KR101254512B1 - Immunochromatogaraphic test instrument and semiquantitative method using the same - Google Patents

Abstract

A novel test tool for easily semi-quantitating a substance to be included in a sample by using an immunochromatography method, and an immunochromatography test tool having a porous body in which a sample is moved by capillary phenomenon. The test instrument is (1) a region in which a labeled antibody or antigen is arranged to be movable in a state where it can specifically bind to a substance to be measured in a sample, and (2) an antibody that can specifically bind to the substance to be measured. Or a region in which an antigen is immobilized on the porous body, and (3) a region in which a standard color corresponding to a color at a specific concentration of the measurement target is immobilized on the porous body. A test tool for semi-quantifying a substance to be measured in a sample by comparing the color generated in the region 2 with the standard color; and A semiquantitative method using this test piece is provided.

Description

Test equipment for immunochromatography and semi-quantitative method using the same {IMMUNOCHROMATOGARAPHIC TEST INSTRUMENT AND SEMIQUANTITATIVE METHOD USING THE SAME}

The present invention relates to a test piece for semi-quantifying a substance to be measured in a sample using immunological chromatography (immune chromatography), and a semi-quantitative method using the test piece.

The immunochromatography test kit is for detecting anti-HBs antigens in serum samples, anti-treponemapallidum antibodies in serum or plasma samples, blood types, using blood or urine or other body fluids including serum or plasma. And detection of human chorionic gonadotropin (hCG) in urine specimens.

FIG. 5 is a conceptual diagram showing an example of a conventional test kit for immunochromatography, FIG. 5A is a plan view of the test kit for immunochromatography, and FIG. 5B is a side view thereof. Although FIG. 5C is the same drawing as FIG. 5A, the test tool for immunochromatography after use is shown.

In the test tool for immunochromatography 11 shown in FIGS. 5A and 5B, a sample pad 13 is formed on the sheet-like substrate 12. When the test tool 11 is used, the sample pad 13 is immersed in the sample to be measured. The sample absorbed by the sample pad 13 moves to the reagent pad 14 formed adjacent to the sample pad 13 by capillary phenomenon. The reagent pad 14 carries a labeled antibody or antigen.

When the sample absorbed in the sample pad 13 reaches the reagent pad 14, the labeled antibody or antigen carried on the reagent pad 14 is eluted. The measurement target in the sample and the labeled antibody or antigen may form a complex. The sample containing these moves to the porous body 15 formed adjacent to the reagent pad 14 by capillary phenomenon. The composite that has reached the porous body 15 moves in the downstream direction (when the sample pad 13 side is upstream in the drawing) by the capillary phenomenon.

On the porous body 15, a reaction region 16 to which an antibody or antigen is immobilized is formed. An antigen, an antibody, or the complex which has moved in the porous body 15 by the capillary phenomenon is captured in the reaction region 16. As shown in FIG. 5C, this capture can be confirmed by coloration 16 ′ in the area 16. In this way, the presence or absence of the measurement target substance in the sample can be visually confirmed.

Further downstream of the test tool 11, an absorbent pad 17 for absorbing other components in the specimen, a substance to be measured, etc. which is not captured is formed.

In addition, the test target for immunochromatography can be used to semi-quantify the substance to be measured in the sample. For example, a plurality of reaction regions 16 having different detection sensitivity with respect to the measurement target substance are formed, and the number of measurement target substances in the sample can be semi-quantified by the number of the regions 16 where the coloration 16 'is generated.

Patent Literatures 1 to 6 describe such an immunochromatography test tool and a method for detecting a substance to be measured in a sample or a semi-quantitative measure of the substance to be measured using the same.

Patent Literature 1 describes a method of measuring an antigen or an antibody in a liquid using a sheet-like test tool based on immunochromatography. However, there is no disclosure or suggestion regarding semi-quantification of antigens or antibodies in the document, and no disclosure or suggestion regarding a test piece having a region showing a standard color on a porous body.

Patent document 2 has description regarding the test tool for measuring the presence or absence of the antigen or antibody in a liquid using a pigment-labeled antigen or antibody. However, there is no disclosure or suggestion regarding semi-quantification of an antigen or an antibody in the document, and no disclosure or suggestion regarding a test instrument having a region showing a standard color on a porous body.

Patent document 3 has description regarding the test tool for quantifying the antigen or antibody in a sample solution. This test zone is characterized by having a plurality of reaction zones in which an increasing amount of capture reagent is immobilized from the upstream to the downstream, and quantifying the analyte by the number of the reaction zones developed. Although it is essential to have a plurality of reaction zones, it is not preferable to form a plurality of reaction zones because the manufacturing process is complicated. In addition, this test sphere does not disclose or suggest a test sphere having a region showing a standard color on the porous body.

Patent document 4 has description regarding the test tool for semi-quantifying the antigen or antibody in a sample solution. This test sphere has a plurality of reaction regions which are arranged in series with respect to the development direction of the particle-labeled antigen or antibody and the sample solution, and the amount of the antigen or antibody can be estimated semi-quantitatively by the principle of the sandwich method. There is a start. Although it is essential to have a plurality of reaction zones, it is not preferable to form a plurality of reaction zones because the manufacturing process is complicated. In addition, this test sphere does not disclose or suggest a test sphere having a region showing a standard color on the porous body.

Patent Document 5 uses an immunological chromat analysis specimen having a portion where a constant color tone appears on the same membrane and a portion where a concentration-dependent color tone appears, and a sample having an unknown concentration on the immunological chromat analysis piece. After the injection and chromatographic expansion, the color tone of the part where the constant color tone appears and the color tone of the part where the concentration-dependent color tone appears is measured by an optical density measuring device or visually. It is described. This method is characterized by the fact that the color region of the control and the color-dependent color region of the sample are on the same membrane, but the color region of the control is colored by using immobilized antibodies and labeling antibodies different from the sample. At the same time, it is not easy to adjust the color to correspond to the concentration of a particular sample.

Patent Literature 6 describes an immunochromatography test tool in which the type indication of a determination line is directly printed on a porous body in which a specimen moves due to capillary phenomenon. As described in Patent Document 6, there is no idea that printing is carried out on a porous body in which a specimen moves due to a capillary phenomenon, but prior to Patent Document 6, an indication useful for measuring the concentration of a substance to be measured is formed on the porous body. There was no idea that there is any conventional.

Patent Document 1: Japanese Patent Application Laid-Open No. H07-55808

Patent Document 2: Japanese Patent Application Laid-Open No. H10-73592

Patent Document 3: Japanese Patent Application Laid-Open No. H05-5743

Patent Document 4: JP H08-278305

Patent Document 5: Japanese Unexamined Patent Publication No. 2001-83153

Patent Document 6: Japanese Unexamined Patent Publication No. 2001-13143

Challenges to be solved by the invention

Accordingly, the present invention provides a novel test apparatus for easy production using an immunochromatography method, and a semi-quantitative method for easily semi-quantifying the concentration of a measurement target contained in a sample, and a semi-quantitative method using the test tool. It aims to do it.

Means for solving the problem

In order to achieve the above object, the present invention provides a test instrument for immunochromatography having a porous body in which a specimen is movable by capillary phenomenon,

(1) a region in which a labeled antibody or antigen capable of specifically binding to a substance to be measured in the sample is arranged in a movable state,

2) a region in which an antibody or antigen that can specifically bind to the measurement target substance is arranged in a fixed state on the porous body, and

(3) has a region in which a standard color corresponding to a color at a specific concentration of the measurement target material is arranged fixed on the porous body,

A test tool (hereinafter referred to as the "test tool of the present invention") for semi-quantifying the measurement target substance in the sample by comparing the color generated in the region 2 with the standard color is provided. do.

As a preferable aspect of the test tool of the present invention, a region in which an antibody or antigen capable of specifically binding to the measurement target material of (2) is disposed on the porous body, and the measurement of (3) above The test instrument in which a region in which a standard color corresponding to a color at a specific concentration of a target substance is fixed on the porous body is arranged in the series or parallel to the developing direction of the developing solvent is mentioned.

As another preferable form of the test tool of the present invention, the labeling pigment used in the region in which the labeled antibody or antigen is movable in a state where the labeled antibody or antigen which can specifically bind to the substance to be measured in the sample of (1) and And the test sphere wherein the standard coloring pigment used in the region where the standard coloring corresponding to the coloring at a specific concentration of the measurement target substance of the above (3) is fixed on the porous body is the same dye. have.

The present invention also provides a method for semi-quantifying a substance to be measured in a sample using the test tool of the present invention.

Effects of the Invention

The test sphere of the present invention compares the standard color immobilized on the porous body of the test chromatography for immunochromatography with the color generated by being captured by the antibody or antigen immobilized on the porous body, thereby being included in the sample. The material can be easily semi-quantified.

In addition, the test apparatus of the present invention corresponds to a region (reaction zone) in which an antibody or antigen that can specifically bind to a substance to be measured is immobilized on a porous body, and to color at a specific concentration of the substance to be measured. Since the standard color has a region arranged in a fixed state on the porous body, it is possible to compare the color of the reaction region with the standard color under the same conditions, thereby obtaining high precision.

In addition, since the test tool of the present invention has at least one reaction region and a region showing standard coloration, semi-quantification is possible. Therefore, the test tool of the present invention can be easily prepared as compared with a test tool having a plurality of reaction regions as essential requirements for semi-quantification. Can be. In addition, since the pigment or the like is directly fixed on the porous body without passing through antibodies, antigens, or the like for the region showing standard coloration, it is easier to prepare the pigments, etc., than the test strips that require fixing with antibodies, antigens, or the like. Can be. In addition, when using this test tool indirectly without a reaction such as an antigen antibody reaction, the color tone of the standard color is not affected at all by the difference in various conditions of use.

1 is a conceptual diagram of one embodiment of a test fixture of the present invention.

FIG. 2 is a side view of the test fixture of FIG. 1. FIG.

Fig. 3 is a graph showing the change over time of the degree of color intensity of standard color 8 and color 6 'after immersing the test sphere of the present invention in hCG adjusted to various concentrations.

4A is a plan view of the test tool used in the example, showing an unused test tool before being immersed in the urine sample.

4B is a plan view of the test tool used in the Example, showing the test tool after semi-quantification using a sample having a hCG concentration of 25 IU / L.

Fig. 4C is a plan view of the test tool used in the example, and shows the test tool after semi-quantification using a sample having a hCG concentration of 1000 IU / L.

FIG. 4D is a plan view of the test sphere used in the Example, and shows the test sphere after semi-quantification using a sample having a hCG concentration of 10000 IU / L.

Fig. 4E is a plan view of the test sphere used in the example, and shows the test sphere after semi-quantification using a sample having a hCG concentration of 0 IU / L (negative).

Fig. 5A is a conceptual diagram showing a test tool for a conventional immunochromatography method, and the test tool is shown by a plan view.

FIG. 5B is a side view of the test fixture shown in FIG. 5A. FIG.

Although FIG. 5C is the same view as FIG. 5A, the test tool after use is shown.

Explanation of symbols

1: Test Specimen 2: Gas

3: sample pad 4: reagent pad

5: porous body 6: reaction zone

6 ': color 7: absorption pad

8: Standard color: 9: Area

9 ': color 11: test sphere

12: gas 13: sample pad

14: reagent pad 15: porous body

16: reaction zone 16 ': color

17: absorbent pad 20: protective film

21: transparent part 22: window

23, 23 ', 23 ": opaque portion

EMBODIMENT OF THE INVENTION Hereinafter, the test tool of this invention is demonstrated with reference to drawings.

1 is a conceptual diagram of one embodiment of a test fixture of the present invention, and FIG. 2 is a side view of the test fixture shown in FIG. 1.

In the test tool 1 shown in FIGS. 1 and 2, a sample pad 3, a reagent pad 4, a porous body 5, and an absorption pad 7 are formed on a sheet-like substrate 2. In the test tool 1, the reagent pad 4, the porous body 5 and the absorption pad 7 are covered with a protective film 20.

Hereinafter, each component of the test tool 1 of this invention is demonstrated.

In the test tool 1 shown in FIG. 1, each component is formed on the sheet | seat base 2. The gas 2 is made of a material which does not absorb or penetrate the sample passing through each component in the immunochromatography method, and does not denature the sample when the sample comes into contact. Examples of such materials include plastic materials used in medical devices, such as vinyl chloride, polyethylene, ethylene vinyl acetate resin, polypropylene, polycarbonate, polyurethane, polystyrene, silicone resin, fluorocarbon resin, polyethylene terephthalate, Polyamide, ABS resin, etc. are mentioned. In addition, the base 2 may be made of paper which has been water-repellent oil-repellent processed on its surface.

The sample pad 3 is a site where a sample is dropped or immersed in the sample, and when the insoluble material is present in the sample, the sample pad 3 also functions as a filter for removing the insoluble material. From this point of view, the sample pad 3 is exemplified by a nonwoven fabric or porous membrane having a particle size of 20 µm to 100 µm, preferably 30 µm to 40 µm, but is not particularly limited thereto. As a material which can be used for the sample pad 3, if a certain amount of sample can be collected, it is hydrophilic, and it has a filter function, it will not specifically limit. Examples include regenerated cellulose, cellulose acetate, nitrocellulose, polyacrylonitrile, ethylene vinyl acetate, polyurethane, polymethyl methacrylate, nylon resin, glass fiber, pulp, cotton, rayon, acrylic, polyester and the like. In particular, it is not limited to these.

The sample dropped on the sample pad 3 is moved downstream by the capillary phenomenon, that is, the reagent pad 4. Hereinafter, in this specification, the side of the sample pad 3 of the test tool 1 shall be an upstream side, and the opposite side shall be a downstream side. In addition, when said sample dropped in the sample pad 3, when the sample pad 3 was immersed in the sample, the sample contained in the sample pad 3 also means.

As a material of the reagent pad 4, although a nonwoven fabric or a porous membrane is illustrated similarly to the sample pad 3, it is not specifically limited to these. The reagent pad 4 carries an antibody or antigen that can be specifically bound to the substance to be measured in the sample in a state labeled with a labeling material such as a dye.

Antibodies or antigens that can specifically bind to the measurement target are appropriately selected depending on the measurement target. When the substance to be measured is human chorionic gonadotropin (hCG) in the urine sample, an anti-hCC antibody, more specifically, an anti-hCC mouse polyclonal antibody, an anti-hCG mouse monoclonal antibody, or the like is used. . If the substance to be measured is alphafetoprotein (AFP), an anti-AFP antibody is used. When the substance to be measured is an anti-HIV antibody, an HIV antigen peptide or the like is used. When the substance to be measured is a prostate specific antigen (PSA), which is a semi-quantitative cancer marker, for example, an anti-human PSA rabbit antibody or the like is used.

The labeling substance is not particularly limited as long as it can be visually recognized. Examples of the labeling substance include metal colloids, non-metal colloids, dye particles such as dye sol or dispersion dyes, latex particles or colored fine particles represented by gold colloids. have. Among them, latex fine particles or polystyrene fine particles colored with dyes or pigments are preferable from the viewpoint of excellent color development, rich color tone and particle diameter, and plural labeling methods for antibodies or antigens. On the other hand, this labeling method is well known by those skilled in the art, and there are a method by adsorption, a method by chemical bonding, and the like. Particularly, spherical or nearly spherical latex fine particles or polystyrene fine particles having a particle size of 0.5 μm or less, preferably a particle size of 0.05 to 0.5 μm, colored with a dye or pigment are preferable. Most preferably blue latex.

The labeled antibody or antigen is carried on the reagent pad 4 in a movable state. Herein, the supported state in the movable state is that the labeled antibody or antigen is retained in the reagent pad 4 at the time of use, that is, when the reagent pad 4 is dried. When the reagent pad 4 is wetted by the arrival, it means that the labeled antibody or antigen becomes mobile. For this purpose, the reagent pad 4 must be a material which does not adsorb the labeled antibody or antigen. If the material is an adsorbent material, the reagent pad 4 can be used by masking it with an inert protein such as another BSA in advance. Thus, the labeled antibody or antigen is held in the reagent pad 4 by, for example, being impregnated with the reagent pad 4 and then lyophilized.

Therefore, in the test fixture 1, the reagent pad 4 forms a region in which a labeled antibody or antigen capable of specifically binding to a substance to be measured in the sample is disposed in a movable state.

The sample dropped on the sample pad 3 moves to the reagent pad 4 by capillary phenomenon. For this reason, it is preferable that the particle diameter of the reagent pad 4 is the same as or smaller than the sample pad 3. The particle diameter of the reagent pad 4 is 1 micrometer-50 micrometers, Preferably, they are 5 micrometers-30 micrometers.

In the test tool 1 shown in FIG. 1 and FIG. 2, the sample pad 3 and the reagent pad 4 are partly laminated | stacked in order to facilitate the movement of a sample by a capillary phenomenon. However, the sample pad 3 and the reagent pad 4 may simply be disposed adjacent to each other and may be the same pad as long as the sample can be properly moved by the capillary phenomenon.

When the sample reaches the reagent pad 4, the substance to be measured in the sample and the labeled antibody or antigen in the pad 4 move downstream, that is, to the porous body 5, by capillary phenomenon. In addition, when the substance to be measured and the labeled antigen or antibody are in a relationship between the antigen and the antibody, some or all of them form an antigen-antibody complex, which also moves downstream, that is, to the porous body 5. . In the test tool 1 shown in FIG. 1 and FIG. 2, in order to make this movement easy, the reagent pad 4 and the porous body 5 are partly laminated | stacked. However, if the sample can be properly moved by the capillary phenomenon, the reagent pad 4 and the porous body 5 may simply be disposed adjacent to each other, and the reagent pad 4 may also serve as the porous body 5. , Or vice versa.

The porous body 5 has a sponge-like pore or a mesh-like shape, and is a medium in which a specimen can be moved by a capillary phenomenon. In the test tool 1 shown to FIG. 1 and FIG. 2, it shows as a sheet-like porous film. The material of the porous body 5 is not particularly limited as long as it can carry out an immunochromatography method. Specifically, for example, cellulose, cellulose derivatives, nitrocellulose, ethylene vinyl acetate, polyurethane, polymethyl methacrylate and nylon resin And woven fabrics made of porous synthetic polymers such as polyvinylidene difluoride (PVDF), and the like, nonwoven fabrics and membranes, glass fiber filters, various kinds of materials, and the like. It is especially preferable to use nitrocellulose. As for the particle size of the porous body 5, 1 micrometer-30 micrometers are illustrated, Preferably they are 1 micrometer-20 micrometers.

The porous body 5 has a region 6 (reaction region) in which an antibody or antigen capable of specifically binding to a substance to be measured is fixed. Antibodies or antigens immobilized in the region 6 can be appropriately selected depending on the substance to be measured. Specifically, when the substance to be measured is hCG, anti-hCG antibodies, more specifically, anti-hCG mouse polyclonal antibodies, anti-hCG mouse monoclonal antibodies and the like can be used. If the substance to be measured is AFP, anti-AFP antibodies can be used. When the substance to be measured is an anti-HIV antibody, an HIV antigen peptide or the like can be used. When the substance to be measured is PSA, an anti-PSA mouse monoclonal antibody, an anti-PSA rabbit antibody, or the like can be used. In addition, these immobilization methods can be easily immobilized by directly applying and drying a solution of the above-described antibody or antigen when the porous body 5 is nitrocellulose, nylon or the like. In the case of a porous fiber such as a glass fiber filter or a filter which cannot be immobilized by direct application and drying, it can be easily immobilized by pretreatment by activation of the porous body, activation of an immobilized antibody or antigen, or the like. The immobilization method of is well known to those skilled in the art.

By the capillary phenomenon, the antigen, the antibody, and / or the complex which have moved in the porous body 5 are captured by the antibody or antigen immobilized in the region 6. When the antigen, antibody, and / or complex containing the label is captured, it can be identified by the color generated in the region 6. The color tone of the color generated in the region 6 here depends on the immunochromatography method (combination of reagents) used and the concentration of the substance to be measured in the sample. For example, in the case of capturing the complex formed by the antigen in the sample and the labeled antibody held in the reagent pad 4 with the immobilized antibody in the reaction region, if the concentration of the substance to be measured in the sample is high, the region ( Since the complex captured in 6) increases, a color tone of strong color is displayed, and when the concentration of the substance to be measured in the sample is low, the color film of weak color tone is displayed because the complex captured in the region 6 decreases. On the other hand, for example, in the competitive reaction between the antigen in the sample and the labeled antigen held in the reagent pad 4, when the labeled antigen is captured by the immobilized antibody in the reaction region, the concentration of the substance to be measured in the sample is measured. If is high, the labeled antigen captured in the area 6 decreases, so that a weak color tone appears. If the concentration of the substance to be measured in the sample is low, the labeled antigen captured in the area 6 increases. Produces the color of.

The porous body 5 has an area in which the standard color 8 corresponding to the color at a specific concentration of the substance to be measured is fixed. That is, in the test zone 1, when a sample containing a specific concentration (hereinafter referred to as "reference concentration") is used as the measurement target substance, an area having the same color tone as that of the color generated in the area 6 is used. It is formed on the porous body 5. In addition, the same immobilization method as that of the reaction zone can be used for the standard immobilization method 8.

In the test sphere of the present invention, the measurement target substance in the sample is easily obtained by comparing the color tone of the color generated in the region 6 existing on the porous body 5 of the test sphere 1 with the color tone of the standard coloration 8, respectively. Can be semiquantified. That is, for example, in the case of capturing the complex formed by the antigen in the sample and the labeled antibody held in the reagent pad 4 with the immobilized antibody in the reaction region, the color tone of the color generated in the region 6 is If it is weaker than the color tone of the standard color (8), the concentration of the substance to be measured in the sample is lower than the reference concentration. When the color tone of the color tone generated in the region 6 is stronger than that of the standard coloration 8, the concentration of the substance to be measured in the sample becomes higher than the reference concentration. When the color tone of the color generated in the region 6 is about the same as the color tone of the standard color 8, it is said that the concentration of the substance to be measured in the sample is about the same as the reference concentration. In addition, when no color is generated in the region 6, it means that the substance to be measured does not exist in the sample or is below the detection sensitivity. Therefore, the test tool 1 of the present invention can also perform qualitative evaluation at a specific sensitivity of the substance to be measured with or without coloration in the region 6.

In addition, by comparing the color tone of the color generated in the region 6 with the color tone of the standard color 8 using an optical density meter or the like, the concentration of the measurement target substance in the sample can be accurately measured.

Since the specimen moves inside the porous body 5 by the capillary phenomenon, it is preferable that the standard color 8 is fixed to the surface of the porous body 5. Herein, the standard color 8 is preferably a labeling material used to label the antibody or antigen carried on the reagent pad 4. Therefore, metal colloids, nonmetal colloids, dye particles such as dye sol or disperse dyes, latex particles, colored fine particles and the like represented by gold colloids are preferably exemplified. Among them, latex fine particles or polystyrene fine particles colored with dyes or pigments are preferable from the viewpoint of excellent color development, rich color tone and particle diameter, and a plurality of methods for labeling antibodies or antigens. In addition, this labeling method is well known by those skilled in the art, and there are a method by adsorption, a method by chemical bonding, and the like. Particularly preferred are spherical or nearly spherical latex fine particles or polystyrene fine particles having a particle size of 0.5 μm or less, preferably a particle diameter of 0.05 to 0.5 μm, colored with a dye or pigment. Most preferably blue latex.

However, the standard color 8 may be formed using other substances such as dyes, dyes, and pigments as long as the color tone is the same as the color tone at a specific concentration of the measurement target substance.

In addition, a region (reaction region 6) in which an antibody or antigen that can specifically bind to a substance to be measured is immobilized on the porous body (reaction region 6), and a standard corresponding to coloration at a specific concentration of the substance to be measured It is preferable that the area | region (standard coloration 8) arrange | positioned in the state which fixed color is fixed on the said porous body is arrange | positioned in series or parallel with respect to the development direction of a developing solvent, and it is most preferable to arrange in series. It is also preferable that both are located in the vicinity. Specifically, the distance between the two is preferably within 20 mm, more preferably within 10 mm.

The developing solvent is not particularly limited as long as it can be contained in a normal sample. For example water (including water contained in the sample); Saline solution; Organic solvents, such as acetone, etc. are mentioned.

The developing direction of the developing solvent is the direction from the sample pad 3 to the absorbent pad 7 in FIG. 1.

In addition, the labeling pigment used in the region (reagent pad 4) arranged in a state where the labeled antibody or antigen is movable in a state capable of specifically binding to the substance to be measured in the sample, and the specific concentration of the substance to be measured. It is preferable that the standard coloring pigment used for the area | region (standard coloring 8) arrange | positioned in the state in which the standard color corresponding to the coloration in the state is fixed on the said porous body is the same dye, and it is more preferable that it is a latex particle.

The standard concentration and the detection sensitivity represented by the standard color (8) can be appropriately selected depending on the kind of the substance to be measured and the purpose of the test fixture. For example, when the test fixture 1 measures the amount of hCG in the urine sample, the detection sensitivity is preferably 25 IU / L and the reference concentration is 1000 IU / L. When the detection sensitivity of hCG is 25 IU / L, pregnancy diagnosis can be performed by the presence or absence of color development of the region 6. If the reference concentration is 1000 IU / L, it is possible to evaluate the prognosis of ectopic pregnancy, oily acid, and chorionic tumor. As a result, based on the hCG concentration of 25 IU / L and 1000 IU / L, the substance to be measured in the sample can be semi-quantified in one measurement. For example, when measuring the amount of PSA in the blood, the detection sensitivity is preferably 1 ng / ml and the reference concentration is 4 ng / ml. When the detection sensitivity of PSA is set to 1 ng / ml, prostate enlargement can be diagnosed by the presence or absence of color development in the region 6, and when the reference concentration is 4 ng / ml, prostate cancer can be diagnosed. do. As a result, it is possible to quantify the PSA concentration in the sample based on the PSA concentrations of 1 ng / ml and 4 ng / ml in one operation.

As described above, part or all of the substance to be measured in the specimen moving in the porous body 5 is captured by the antibody or antigen immobilized in the region 6. On the other hand, the other components in the sample, the substance to be captured, and the like that are not captured move in the porous body 5 in the downstream direction. In the test tool 1 shown in FIGS. 1 and 2, an absorption pad 7 for absorbing these is formed. The absorbent pad 7 is not particularly limited as long as the absorbent pad 7 can absorb and retain the sample that has moved in the porous body 5. Therefore, the absorbent pad 7 can be widely used in the conventionally used for the purpose of absorbing a liquid such as filter paper, nonwoven fabric, porous membrane, and retaining therein. The absorbent pad 7 may contain a substance having absorbency and hygroscopicity such as silica gel.

As described above, when the measurement target material is not included in the sample or is less than the detection sensitivity even when it is included, coloration does not occur in the region 6, and the test fixture 1 is unchanged in appearance. . For this reason, it is preferable that the test tool 1 of this invention has a mechanism for confirming completion | finish of a test. In the test tool 1 shown to FIG. 1 and FIG. 2, the area | region 9 which produces color when a sample reaches | attains downstream from the area | region where the standard color 8 of the porous body 5 was fixed is formed. The transparent window 22 is formed in the part which covers the area | region 9 among the protective films 20 which coat | cover the porous body 5. 1, the parts 21 and 22 of the protective film 20 are transparent, and the parts 23, 23 ', and 23 "are opaque.

In the test tool 1 shown in FIG. 1 and FIG. 2, the area | region 9 and the window 22 comprise the mechanism which confirms completion | finish of a test. That is, when the sample which moved in the porous body 5 reaches the area | region 9, color development arises and this color development can be confirmed by the window 22. FIG. This confirms the end of the test.

For this purpose, various coloring agents are impregnated or immobilized in the region 9. As a colorant impregnated or immobilized in the region 9, a colorant developed by contact with a liquid sample of a predetermined pH, specifically, eosin B, phloxine B, benzo perpurine 4B, congo red, bromine phenol blue, Bromcresol green, 2,5-dinitrophenol, or a related substance thereof. The coloring agent may be colored by the moisture when the sample reaches the region 9.

In addition, a water-soluble pigment is impregnated so as to dissolve when the sample reaches an area upstream of the window 22 of the porous body 5, for example, in an area covered with the opaque portion 23 '. The pigment 22 which melts and moves may be confirmed by the window 22.

In the case of using a component that specifically reacts with a labeled antigen or antibody, for example, a labeled anti-hCG mouse monoclonal antibody, even if the anti-mouse antibody or the like is immobilized in the region (9), Can be achieved.

In addition, the antibody or antigen, avidin, or anti-keyhole hemocyanin antibody, which specifically reacts with the label antibody, is retained on the reagent pad 4 by retaining the label antibody, label biotin, keyhole hemocyanate, etc., which are not involved in the measurement. Can be achieved in the region (9).

As mentioned above, although the test tool of this invention was demonstrated in detail with reference to drawings, the test tool of this invention is not limited to the form of illustration. The test tool of the present invention can be widely used as a test tool for immunochromatography having a porous body in which a specimen can move by capillary phenomenon.

(1) a region in which a labeled antibody or antigen is arranged in a movable state that can specifically bind to a substance to be measured in the sample,

(2) a region in which an antibody or antigen that can specifically bind to the measurement target substance is arranged in a fixed state on the porous body, and

(3) A region in which a standard color corresponding to a color at a specific concentration of the substance to be measured is fixed on the porous body.

Therefore, the test tool of the present invention is composed of a porous body and the like as the test apparatus for immunochromatography described in Japanese Patent Application Laid-Open No. H01-503174, Japanese Patent Application Laid-Open No. H06-180320, and Japanese Patent Application Laid-open No. H06-160388. It may have a casing to receive the element. In addition, the protective film 20 illustrated in the drawings of the present invention is not essential to the test sphere of the present invention.

The shape of the reaction region and / or the standard color to be fixed on the porous body is not limited to the line shape as shown, but may be another shape. In addition, a plurality of reaction zones and / or standard colors may be fixed on the porous body, each corresponding to a color at a different concentration.

The test tool of the present invention can be applied to various biological samples derived from living organisms. Specific examples of such liquid samples include blood, plasma, serum, saliva, urine, fecal extract, tissue extract, and the like. As the measurement targets contained in these liquid samples, various proteins, peptides, complex proteins, lipids, complex fats, nucleic acids, lectins, sugar chains or compounds having the same, biotin, avidin, hormones, enzymes, enzyme substrates, which function as antigens or antibodies , Enzyme inhibitors, receptors, ligands, drugs, drug metabolites, derivatives of analytes, and more specifically, human chorionic gonadotropin (hCG), luteinizing hormone (LH), alphafetoprotein (AFP) , Cancerous fetal antigen (CEA), human placental lactogen (hPL), beta 2 microglobulin (β2m), ferritin, HBs antigen, estrogen, PSA and the like and antibodies to them are exemplified. In the present invention, antibodies, antigens, nucleic acids, avidins and biotin are preferably used, and antibodies or antigens are particularly preferable.

In addition, the test tool of the present invention can also be applied to a solution prepared by dissolving a standard product of a measurement target as exemplified above in physiological saline or the like.

Example

Hereinafter, the test tool of the present invention will be further described with reference to Examples.

In the present Example, the test tool 1 shown to FIG. 1 and FIG. 2 was used.

Each structure of the test fixture 1 is as follows.

Base (2): polyester, vertical 125 mm x 8 mm x 0.25 mm thick

Sample pad (3): A nonwoven fabric (Benley, manufactured by Asahi Kasei Co., Ltd.) of 30 mm long x 8 mm wide and 0.3 mm thick was adhered.

Reagent pad (4): Latex particles (particle size 0.3 μm) colored with a blue dye as a labeled antibody (labeled as a labeling substance) on a nonwoven fabric (glass fiber, manufactured by Millipore) having a length of 15 mm × 8 mm × 0.3 mm Lyophilized, filled with anti-hCG mouse monoclonal antibody bound to the particles) was used, which was adhered onto the substrate (2).

End portions of the sample pad 3 and the reagent pad 4 are laminated (for about 4 mm).

Porous body 5: A nitrocellulose film (length 25 mm x 8 mm x thickness 235 micrometers, particle size about 8 micrometers) was adhere | attached on the base | substrate 2.

The ends of the porous body 5 and the reagent pad 4 are laminated (for 2 mm type).

The monoclonal antibody was immobilized in the region 6 by applying 1.5 mg / ml anti-hCG mouse monoclonal antibody on the porous body 5 and drying.

Standard color (8): The same latex particles as those used as labeling in standard color (8) so that hCG at a concentration of 1000 IU / L (reference concentration) is the same color as the color when captured by the antibody in region (6) (Colored with blue dye) was fixed to the surface of the porous body 5.

As an indicator of completion of the test, Eosin B was impregnated as a color developer in the region 9 on the downstream side of the standard color 8 of the porous body 5 and dried.

Absorbent pad 7: A nonwoven fabric (glass fiber, manufactured by Whatman Co., Ltd.) having a length of 20 mm × 8 mm × 0.4 mm in thickness was adhered on the base 2.

Protective film: Except for the sample pad 3, a protective film (made of polyethylene terephthalate) having a transparent portion 21, 22 and an opaque portion 23, 23 ', 23' 'on the upper part of the test fixture 1 It was coated using.

Using this test fixture (1), the seminal quantitative measurement was carried out using the target substance as chorionic gonadotropin (hCG).

Immerse the sample pad (3) portion of the test fixture (1) in a solution diluted with physiological saline containing 0.1% BSA to contain hCG (manufactured by the National Institute of Pharmaceutical Food Hygiene) at various concentrations below. I was. Next, the test tool 1 was allowed to stand in the horizontal direction for about 3 minutes so that the protective film 20 turned upward. When the coloration 9 'clearly indicating that the reaction was completed in the window 22 occurred, the coloration 6' of the standard coloration 8 and the region 6 was measured with a densitometer.

Here, the samples used hCG concentrations of 0 IU / L (negative), 25 IU / L, 100 IU / L, 250 IU / L, 500 IU / L, 1000 IU / L and 10000 IU / L.

Below, the grade of the chromaticity of the color 6 'of the area | region 6 of the sample of each density | concentration is shown by the numerical value obtained by the densitometer.

Concentration (IU / L) Color intensity 0 10 25 240 100 1160 250 3400 500 5900 1000 10000 10000 17000

On the other hand, the value measured by the densitometer of the degree of color intensity of the standard color tone 8 was 9800.

Negative samples hardly developed, and color development also increased with increasing color and concentration at 25 IU / L, resulting in a color almost equivalent to standard color at 1000 IU / L. It was useful as a semiquantitative test.

Moreover, using this test tool 1, the presence or absence of the change of the color intensity over time was confirmed. That is, about 3 seconds was immersed in the sample which adjusted the density | concentration so that the sample pad 3 might be locked. Next, the test fixture 1 is left in the horizontal direction for about 3 to 30 minutes so that the protective film 20 is turned upward, and the standard color 8 of each time and the color 6 'of the region 6 are densito. Measured by meter.

Here, the sample used was that the hCG concentration of 0 IU / L (negative), 25 IU / L, 500 IU / L, 1000 IU / L and 10 ⁶ IU / L.

In FIG. 3 and the following table, the degree of color intensity is shown with the change with the numerical value obtained with the densitometer.

Standard color hCG concentration 0 IU / L 25 IU / L 500 IU / L 1000 IU / L 10 ⁶ IU / L 3 minutes 9,174 35 361 4,543 8,565 13,284 5 minutes 9,298 32 394 5,928 10,181 13,783 10 minutes 9,309 36 507 6,586 11,077 13,720 15 minutes 8,938 54 581 6,789 9,084 11,994 30 minutes 6,578 36 473 4,480 6,550 8,276

In addition, color intensity is the average value measured 3 times of each density. The numerical value of the standard color is an average value of 3 times each concentration (15 times in total).

With the change over time up to 30 minutes, the color development in the standard coloration 8 or coloration 6 'of the region 6 at each concentration shows a peak at 5 to 10 minutes after the start of the test, after which the porous body ( The color development decreased due to drying of the porous body 5 due to the decrease in the amount of the sample supplied to 5). Even after a period of time from the start of measurement, almost no color development occurred in the negative sample, color development at 25 IU / L, color development almost equivalent to standard coloration at 1000 IU / L, and a tendency to develop color stronger than standard color at higher concentrations did not change. It was useful as a semiquantitative test.

In addition, although the test results shown in Table 1, Table 2, and FIG. 3 were shown as numerical values as a result measured with a densitometer, in the visual observation, from the viewpoint of the presence or absence of coloration and the strength and weakness with standard coloration, The same result was obtained.

4 is a plan view showing the test fixture 1 before and after performing the semi-quantification. Fig. 4A shows an unused test piece before immersion in a sample, Fig. 4B shows a test piece after semi-quantification using a urine sample with a hCG concentration of 25 IU / L, and Fig. 4C shows a sample with an hCG concentration of 1000 IU / L. After the semi-quantitative test, FIG. 4D shows the test sample after the semi-quantification using a sample having an hCG concentration of 10000 IU / L, and FIG. The test tool after implementation is shown, respectively.

As can be seen from FIG. 4, the test tool 1 after the semi-quantification can be confirmed by the coloration 9 ′ generated in the window 22 (FIGS. 4B to E). In the example in which the negative sample was used, no coloration occurred in the region 6, and only the standard coloration 8 was confirmed in the transparent portion 21. On the other hand, in the sample containing hCG, the coloration 6 'which shows that the complex of hCG and the labeled antibody was captured by the antibody in the area | region 6 was confirmed. This coloration 6 'differed in color tone by the hCG concentration in the sample. Specifically, in the example in which the sample of 25 IU / L whose hCG concentration was lower than the reference concentration was used, the color tone 6 'was much weaker than the standard color S (Fig. 4B). In the example using a 1000 IU / L sample whose hCG concentration was equal to the reference concentration, the color tone of the color tone 6 'was almost the same as that of the standard color 8 (FIG. 4C). In the example in which the sample of 10000 IU / L whose hCG concentration was higher than the reference concentration was used, the color tone of the color tone 6 'was stronger than that of the standard color 8 (FIG. 4D).

The present invention relates to a test piece for semi-quantifying a substance to be measured in a sample using immunological chromatography (immune chromatography) and a semi-quantitative method using the test piece.

Claims (6)

A test instrument for immunochromatography having a porous body in which a specimen is movable by capillary phenomenon, The test sphere for immunochromatography, (1) a region in which a labeled antibody or antigen is disposed in a movable state, capable of specifically binding to human chorionic gonadotropin (hCG) in the sample, (2) a region in which an antibody or antigen capable of specifically binding to human chorionic gonadotropin (hCG) is immobilized on the porous body, and (3) has a region in which a standard color corresponding to coloration at a specific concentration of human chorionic gonadotropin (hCG) is arranged fixed on the porous body, A test tool for semi-quantifying the human chorionic gonadotropin (hCG) in the sample by comparing the color generated in the region (2) with the standard color. The method of claim 1, A region in which an antibody or antigen capable of specifically binding to the human chorionic gonadotropin (hCG) of (2) is arranged in a fixed state on the porous body, and the human chorionic gonadotropin of (3) A test piece in which a region in which a standard color corresponding to a color at a specific concentration of a stimulating hormone (hCG) is fixed on the porous body is arranged in series or in parallel with respect to the developing direction of the developing solvent. The method of claim 1, A labeling pigment for use in a region in which a labeled antibody or antigen is arranged in a movable state, capable of specifically binding to human chorionic gonadotropin (hCG) in the sample of (1), and (3) The standard color corresponding to the coloration at a specific concentration of the human chorionic gonadotropin (hCG) of the) is a pigment for the same standard color for use in the region arranged in a fixed state on the porous body. The test zone of claim 1, wherein the standard coloration is coloration at 1000 IU / L of human chorionic gonadotropin (hCG). A method of semi-quantifying human chorionic gonadotropin (hCG) in a sample using the test apparatus according to any one of claims 1 to 4. delete

Images