JP4988546B2 - Immunochromatographic test device and semi-quantitative method using the same - Google Patents

Description

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

  The immunochromatographic test device uses serum or plasma-containing blood, urine and other body fluids as samples, detection of anti-HBs antigens in serum samples, detection of anti-Treponema paridum antibodies in serum or plasma samples, determination of blood type It is used for detecting human chorionic gonadotropin (hCG) in urine samples.

  FIG. 5 is a conceptual diagram showing an example of a conventional immunochromatographic test device, FIG. 5 (a) is a plan view of the immunochromatographic test device, and FIG. 5 (b) is a side view thereof. FIG. 5 (c) is a view similar to FIG. 5 (a), but shows the immunochromatographic test device after use.

  In the immunochromatographic test device 11 shown in FIGS. 5A and 5B, a sample pad 13 is provided on a sheet-like substrate 12. When the test tool 11 is used, the sample pad 13 is immersed in a specimen to be measured. The specimen absorbed by the sample pad 13 moves to a reagent pad 14 provided adjacent to the sample pad 13 by capillary action. 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 substance to be measured in the sample and the labeled antibody or antigen may form a complex. The specimen containing these moves to the porous body 15 provided adjacent to the reagent pad 14 by capillary action. The composite that has reached the porous body 15 moves in the downstream direction (when the sample pad 13 side is the upstream side in the figure) by capillary action.

On the porous body 15, a reaction region 16 to which an antibody or an antigen is immobilized is provided. The antigen, antibody, or the complex that has moved through the porous body 15 by capillary action is captured in the reaction region 16. As shown in FIG. 5 (c), this capture can be confirmed by the coloration 16 ′ in the region 16. In this way, the presence or absence of the measurement target substance in the sample can be confirmed visually.
An absorption pad 17 for absorbing other components in the specimen and a substance to be measured that has not been captured is provided further downstream of the test device 11.

  In addition, the substance to be measured in the sample can be semi-quantified using the illustrated immunochromatographic test device. For example, a plurality of reaction regions 16 each having different detection sensitivities for the measurement target substance can be provided, and the measurement target substance in the sample can be semi-quantified by the number of the regions 16 where the coloration 16 'occurs.

Patent Documents 1 to 6 describe such an immunochromatographic test device and a method for detecting a measurement target substance in a specimen or a method for semi-quantifying the measurement target substance using the same.
Patent Document 1 describes a method for measuring an antigen or antibody in a liquid using a sheet-like test tool based on the immunochromatography method. However, this document has no disclosure or suggestion regarding semi-quantification of an antigen or antibody, nor is there any disclosure or suggestion regarding a test device having a region exhibiting a standard color on a porous body.
Patent Document 2 describes a test device for measuring the presence or absence of an antigen or antibody in a liquid using a dye-labeled antigen or antibody. However, there is no disclosure or suggestion regarding semi-quantification of an antigen or antibody in this document, nor is there any disclosure or suggestion regarding a test device having a region exhibiting a standard color on a porous body.
Patent Document 3 describes a test device for quantifying an antigen or antibody in a sample solution. The test device is characterized by having a plurality of reaction regions on which an increasing amount of a capture reagent is immobilized from upstream to downstream, and quantifies the analysis target based on the number of colored reaction regions. A plurality of reaction regions is an essential requirement, but providing a plurality of reaction regions is not preferable because the manufacturing process becomes complicated. In addition, the test device does not disclose or suggest a test device having a region exhibiting a standard color on the porous body.
Patent Document 4 describes a test device for semi-quantifying an antigen or antibody in a sample solution. The test device has a plurality of reaction regions arranged in series in the development direction of the particle-labeled antigen or antibody and specimen solution, and the amount of the antigen or antibody is semi-quantitatively determined by the principle of the sandwich method. There is a disclosure that it can be inferred. A plurality of reaction regions is an essential requirement, but providing a plurality of reaction regions is not preferable because the manufacturing process becomes complicated. In addition, the test device does not disclose or suggest a test device having a region exhibiting a standard color on the porous body.

In Patent Literature 5, an immunological chromatographic analysis piece having a portion where a constant color tone appears on the same film and a portion where a concentration-dependent color tone of a specimen appears is used. After the sample was injected and chromatographed, the color tone of the part where a certain color tone appears and the color tone of the part where the concentration-dependent color tone of the sample appear were read by an optical density measuring device or visually, and the concentration of the unknown sample was determined. A method of measuring is described. The method is characterized in that the control color region and the concentration-dependent color region of the sample are on the same film, but the control color region is immobilized differently from the sample. The coloration is performed using an antibody and a labeled antibody, which is complicated and difficult to adjust to a color corresponding to the concentration of a specific specimen.
Patent Document 6 describes an immunochromatographic test device in which a classification line type display is directly printed on a porous body on which a specimen moves by capillary action. As described in Patent Document 6, there is no idea that printing is performed on a porous body in which a specimen moves due to capillary action before Patent Document 6, and the concentration of a measurement target substance is measured on the porous body. In the past, there was no idea of providing a useful display.

JP-A-7-55808 JP-A-10-73592 JP-A-5-5743 JP-A-8-278305 JP 2001-83153 A JP 2001-13143 A

  Therefore, the present invention is a novel test device that is easy to manufacture and that can easily semi-quantify the concentration of a substance to be measured contained in a specimen using an immunochromatography method, and a semi-quantitative amount using this test device. It aims to provide a method.

In order to achieve the above object, the present invention is an immunochromatographic test device having a porous body in which a specimen can move by capillary action,
The immunochromatographic test tool is:
(1) a region where a labeled antibody or antigen capable of specifically binding to a measurement target substance in the specimen is arranged in a movable state;
(2) An area where an antibody or antigen that can specifically bind to the measurement target substance is arranged in a state of being fixed on the porous body, and (3) a coloration at a specific concentration of the measurement target substance The corresponding standard color has a region arranged in a fixed state on the porous body,
By comparing the coloration generated in the region (2) with the standard coloration, a test device for semi-quantifying the measurement target substance in the sample (hereinafter referred to as “test device of the present invention”). .)I will provide a.
As a preferred embodiment of the test device of the present invention, the region in which the antibody or antigen capable of specifically binding to the substance to be measured of (2) is fixed on the porous body, and (3) And a region in which a standard color corresponding to the color at a specific concentration of the measurement target substance is fixed on the porous body is in series or parallel to the development direction of the development solvent. Examples of the test device arranged in the above.
In another preferred embodiment of the test device of the present invention, a region in which a labeled antibody or antigen capable of specifically binding to the measurement target substance in the specimen of (1) is arranged in a movable state. Standard coloration used for the region where the labeling dye used in the above and the standard coloration corresponding to the coloration at the specific concentration of the substance to be measured in (3) are fixed on the porous body Examples thereof include the above-mentioned test device in which the dye for use is the same dye.
The present invention also provides a method for semi-quantifying a measurement target substance in a specimen using the test device of the present invention.

The test device of the present invention compares the standard coloration fixed on the porous body of the immunochromatographic test device with the coloration generated when the substance to be measured is captured by the antibody or antigen fixed on the porous body. By doing so, the substance to be measured contained in the sample can be easily semi-quantified.
In addition, the test device of the present invention includes a region (reaction region) in which an antibody or an antigen that can specifically bind to a measurement target substance is fixed on the porous body, and a specific substance of the measurement target substance. Since the standard coloration corresponding to the coloration at the concentration has a region arranged in a fixed state on the porous body, it is possible to compare the coloration of the reaction region with the standard coloration under the same conditions. It is possible and high accuracy is obtained.
In addition, since the test device of the present invention can be semi-quantified if there is at least one reaction region and a region exhibiting standard coloration, a test in which a plurality of reaction regions are essential for semi-quantification. Compared to tools, it can be easily manufactured. In addition, for areas showing standard coloration, it is an essential requirement to fix dyes or the like directly on the porous body without using antibodies or antigens. Compared to a test device, it can be easily manufactured. In addition, since the reaction such as an antigen-antibody reaction is not indirectly performed, the color tone of the standard coloring is not affected at all by the difference in various use conditions when using this test device.

FIG. 1 is a conceptual diagram of an embodiment of a test device of the present invention. FIG. 2 is a side view of the test device of FIG. FIG. 3 is a graph showing changes over time in the degree of coloration of the standard color 8 and the color 6 ′ after the test device of the present invention is immersed in hCG adjusted to various concentrations. 4 (a) to 4 (e) are plan views of the test device used in the example, FIG. 4 (a) is an unused test device before being immersed in a urine specimen, and FIG. FIG. 4 (c) shows a test device after semi-quantification using a sample with hCG concentration of 25 IU / L. FIG. 4 (d) shows a test device after semi-quantification using a sample with hCG concentration of 1000 IU / L. ) Is a test device after semi-quantification was performed using a sample having an hCG concentration of 10000 IU / L. FIG. 4E is a test device after semi-quantification was performed using a sample having an hCG concentration of 0 IU / L (negative). Is shown. FIG. 5A is a conceptual diagram showing a conventional immunochromatographic test device, and the test device is shown in a plan view. FIG. 5B is a side view of the test device shown in FIG. FIG.5 (c) is the same figure as Fig.5 (a), but has shown the test device after use.

Explanation of symbols

1: Test tool 2: Substrate 3: Sample pad 4: Reagent pad 5: Porous body 6: Reaction region 6 ′: Color 7: Absorption pad 8: Standard color 9: Region 9 ′: Color 11: Test tool 12 : Substrate 13: Sample pad 14: Reagent pad 15: Porous body 16: Reaction region 16 ': Color 17: Absorption pad 20: Protective film 21: Transparent part 22: Window 23, 23', 23 ": Opaque part

Hereinafter, the test device of the present invention will be described with reference to the drawings.
FIG. 1 is a conceptual diagram of an embodiment of the test device of the present invention, and FIG. 2 is a side view of the test device shown in FIG.
In the test device 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. Of the test device 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 device 1 of the present invention will be described.
In the test device 1 shown in FIG. 1, each component is formed on a sheet-like substrate 2. The substrate 2 is made of a material that does not absorb or permeate the specimen passing through each component during the immunochromatography and does not denature the specimen when the specimen comes into contact. Examples of such materials include plastic materials used for medical devices, such as vinyl chloride, polyethylene, ethylene vinyl acetate resin, polypropylene, polycarbonate, polyurethane, polystyrene, silicone resin, fluororesin, polyethylene terephthalate, polyamide. , ABS resin and the like. The substrate 2 may be made of paper having a surface subjected to water / oil repellent finish.

  The sample pad 3 is a part where the specimen is dropped or immersed in the specimen, and when an insoluble substance is present in the specimen, the sample pad 3 also functions as a filter for removing the insoluble substance. From this viewpoint, the sample pad 3 is exemplified by a nonwoven fabric or a porous membrane having a pore diameter of 20 μm to 100 μm, preferably 30 μm to 40 μm, but is not particularly limited thereto. The material that can be used for the sample pad 3 is not particularly limited as long as it can collect a certain amount of specimen, is hydrophilic, and has a filter function. For example, regenerated cellulose, cellulose acetate, nitrocellulose, polyacrylonitrile Examples thereof include, but are not limited to, ethylene vinyl acetate, polyurethane, polymethyl methacrylate, nylon resin, glass fiber, pulp, cotton, rayon, acrylic, and polyester.

  The specimen dropped on the sample pad 3 moves in the downstream direction, that is, toward the reagent pad 4 by capillary action. Hereinafter, in this specification, the sample pad 3 side of the test device 1 is an upstream side, and the opposite side is a downstream side. In addition, the term “sample dropped onto the sample pad 3” also means a sample contained in the sample pad 3 as a result of immersing the sample pad 3 in the sample.

The material of the reagent pad 4 is exemplified by a non-woven fabric or a porous film as in the case of the sample pad 3, but is not particularly limited thereto. The reagent pad 4 carries an antibody or an antigen that can specifically bind to the measurement target substance in the sample in a state of being labeled with a labeling substance such as a dye.
The antibody or antigen that can specifically bind to the measurement target substance is appropriately selected according to the measurement target substance. When the substance to be measured is human chorionic gonadotropin (hCG) in a urine sample, an anti-hCG antibody, more specifically, an anti-hCG mouse polyclonal antibody, an anti-hCG mouse monoclonal antibody, or the like is used. When the substance to be measured is alpha fetoprotein (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 prostate specific antigen (PSA) which is a cancer marker that requires semi-quantification, for example, an anti-human PSA rabbit antibody or the like is used.
The labeling substance is not particularly limited as long as visible coloring is recognized, but for example, metal colloid represented by gold colloid, non-metal colloid, dye particles such as dye sol or disperse dye, latex particles or Examples thereof include colored fine particles. Of these, latex fine particles or polystyrene fine particles colored with dyes or pigments are preferred from the viewpoints of excellent color developability, abundant color tone and particle diameter, and a plurality of methods for labeling antibodies or antigens. This labeling method is well known by those skilled in the art and includes a method using adsorption and a method using chemical bonding. In particular, latex fine particles or polystyrene fine particles colored with a dye or pigment and having a particle size of 0.5 μm or less, preferably 0.05 to 0.5 μm and having a spherical or substantially spherical shape are preferable. Most preferred is blue latex.

The labeled antibody or antigen is supported on the reagent pad 4 in a movable state. Here, “supported in a movable state” means that the labeled antibody or antigen is retained in the reagent pad 4 when not in use, that is, when the reagent pad 4 is dry. This means that when the reagent pad 4 is wetted due to the arrival of the specimen, that is, the labeled antibody or antigen becomes movable. For this purpose, the reagent pad 4 must be a material that does not adsorb the labeled antibody or antigen, but if it is an adsorbing material, it can be masked with an inactive protein such as another BSA in advance. Can be used. The antibody or antigen thus labeled is retained on the reagent pad 4 by, for example, impregnating the reagent pad 4 and then lyophilizing.
Therefore, in the test device 1, the reagent pad 4 forms an area in which the labeled antibody or antigen that can specifically bind to the measurement target substance in the sample is movable.

  The specimen dropped on the sample pad 3 moves to the reagent pad 4 by capillary action. For this reason, the hole diameter of the reagent pad 4 is preferably the same or smaller than that of the sample pad 3. The pore size of the reagent pad 4 is exemplified by 1 μm to 50 μm, preferably 5 μm to 30 μm.

  In the test device 1 shown in FIGS. 1 and 2, the sample pad 3 and the reagent pad 4 are partially stacked in order to facilitate the movement of the specimen due to capillary action. However, the sample pad 3 and the reagent pad 4 may be simply arranged adjacent to each other as long as the specimen can be appropriately moved by capillary action, or may be the same pad.

  When the specimen reaches the reagent pad 4, the substance to be measured in the specimen and the labeled antibody or antigen in the pad 4 move downstream, that is, toward the porous body 5 by capillary action. In addition, when the measurement target substance and the labeled antigen or antibody are in a relationship between the antigen and the antibody, part or all of both forms an antigen-antibody complex, which is also downstream, that is, porous. Move to body 5. In the test device 1 shown in FIGS. 1 and 2, the reagent pad 4 and the porous body 5 are partially laminated to facilitate this movement. However, the reagent pad 4 and the porous body 5 may simply be disposed adjacent to each other as long as the specimen can be appropriately moved by capillary action, and the reagent pad 4 may also serve as the porous body 5. Or vice versa.

  The porous body 5 is a medium whose structure has sponge-like pores or has a mesh shape, and the specimen can move by capillary action. The test device 1 shown in FIGS. 1 and 2 is shown as a sheet-like porous film. The material of the porous body 5 is not particularly limited as long as the immunochromatography method can be performed. Specifically, for example, cellulose, cellulose derivative, nitrocellulose, ethylene vinyl acetate, polyurethane, polymethyl methacrylate, nylon resin, polyvinylidene Examples include woven fabrics, non-woven fabrics, membranes, glass fiber filters, various filter papers, etc. made of porous synthetic polymers such as difluoride (PVDF), and those using cellulose, cellulose derivatives, and porous synthetic polymers are preferred. Those using nitrocellulose are particularly preferred. The pore diameter of the porous body 5 is exemplified by 1 μm to 30 μm, preferably 1 μm to 20 μm.

  The porous body 5 has a region 6 (reaction region) to which an antibody or antigen that can specifically bind to the measurement target substance is immobilized. The antibody or antigen immobilized on the region 6 can be appropriately selected according to the substance to be measured. Specifically, when the substance to be measured is hCG, an anti-hCG antibody, more specifically, an anti-hCG mouse polyclonal antibody, an anti-hCG mouse monoclonal antibody, or the like can be used. When the substance to be measured is AFP, an anti-AFP antibody 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. For example, when the porous body 5 is nitrocellulose or nylon, these immobilization methods can be easily immobilized by directly applying and drying the antibody or antigen solution described above. In the case of porous materials such as glass fiber filters and filter paper that cannot be fixed by direct application and drying, it is easy to activate the porous material or pretreatment by activating the immobilized antibody or antigen. These immobilization methods are techniques well known to those skilled in the art.

  Antigens, antibodies and / or complexes that have migrated through the porous body 5 due to capillary action are captured by the antibodies or antigens immobilized in the region 6. When the antigen, antibody and / or complex containing the labeling substance is captured, it can be confirmed as a coloration generated in the region 6. Here, the color tone generated in the region 6 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 where the complex formed by the labeled antibody held in the reagent pad 4 with the antigen in the specimen is captured by the immobilized antibody in the reaction region, when the concentration of the measurement target substance in the specimen is high, Since the complex captured in the region 6 increases, a strong color tone is generated. When the concentration of the substance to be measured in the specimen is low, the complex captured in the region 6 decreases, and thus the weak color tone is exhibited. Produce color. On the other hand, for example, when the labeled antigen is captured by the immobilized antibody in the reaction region in the competitive reaction between the antigen in the sample and the labeled antigen held in the reagent pad 4, the concentration of the measurement target substance in the sample When the value is high, the labeled antigen captured in the region 6 decreases, so that a weak color tone is generated. When the concentration of the measurement target substance in the specimen is low, the labeled antigen captured in the region 6 increases. This produces a strong color tone.

  The porous body 5 has a region in which a standard coloration 8 corresponding to the coloration at a specific concentration of the measurement target substance is fixed. That is, in the test device 1, a region having a color tone equal to the coloration generated in the region 6 is formed on the porous body 5 when a specimen containing a specific concentration (hereinafter referred to as “reference concentration”) of the measurement target substance is used. Has been. Even in the standard coloration 8 immobilization method, the same immobilization method as in the reaction region can be used.

In the test device of the present invention, by comparing the color tone generated in the region 6 and the color tone of the standard color 8 existing on the porous body 5 of the test device 1, the substance to be measured in the sample is compared. It can be easily semi-quantified. That is, for example, when the complex formed by the labeled antibody held in the reagent pad 4 with the antigen in the specimen is captured by the immobilized antibody in the reaction region, the color tone of the color produced in the region 6 is standard. When the color tone is weaker than the color tone 8, the concentration of the measurement target substance in the sample is lower than the reference concentration. When the color tone generated in the region 6 is stronger than the color tone of the standard color 8, the concentration of the measurement target substance in the sample is higher than the reference concentration. When the color tone of the color generated in the region 6 is approximately the same as the color tone of the standard color 8, the concentration of the measurement target substance in the sample is approximately the same as the reference concentration. Further, when no coloration occurs in the region 6, it means that the measurement target substance does not exist in the specimen or is below the detection sensitivity. Therefore, the test device 1 of the present invention can also perform qualitative evaluation at a specific sensitivity of the measurement target substance based on the presence or absence of coloration in the region 6.
Furthermore, by comparing the color tone generated in the region 6 with the standard color tone 8 using an optical densitometer 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 capillary action, the standard color 8 is preferably fixed on the surface of the porous body 5. Here, the standard color 8 is preferably a labeling substance used to label the antibody or antigen carried on the reagent pad 4. Accordingly, preferred examples include metal colloids represented by gold colloids, non-metal colloids, dye particles such as dye sols or disperse dyes, latex particles, and colored fine particles. Of these, latex fine particles or polystyrene fine particles colored with dyes or pigments are preferred from the viewpoints of excellent color developability, abundant color tone and particle diameter, and a plurality of methods for labeling antibodies or antigens. This labeling method is well known by those skilled in the art and includes a method using adsorption and a method using chemical bonding. In particular, latex fine particles or polystyrene fine particles colored with a dye or pigment and having a particle size of 0.5 μm or less, preferably 0.05 to 0.5 μm and having a spherical or substantially spherical shape are preferable. Most preferred is blue latex.
However, the standard coloration 8 may be formed using other substances such as pigments, dyes, and pigments as long as the color tone is the same as the coloration at a specific concentration of the measurement target substance.
In addition, an antibody or antigen that can specifically bind to the measurement target substance corresponds to a region (reaction region 6) arranged in a state of being fixed on the porous body, and coloration at a specific concentration of the measurement target substance. The region where the standard coloration is arranged in a fixed state on the porous body (standard coloration 8) is preferably arranged in series or in parallel with the development direction of the development solvent, Most preferably, they are arranged in series. Moreover, it is preferable that both are located in the vicinity. Specifically, the distance between the two is preferably within 20 mm, and more preferably within 10 mm.
The developing solvent is not particularly limited as long as it can be normally contained in a specimen. Examples thereof include water (including water contained in the specimen); physiological saline; organic solvents such as acetone.
The developing direction of the developing solvent is the direction from the sample pad 3 to the absorption pad 7 in FIG.
In addition, a labeling dye used for a region (reagent pad 4) in which a labeled antibody or antigen that can specifically bind to a measurement target substance in a specimen is movable, and identification of the measurement target substance It is preferable that the standard coloring dye used in the region (standard coloring 8) arranged in a state where the standard coloring corresponding to the coloration at the density is fixed on the porous body is the same dye. More preferred are latex particles.

  The reference concentration and detection sensitivity shown as the standard coloration 8 can be appropriately selected according to the type of the substance to be measured and the use of the test device. For example, when the test device 1 measures the amount of hCG in the urine sample, the detection sensitivity is preferably 25 IU / L and the reference concentration is preferably 1000 IU / L. When the hCG detection sensitivity is 25 IU / L, pregnancy diagnosis is possible depending on the presence or absence of coloration in the region 6. When the reference concentration is 1000 IU / L, evaluation such as ectopic pregnancy, premature labor, prognosis determination of choriocarcinoma, and the like can be performed. As a result, the substance to be measured in the sample can be semi-quantified in one measurement based on the hCG concentrations of 25 IU / L and 1000 IU / L. For example, when measuring the amount of PSA in blood, the detection sensitivity is preferably 1 ng / mL and the reference concentration is preferably 4 ng / mL. When the PSA detection sensitivity is 1 ng / mL, prostate hypertrophy 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. As a result, it is possible to semi-quantify the PSA concentration in the specimen based on the PSA concentrations of 1 ng / mL and 4 ng / mL in a single operation.

  As described above, part or all of the substance to be measured in the specimen moving through the porous body 5 is captured by the antibody or antigen immobilized on the region 6. On the other hand, the other components in the specimen and the measurement target substance not captured move in the porous body 5 in the downstream direction. The test device 1 shown in FIGS. 1 and 2 is provided with an absorbent pad 7 for absorbing these. The absorption pad 7 is not particularly limited as long as it can absorb and hold the specimen that has moved through the porous body 5. Therefore, the absorption pad 7 can use widely what is conventionally used in order to absorb liquid and hold | maintain inside it, such as a filter paper, a nonwoven fabric, and a porous membrane. The absorbent pad 7 may include a water-absorbing / hygroscopic material such as silica gel.

As described above, when the measurement target substance is not contained in the specimen or when it is contained, the color is not generated in the region 6 when the specimen is below the detection sensitivity. no change. For this reason, it is preferable that the test device 1 of the present invention has a mechanism for confirming the completion of the test. In the test device 1 shown in FIGS. 1 and 2, a region 9 that develops color when the specimen reaches is provided downstream of the region where the standard color 8 of the porous body 5 is fixed. A transparent window 22 is provided in a portion of the protective film 20 that covers the porous body 5 that covers the region 9. In FIG. 1, portions 21 and 22 of the protective film 20 are transparent, and portions 23, 23 ', and 23 "are opaque.
In the test device 1 shown in FIGS. 1 and 2, the region 9 and the window 22 form a mechanism for confirming the end of the test. That is, when the specimen that has moved through the porous body 5 reaches the region 9, color development occurs, and this color development can be confirmed by the window 22. Thereby, it can confirm that the test was complete | finished.

For this purpose, the area 9 is impregnated or fixed with various color formers. Examples of color formers impregnated or immobilized in the region 9 are those that develop color when contacted with a liquid sample having a predetermined pH, such as eosin B, phloxine B, benzoperpurine 4B, Congo red, bromophenol. Blue, bromcresol green, 2,5-dinitrophenol, or related substances thereof. Further, the color former may be one that develops color due to its moisture when the specimen reaches the region 9.
Furthermore, the area upstream of the window 22 of the porous body 5 is impregnated with a water-soluble dye so as to dissolve when the specimen reaches, for example, an area covered with the opaque portion 23 ′. The dye that dissolves and moves in the specimen may be confirmed by the window 22.
In addition, when a component that specifically reacts with a labeled antigen or antibody, for example, a labeled anti-hCG mouse monoclonal antibody, an anti-mouse sheep antibody or the like is immobilized in the region 9, the object can be achieved.
In addition, a labeled antibody, labeled biotin, keyhole hemocyanin, or the like that is not involved in the measurement is held in the reagent pad 4, and an antibody that reacts specifically with the labeled antibody or an antigen, avidin, or anti-keyhole hemocyanin antibody is added to the region 9. The object can be achieved even if it is fixed to the above.

As mentioned above, although the test tool of this invention was demonstrated in detail using the figure, the test tool of this invention is not limited to the form shown in figure. The test device of the present invention is an immunochromatographic test device having a porous body in which a specimen can move by capillary action, and those having the following areas (1) to (3) can be widely used.
(1) a region where a labeled antibody or antigen capable of specifically binding to a measurement target substance in the specimen is arranged in a movable state;
(2) An area where an antibody or antigen that can specifically bind to the measurement target substance is arranged in a state of being fixed on the porous body, and (3) a coloration at a specific concentration of the measurement target substance The area | region where the corresponding standard color is arrange | positioned in the state fixed on the said porous body.

Therefore, the test device of the present invention is a porous material such as a test apparatus for immunochromatography described in JP-A-1-503174, JP-A-6-180320, JP-A-6-160388. You may have the casing which accommodates these components. Moreover, the protective film 20 illustrated in the figure of this invention is not essential for the test device of this invention.
The shape of the reaction region and / or standard coloration fixed on the porous body is not limited to a linear shape as illustrated, and may be other shapes. In addition, a plurality of reaction regions and / or standard colors corresponding to colors at different concentrations of the measurement target substance may be fixed on the porous body.

The test device of the present invention can be applied to various liquid samples derived from living organisms. Specific examples of such a liquid sample include blood, plasma, serum, saliva, urine, fecal extract, tissue extract and the like. The measurement target substances contained in these liquid samples include various proteins and peptides that function as antigens or antibodies, complex proteins, lipids, complex lipids, nucleic acids, lectins, sugar chains or compounds having these, biotin, avidin, hormones , Enzymes, enzyme substrates, enzyme inhibitors, receptors, ligands, drugs, drug metabolites, analyte derivatives, etc. More specifically, human chorionic gonadotropin (hCG), luteinizing hormone (LH) , Alphafetoprotein (AFP), carcinoembryonic antigen (CEA), human placental lactogen (hPL), beta2 microglobulin (β2m), ferritin, HBs antigen, estrogen, PSA and the like, and antibodies thereto. In the present invention, antibodies, antigens, nucleic acids, avidin, and biotin are preferably used, and antibodies or antigens are particularly preferable.
Note that the test device of the present invention can also be applied to a solution prepared by dissolving a standard sample of a measurement target substance as exemplified above in physiological saline or the like.

Hereinafter, the test device of the present invention will be further described with reference to examples.
In this example, the test device 1 shown in FIGS. 1 and 2 was used.
Each configuration of the test device 1 was as follows.
Base body 2: made of polyester, length 125 mm × width 8 mm × thickness 0.25 mm
Sample pad 3: A non-woven fabric (Benlyse, manufactured by Asahi Kasei Co., Ltd.) having a length of 30 mm, a width of 8 mm, and a thickness of 0.3 mm was adhered.
Reagent pad 4: Latex particles colored with a blue dye as a labeling substance (particle size: 0.3 μm) on a nonwoven fabric (glass fiber, manufactured by Millipore) 15 mm long × 8 mm wide × 0.3 mm thick The anti-hCG mouse monoclonal antibody) to which the spherical particles) were bound and freeze-dried were used. This was adhered on the substrate 2.
The sample pad 3 and the reagent pad 4 are laminated at the ends (about 4 mm in length).
Porous body 5: A nitrocellulose film (length 25 mm × width 8 mm × thickness 235 μm, pore diameter about 8 μm) was adhered on the substrate 2.
The ends of the porous body 5 and the reagent pad 4 are laminated (for 2 mm in length).
On the porous body 5, 1.5 mg / mL anti-hCG mouse monoclonal antibody was applied and dried to immobilize the monoclonal antibody in region 6.
Standard coloration 8: Standard coloration 8 was used as a labeling substance so that hCG having a concentration of 1000 IU / L (reference concentration) had the same color tone as that when the antibody was captured in region 6. The same latex particles (colored with a blue dye) were fixed on the surface of the porous body 5.
As an index indicating the end of the test, eosin B was impregnated as a color former in the region 9 downstream of the standard coloration 8 of the porous body 5 and dried.
Absorption pad 7: Nonwoven fabric (glass fiber, manufactured by Whatman) having a length of 20 mm, a width of 8 mm, and a thickness of 0.4 mm was bonded onto the substrate 2.
Protective film: Except for the sample pad 3, the upper part of the test device 1 was covered with a protective film (made of polyethylene terephthalate) having transparent portions 21, 22 and opaque portions 23, 23 ', 23 ".

Using this test device 1, semi-quantification was carried out using the substance to be measured as human chorionic gonadotropin (hCG).
The sample pad 3 part of the test device 1 is immersed for about 3 seconds in a solution diluted with physiological saline containing 0.1% BSA so as to contain hCG (manufactured by the National Institute of Health Sciences) at the following various concentrations. I let you. Next, the test device 1 was left in the horizontal direction for about 3 minutes so that the protective film 20 was on top. When the coloration 9 'indicating that the reaction was completed in the window 22 was clearly generated, the standard coloration 8 and the coloration 6' in the region 6 were measured with a densitometer.
Here, samples with 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 were used.
In the following, the degree of coloration of the color 6 ′ of the region 6 of the specimen of each concentration is indicated by a numerical value obtained from the densitometer.

On the other hand, the value of the degree of coloration of standard coloration 8 measured with a densitometer was 9800.
The negative sample hardly develops color, the color develops at 25 IU / L, and the color develops as the density increases. At 1000 IU / L, the color develops almost the same as the standard color. At higher concentrations, the color develops stronger than the standard color. It was useful as a semi-quantitative test device.

Furthermore, using this test tool 1, the presence or absence of a change in coloration with time was confirmed. That is, the test device 1 was immersed for about 3 seconds in the specimen whose concentration was adjusted so that the sample pad 3 was immersed. Next, the test device 1 was left to stand for about 3 to 30 minutes in the horizontal direction so that the protective film 20 was on top, and the standard coloration 8 and the coloration 6 'of the region 6 at each time were measured with a densitometer.
Here, samples with hCG concentrations of 0 IU / L (negative), 25 IU / L, 500 IU / L, 1000 IU / L and 10 ⁶ IU / L were used.
In FIG. 3 and the table below, the degree of coloration is shown as the change with time of the numerical value obtained from the densitometer.

The coloration degree is an average value measured three times for each density. The numerical value of the standard coloration is an average value of each density 3 times (15 times in total).
Looking at the change over time up to 30 minutes, the color development in the standard coloration 8 and the coloration 6 'in the region 6 at each concentration peaked 5 to 10 minutes after the start of the test, and thereafter the sample was supplied to the porous body 5 The degree of color development decreased due to the drying of the porous body 5 accompanying the decrease in the amount. Even if time elapses from the start of measurement, the negative sample hardly develops color, color develops at 25 IU / L, color develops almost equal to the standard color at 1000 IU / L, and color develops stronger than the standard color at higher concentrations. The trend did not change, and it was useful as a semi-quantitative test device.
In addition, although this test result shown in Table 1, Table 2, and FIG. 3 was shown by the numerical value as a result measured with a densitometer, also in visual judgment, from the viewpoint of the presence or absence of coloring and the strength of standard coloring The same result was obtained.

FIG. 4 is a plan view showing the test device 1 before and after the semi-quantitative measurement. 4A shows an unused test device before immersion in the specimen, FIG. 4B shows a test instrument after semi-quantification using a urine specimen having an hCG concentration of 25 IU / L, and FIG. 4C shows an hCG concentration of 1000 IU. Test device after semi-quantification using / L sample, (d) Test device after semi-quantification using sample with hCG concentration of 10000 IU / L, (e) hCG concentration 0 IU / L Each of the test devices after semi-quantification using a (negative) specimen is shown.
As is clear from FIG. 4, it can be confirmed by the color 9 ′ generated in the window 22 that the test device 1 has been subjected to the semi-quantitative measurement (FIGS. 4B to 4E). In the example using a negative sample, no coloration occurred in the region 6, and only the standard coloration 8 was confirmed from the transparent portion 21. On the other hand, in the specimen containing hCG, a color 6 ′ indicating that the complex of hCG and the labeled antibody was captured by the antibody in region 6 was confirmed. The color tone 6 ′ was different in color tone depending on the hCG concentration in the specimen. Specifically, in the example using a 25 IU / L sample whose hCG concentration was lower than the reference concentration, the color 6 ′ was much weaker than the standard color 8 (FIG. 4B). In an example in which a specimen having a hCG concentration equal to the reference concentration of 1000 IU / L was used, the color tone of the color 6 ′ was almost the same as that of the standard color 8 (FIG. 4C). In an example in which a sample having a hCG concentration of 10,000 IU / L higher than the reference concentration was used, the color tone of the color 6 ′ was stronger than the standard color 8 (FIG. 4D).

  The present invention relates to a test device for semiquantitating a substance to be measured in a specimen using immunological chromatography (immunochromatography), and a semiquantitative method using the test device.

Claims (5)

An immunochromatographic test device having a porous body in which a specimen can move by capillary action, the immunochromatographic test device,
(1) a region where a labeled antibody or antigen capable of specifically binding to human chorionic gonadotropin (hCG) in the specimen is arranged in a movable state;
(2) a region where an antibody or antigen capable of specifically binding to the human chorionic gonadotropin (hCG) is arranged in a state of being immobilized on the porous body, and (3) the human chorionic gonad stimulation Standard coloration corresponding to coloration at a specific concentration of hormone (hCG) has a region arranged in a fixed state on the porous body,
A test device for semi-quantifying the measurement target substance in the specimen by comparing the coloration generated in the region (2) with the standard coloration. A region in which an antibody or an antigen capable of specifically binding to the human chorionic gonadotropin (hCG) in (2) is fixed on the porous body; and the human in (3) An area where standard coloration corresponding to coloration at a specific concentration of chorionic gonadotropin (hCG) is fixed on the porous body is arranged in series with the development direction of the development solvent. Or the test device of Claim 1 arrange | positioned in parallel. (1) a labeling dye used for a region where a labeled antibody or antigen capable of specifically binding to human chorionic gonadotropin (hCG) in the specimen is arranged in a movable state; The standard coloration corresponding to the coloration at a specific concentration of the human chorionic gonadotropin (hCG) in (3) is used for a standard coloration used in a region arranged in a fixed state on the porous body. The test device according to claim 1 or 2, wherein the pigment is the same pigment. The test device according to any one of claims 1 to 3 , wherein the standard coloration is a coloration in human chorionic gonadotropin (hCG) 1000 IU / L. How to semi-quantitative measurement object substance in a sample by using a test device according to any one of claims 1 to 4.

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