so that the solid content concentration was 2% by weight.

3) Preparation of test sample for test liquid drop containing labeled secondary antibody Nitrocellulose membrane (Whatman, pore size 12 μm
m, 6 mm x 10 mm) (water-absorbent substrate 2) in an aqueous solution of bovine serum albumin (1% by weight) and polyoxyethylene (10) octylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd., 0.1% by weight) After soaking for 1 minute, it was dried at 40 ° C. for 2 hours. Next, the labeled secondary antibody prepared in 2) above was dispersed in a solution containing 1% sucrose so that the final concentration was 0.1%, and 1 μl of the solution was spotted on the center of the test piece at 40 ° C. It was dried for 2 hours and fixed. Then, a polyester film (25 μm
(Thickness) was adhered to the place of both ends 0 to 3 mm of the test piece by using a spray glue. Furthermore, on the bottom surface, 2
A polyester film (thickness of 25 μm) was attached to a place of ~ 8 mm. Then, a polyester non-woven fabric (6 mm × 4 mm, thickness 2.5 mm) was placed on the exposed surface of the membrane on the upper surface of 3 to 7 mm to prepare a member containing the labeled second antibody for dropping the test liquid.

4) Preparation of test piece for impregnation with buffer solution for deployment Nitrocellulose membrane (Whatman, pore size 12μ
m, 6 mm x 10 mm) (water-absorbent substrate 3) in an aqueous solution of bovine serum albumin (1% by weight) and polyoxyethylene (10) octylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd., 0.1% by weight) After soaking for 1 minute, it was dried at 40 ° C. for 2 hours. Next, a polyester film (100 μm thick) was attached to one side of this membrane using spray glue. On the opposite side of the film, the polyester non-woven fabric (6m
m × 6 mm, thickness 2.5 mm) were bonded to each other to prepare a test piece for impregnating with the developing buffer solution.

5) Measurement The member of the test piece prepared in 1), 3) and 4) above is shown in FIG.
In combination with the form shown in (a), the test piece of the present invention,
The measurement was performed as shown in the order of FIGS. 0.1M
A test solution was prepared by dissolving human CRP (manufactured by Nippon Biotest Co., Ltd.) as a test sample at an arbitrary concentration in a phosphate buffer solution (containing 0.9% by weight NaCl, pH 7.4). As a developing buffer
0.1M phosphate buffer (containing 0.9 wt% NaCl, pH 7.4)
60μl on the polyester nonwoven fabric part of the test piece prepared in 4)
It was dripped and impregnated. Next, 20 μl of the test liquid of each concentration was dropped on the polyester non-woven fabric of the test piece member prepared in 3), absorbed by the water-absorbing base material containing the labeled second antibody at the bottom until it was saturated, and dried and fixed. The labeled second antibody was detached and mixed with the test solution. Next, the test piece having the antibody solid phase portion prepared in 1) and the test piece containing the developing buffer prepared in 4), which are separated and arranged at an interval of 6 mm, are attached to the labeled second antibody described above. And contact crosslinking using a test piece containing a mixture of the test liquid and the test liquid. Upon contact, liquid transfer to the direction of the test piece having the solid phase portion of the antibody is started, and when the mixed solution of the test liquid and the labeled second antibody passes through the solid phase portion on which the first antibody is immobilized, it is complexed. The body is formed and a detection signal derived from the labeling substance is generated. After 10 minutes of development, this detection signal was sent to a chromatoscanner (Shimadzu Corporation, model number CS-9
00) was used for quantitative measurement.

Comparative Example Detection of C-reactive protein (CRP) 1) Preparation of first antibody-immobilized test piece Nitrocellulose membrane (pore size 8 μm, 6 mm × 60 mm)
1.5μ of anti-human CRP antibody (DAKO, rabbit IgG, 1mg / ml, 0.1M-phosphate buffer pH7.4) at 25mm from the upper end of
l, was applied in a line using a dispenser. This membrane was immersed in an aqueous solution consisting of bovine serum albumin (1% by weight) and polyoxyethylene (10) octylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd., 0.1% by weight) for 10 minutes and then at 40 ° C for 2 hours. Dried. Then, a polyester film (thickness of 100 μm) was attached to the back side of the membrane (the side opposite to the antibody-coated surface) using a spray glue.

2) Preparation of labeled secondary antibody Blue-colored carboxylated polystyrene latex particle dispersion (solid content concentration 5% by weight, average particle diameter 0.1 μm, 0.01 M-
Water-soluble carbodiimide (1mg / in 3ml of borate buffer pH8)
ml, 0.01 M-borate buffer pH8) 1 ml and anti-human CRP antibody (DAKO, rabbit IgG) 1 mg / ml, 0.01 M-borate buffer p
H8) 1 ml was added and reacted at 10 ° C for 3 hours, followed by centrifugation and washing using a borate buffer (pH 8) as a washing solution,
A blue-colored latex particle-labeled anti-human CRP antibody was prepared. Next, the latex particle-labeled antibody was suspended in 0.01 M-borate buffer (pH 8) so that the solid content concentration was 2% by weight.

3) Preparation of lower part of test liquid drop containing labeled second antibody In the part 50 mm from the upper end of the test piece prepared in 1), the above 2)
The labeled secondary antibody prepared in step 1 was dispersed in a solution containing 1% sucrose to a final concentration of 0.1%, and
It was spotted and dried at 40 degrees for 2 hours. Next, a polyester non-woven fabric (6 mm × 4 mm, thickness 2.5 mm) was placed at a location (47 to 53 mm from the upper end) where the labeled second antibody was immobilized, and a lower portion of the test liquid droplet was prepared.

4) Preparation of lower part of developing buffer droplets A polyester non-woven fabric (6 mm × 6 mm, thickness 2.5 mm) was attached to the lower end portion of the test piece prepared in 3) at a position of 0 to 6 mm, and Parts were produced.

5) Measurement Using the test piece shown in FIG. 2 (a) prepared above as an inspection piece, measurement was performed in the order shown in FIGS. 2 (b) to (d). 0.1M phosphate buffer (containing 0.9% by weight NaCl, pH 7.4)
In addition, human CRP (manufactured by Japan Biotest) as a test sample
A test liquid in which was dissolved at an arbitrary concentration was prepared. 20 μl of the test liquid of each concentration was dropped on the polyester nonwoven fabric under the test liquid droplet prepared in 3), and the water-absorbent substrate containing the labeled second antibody in the lower portion was absorbed and dried and fixed to the labeled second liquid. The antibody was released and mixed with the test solution. Then, 0.1M phosphate buffer (containing 0.9 wt% NaCl, p
60 μl of H7.4) was dropped onto the polyester non-woven fabric portion of the dropping portion prepared in 4) and developed. The mixed solution of the test liquid and the labeled second antibody forms a complex when passing through the solid phase portion on which the first antibody is immobilized, and generates a detection signal derived from the labeling substance. Ten minutes after development, this detection signal was quantitatively measured using a chromatoscanner (Shimadzu Corporation, model number CS-900).

Results (CV indicates a coefficient of variation.) Example (n = 10) CV 10% or less Comparative example (n = 10) CV 10% or more In the example, there is little variation in quantitative measurement values and the target test substance is highly accurate. Was able to be detected.

[0044]

According to the test piece and the test method of the present invention, the water-absorbent substrate 1 on which the first binding substance is immobilized and the water-absorbent substrate 3 impregnated with the developing buffer are separated and positioned. A water-absorbent substrate 2 containing a labeled second binding substance, which has absorbed a test liquid
By contact-crosslinking between the water-absorbent substrate 1 and the water-absorbent substrate 3, the detection signal corresponding to the amount of the test substance can be obtained. That is, the test piece of the present invention contains a fixed amount of the test liquid when the test liquid is dropped onto the water-absorbent substrate 2 containing the labeled second binding substance to be absorbed and saturated, and Then, the test liquid and the labeled second binding substance are uniformly mixed. When the water-absorbing substrate 2 on which the first binding substance is immobilized and the water-absorbing substrate 3 impregnated with the developing buffer solution are contact-crosslinked by the water-absorbing substrate 2, liquid transfer starts at that point. Then, the test liquid and the labeled second binding substance pass through the solid phase portion of the water-absorbent substrate 1 in a constant mixed state or concentration distribution to cause a reaction for complex formation, and as a result, depending on the amount of the test substance. Signal can be obtained with high accuracy.

[Brief description of drawings]

FIG. 1 is a cross-sectional view showing a structure of an inspection piece of an embodiment, showing a developing direction.

FIG. 2 is a cross-sectional view showing a configuration of a test piece of a comparative example, showing a developing direction.

[Explanation of symbols]

1 Water-absorbent substrate 1 2 Water-absorbent substrate 2 3 Water-absorbent substrate 3

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Quantum Asai             1-2 1-2 Shimohozumi, Ibaraki City, Osaka Prefecture Nitto             Electric Works Co., Ltd.

Claims (6)

[Claims] 1. A water-absorbent substrate 1 on which a substance capable of specifically binding to a test substance is immobilized separately, a water-absorbent substrate 3 for impregnating a developing buffer, and a test substance. A test piece containing a water-absorbent substrate 2 for adding a test liquid containing a labeled substance capable of specifically binding. 2. The test piece according to claim 1, wherein the labeling substance of the labeled substance capable of specifically binding to the test substance is a dye-colored water-dispersible polymer or gold colloid. 3. The test piece according to claim 1, wherein a labeled substance capable of specifically binding to a test substance is dry-immobilized on the water-absorbent substrate 2. 4. A blood cell separation membrane is provided on the water-absorbent substrate 2,
The test piece according to claim 1. 5. A substance capable of specifically binding to a test substance is an immunochemical component, and a labeled substance capable of specifically binding to a test substance is a labeled immunochemical component. Item 5. The inspection piece according to any one of Items 1 to 4. 6. The test piece according to any one of claims 1 to 5, wherein the water-absorbent substrate 2 is made to absorb a test liquid and then is impregnated with a developing buffer solution, and water-absorbent substrate 3. A method for inspecting a test substance in a test liquid, which comprises subjecting a hydrophilic substrate 1 to contact crosslinking with a water-absorbent substrate 2.