Inspecting base material and manufacturing method thereof

Abstract

To provide an inspecting base material excellent in handling, which has both antibody fixability and flow path performance as a replacement for a nitrocellulose membrane.SOLUTION: An inspecting base material 1 is a base material for inspection, and includes a tabular flow path part 10 which allows liquid to pass by a capillary phenomenon and which has an antibody fixation function. The flow path part 10 comprises a main body part 11 and a detection part 12, and contains fibrous members 14 and a fixing component M capable of bonding to antibodies. The base material for inspection improves mechanical strength while exerting antibody fixability and flow path performance.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a testing substrate and a method for producing the same, and more particularly to a testing substrate used in tests such as immunoassays and a method for producing the same. [Background technology]

[0002] Immunoassays are used to test for viruses, bacteria, hormones, etc. in samples. Among immunoassays, immunochromatography is a commonly used rapid and simple test method. This method uses chromatography to visually determine the presence or absence of viruses, etc. (hereinafter referred to as antigens) in a sample based on differences in color development.

[0003] Specifically, a sample is supplied to the sample dropping portion of the test kit, and the antigen in the sample forms a complex with a labeled antibody labeled with gold colloid or the like placed upstream of the stationary phase, and moves through the stationary phase to the test line by capillary action. Another antibody (hereinafter referred to as the immobilized antibody) that binds to the antigen is placed on this test line. When the complex arrives at the test line, it is captured by the immobilized antibody fixed to the test line and develops color (coloration). Because this color development can be easily determined visually, testing methods using immunochromatography are widely used in pregnancy diagnosis, influenza diagnosis, and the like. For example, Patent Documents 1 and 2 disclose test kits that use a nitrocellulose membrane as the stationary phase, on which a desired solid-phase antibody is fixed as a test line. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4372349 [Patent Document 2] Special Publication No. 2020-515365 Summary of the Invention [Problem to be solved by the invention]

[0005] As shown in Patent Documents 1 and 2, conventional immunochromatography test kits typically use a nitrocellulose membrane as the stationary phase. This is because the nitrocellulose membrane is a unique material that combines antibody fixation and flow path performance. However, nitrocellulose membranes have the disadvantage of being brittle and easily broken. This makes them difficult to handle because they crack or collapse when pressure or bending forces are applied. However, the reality is that currently there is no material that can replace the nitrocellulose membrane as the stationary phase in immunochromatography test kits. Furthermore, conventional immunochromatography-based test kits are manufactured by combining multiple parts, which makes the assembly process complicated and increases costs.

[0006] In view of the above circumstances, an object of the present invention is to provide a testing substrate that is easy to handle and can replace a nitrocellulose membrane, and that has both antibody fixability and flow path performance. [Means for solving the problem]

[0007] The testing substrate of the present invention is a testing substrate having a flat flow path section with an antibody immobilization function that allows liquid to pass through by capillary action, and is characterized in that the flow path section has a main body section and a detection section, and contains a fibrous member and a fixing compound that can bind to the antibody. The method for manufacturing a substrate for testing of the present invention is a method for manufacturing a substrate for testing, and includes the steps of preparing a mixed liquid color containing a forming member and a fixing compound, applying the mixed liquid color in a sheet form to form a sheet member, drying the applied sheet member, and cutting the dried sheet member into strips, wherein the forming member contains a fibrous member. [Effects of the Invention]

[0008] According to the testing substrate of the present invention, it is possible to provide a testing substrate that exhibits antibody fixability and flow path performance while improving mechanical strength. By using the method for producing a testing substrate of the present invention, the testing substrate of the present invention can be produced by a simple operation. [Brief explanation of the drawings]

[0009] [Figure 1] 1(B) is a schematic cross-sectional view taken along line BB in FIG. 1(A), and FIG. 1(C) is a schematic cross-sectional view of the enlarged view on the left of FIG. 1(B) as seen from the liquid advance direction. [Figure 2] FIG. 2(B) is a schematic explanatory diagram of the flow path portion 10 of the testing substrate 1 of this embodiment, in which fibers 14, granular members 15, and binder members 16 are used as the forming member 13, and FIG. 2(B) is a schematic explanatory diagram of the enlarged view on the left of FIG. 2(A) viewed from the liquid advance direction. [Figure 3] FIG. 2 is a schematic explanatory view of the inspection situation of the inspection substrate 1 of the present embodiment. [Figure 4] FIG. 10 is a diagram showing experimental results. [Figure 5] FIG. 10 is a diagram showing experimental results. [Figure 6] FIG. 10 is a diagram showing experimental results. [Figure 7] FIG. 10 is a diagram showing experimental results. [Figure 8] FIG. 10 is a diagram showing experimental results. [Figure 9] FIG. 10 is a diagram showing experimental results. [Figure 10] FIG. 10 is a diagram showing experimental results. [Figure 11] FIG. 10 is a diagram showing experimental results. [Figure 12] FIG. 10 is a diagram showing experimental results. [Figure 13] FIG. 10 is a diagram showing experimental results. DETAILED DESCRIPTION OF THE INVENTION

[0010] Next, an embodiment of the present invention will be described with reference to the drawings. The testing substrate of this embodiment is a testing substrate, and is characterized by improved mechanical strength in addition to antibody fixability and flow channel performance. First, the testing substrate of this embodiment will be outlined, and then the details will be described.

[0011] The test for which the testing substrate of the present embodiment is used is not particularly limited as long as it is a test used for diagnosis, etc. Examples of such tests include immunoassays that utilize antigen-antibody reactions, immunostaining methods, and immunoaffinity methods.

[0012] As shown in FIG. 1, the testing substrate 1 of this embodiment includes a flow path portion 10. The flow path section 10 is a porous, flat testing member with many mesh-like voids 10h formed inside. These voids 10h form a mesh-like network, thereby forming a plurality of fine flow paths for liquid to pass through within the flow path section 10. Therefore, when liquid is supplied to the flow path section 10, it can be passed through the mesh-like flow paths by capillary action. The flow path section 10 contains a fibrous member 14 (hereinafter simply referred to as fiber 14) and a fixing compound M. This fixing compound M is a compound that can bind to an antibody A, which is applied as described below, and fix the antibody A within the flow path section 10, and is arranged in a fixed state on the inner surface of the gap 10h, within the gap 10h, or on the surface of the forming member 13 (for example, on the fiber 14 in FIG. 1, or on the surface of the fiber 14, granular member 15, and binder member 16 in FIG. 2). In other words, the fixing compound M has an antibody immobilization function that can bind to antibody A, and is a compound that can retain the bound antibody A within the flow path section 10, making it difficult for it to move. 1, in the flow path section 10, the portion having the region 12a where the immobilized antibody AI of this antibody A is applied is the detection section 12, and the downstream portion connected to the detection section 12 is the main body section 11. In other words, the flow path section 10 of the testing substrate 1 of this embodiment is formed so as to have, from the upstream side to the downstream side in the direction of advancement of the test liquid, the main body section 11 and the detection section 12 connected to this main body section 11. As shown in FIG. 1, the detection section 12 may have an area 12b to which a control antibody AC of antibody A is applied.

[0013] The downstream and upstream sides of the flow path section 10 refer to the direction in which the test liquid is developed when supplied to the main body 11 of the flow path section 10, and the side on which the test liquid is supplied refers to the upstream side. In Fig. 1, the direction of the arrow indicates the liquid direction in which the test liquid moves when it is developed, and the region 11a in this liquid advance direction where the test liquid is supplied is the upstream side, and the opposite side is the downstream side.

[0014] The test liquid is a liquid to be tested. As described above, the testing substrate 1 of this embodiment is a substrate used for testing, and is configured to capture antigens in the test liquid in the flow path section 10. Therefore, the test liquid can be any liquid that is expected to contain antigens, such as an undiluted solution, a diluted solution thereof, or a filtered solution thereof. For example, various liquids can be used as the test liquid or raw materials for the test liquid, including body fluids such as blood, plasma, serum, urine, saliva, sweat, and tears, river water, seawater, and groundwater, as well as aqueous solutions in which soil or food has been dissolved. Furthermore, the term "antigen" as used herein refers to a target substance to be tested, and is not particularly limited as long as it can be captured by, for example, immobilized antibody AI of antibody A bound to fixing compound M using an antigen-antibody reaction. Examples of antigens include viruses, hormones, allergens, bacteria, and metals.

[0015] The antibody A is not particularly limited as long as it can bind to the fixing compound M and, in the bound state, capture an antigen in the test solution described below or a conjugate LC (sometimes referred to as an immune complex or an antigen-binding antibody), which is a complex of an antigen and a labeled antibody AL. Examples of the antibody A include an immobilized antibody AI (sometimes referred to as a solid-phase antibody or a capture antibody) and a control antibody AC, as shown in Figures 1 to 3. The antibody A is selected appropriately depending on the type of antigen, etc., and is fixed to the surface 10US of the detection section 12 of the flow path section 10 of the testing substrate 1 of this embodiment in advance of the test by coating or the like, and is then bound to the fixing compound M. In other words, at the time of the test, the antibody A is supported on the detection section 12 and is in an immobilized state.

[0016] 1(C) is a schematic explanatory diagram of the enlarged view of the left side of FIG. 1(B) (a schematic cross-sectional view taken along line BB in FIG. 1(A)) as viewed from the liquid advance direction. FIG. 2 is a schematic cross-sectional view of the case where the forming member 13 of the flow path section 10 contains granular members 15 and a binder member 16 in addition to fibers 14, and FIG. 2(B) is a schematic explanatory diagram of the enlarged view of the left side of FIG. 2(A) as viewed from the liquid advance direction.

[0017] As described above, when a test liquid is supplied to the flow path section 10 of the testing substrate 1 of this embodiment, the test liquid spreads in the internal mesh-like voids 10h in the direction of flow due to capillary action. When the spread test liquid reaches the region 12a of the detection section 12 where the immobilized antibody AI is applied, the antigen or conjugate LC in the test liquid is captured by the immobilized antibody AI (see FIG. 3). Then, color develops in the application region 12a depending on the amount of antigen, etc. captured by the immobilized antibody AI, making it possible to visually confirm the presence or absence of the antigen in the test liquid. In other words, the testing substrate 1 of this embodiment is formed to exhibit both the function of transporting the test liquid (flow path performance) and antibody fixation properties.

[0018] Moreover, the flow path section 10 of the testing substrate 1 of this embodiment contains a large number of fibrous members 14 (fibers 14) as the forming members 13. The above-mentioned gaps 10h are formed between the large number of fibers 14 (see FIGS. 1 and 2), and the gaps 10h formed between the fibers 14 form a flow path through which the test liquid moves. In other words, the gaps 10h formed between adjacent fibers 14 form a mesh-like network inside the flow path section 10, thereby forming a plurality of fine flow paths through which the liquid passes. Therefore, even when pressure such as pressurization or bending is applied to the flow path section 10, the contained fibers 14 can suppress plastic deformation such as cracking or crushing in the thickness direction, thereby appropriately maintaining the shape of the flow path for the movement of the test solution. As a result, even if pressure or the like is applied during transportation or handling, the shape of the flow path can be appropriately maintained, so that antigens and the like can be appropriately accumulated in the detection section 12.

[0019] However, conventional products have low strength and are prone to plastic deformation such as peeling and denting when pressure is applied. This causes the internal flow path shape to collapse or deform, making it impossible to properly move the test liquid. In particular, even slight deformations that are not discernible from the outside can cause the flow path to collapse, making proper testing difficult.

[0020] Therefore, by incorporating fibers 14 as the forming member 13 in the flow path portion 10, the testing substrate 1 of this embodiment can improve its mechanical strength compared to conventional products, thereby improving the reliability of analysis. Furthermore, conventional products require packaging the testing substrate with an excess protective sheet or the like during transportation to prevent a sudden delay in the test liquid advancement time or complete cessation of the test liquid advancement due to a slight pressure, etc. In contrast, the testing substrate 1 of this embodiment can improve its mechanical strength as described above, thereby significantly improving its handleability compared to conventional products. Furthermore, improving the strength characteristics of the flow path portion 10 improves the handleability during the manufacturing process of the testing substrate 1 of this embodiment, thereby improving the productivity of the testing substrate 1 of this embodiment and improving the yield in the manufacturing process, thereby reducing manufacturing costs.

[0021] Next, the testing substrate 1 of this embodiment will be described in detail.

[0022] (flow path section 10) As shown in FIGS. 1 and 2, the flow path section 10 contains fibers 14, thereby improving the mechanical strength.

[0023] This mechanical strength can be expressed by the change in thickness of the flow path portion 10 when a predetermined load is applied. Specifically, this change in thickness is a value calculated based on the ratio of the thickness before pressing to the thickness after pressing (thickness after pressing / thickness before pressing) when pressing with a predetermined press load. For example, the thickness change of the flow path section 10 when the press load is 23.1 MPa is 0.3 or more, preferably 0.4 or more, and more preferably 0.5 or more. Furthermore, when the press load is 9.9 MPa, the thickness change is 0.4 or more, preferably 0.5 or more, more preferably 0.6 or more, and even more preferably 0.7 or more. Furthermore, when the press load is 3.3 MPa, the thickness change is 0.75 or more, preferably 0.8 or more, and more preferably 0.9 or more.

[0024] Since the thickness change of the flow path section 10 is within the above value, the shape of the gap 10h (i.e., the flow path through which the test liquid passes) can be appropriately maintained. Therefore, even when a load is applied to the flow path section 10, the test liquid can be appropriately passed through.

[0025] The mechanical strength of the flow path section 10 can also be expressed as the rate at which the shape of the voids is maintained, and this rate can be expressed as the time required for the test liquid to move through the flow path section 10 (referred to as the liquid advance time change). Specifically, this liquid advance time change is a value calculated based on the ratio of the liquid advance time of the test liquid before pressing to the liquid advance time of the test liquid after pressing (liquid advance time after pressing / liquid advance time before pressing) when a predetermined load is applied to the flow path section 10 as described above, and the smaller the liquid advance time change, the more the shape of the voids 10h (flow paths) is maintained. The time required for the test solution to advance can be measured using the half-strip method (HS). Details are given in the "Measurement of the advancement rate" section of the Examples. The test solution used in HS is a 1% diluted nickel dye. For example, in the flow path section 10, when the press load is 23.1 MPa, the liquid advance time change is 5 or less, preferably 4 or less, more preferably 3 or less, and even more preferably 2 or less. Furthermore, when the press load is 9.9 MPa, for example, the liquid advance time ratio is 3 or less, preferably 2 or less.

[0026] Since the flow path section 10 of the testing substrate 1 of this embodiment has the above-mentioned mechanical strength, it is possible to control the rate at which the test liquid advances depending on the type of test liquid, the target substance, etc. In other words, it is possible to control the rate at which the test liquid advances by applying a predetermined press load to the flow path section 10.

[0027] The forming member 13, which is a factor in determining the mechanical strength of the flow path portion 10, will be specifically described below.

[0028] (Forming member 13) The forming member 13 of the flow path section 10 may contain other materials in addition to the fibers 14. Note that the forming member 13 does not contain the fixing compound M. For example, as shown in Fig. 2, the forming member 13 may contain granular members 15 and a binder member 16. The granular members 15 are granular members arranged between the fibers 14 (i.e., within the voids 10h), and the binder member 16 is a member that has the function of connecting the fibers 14 to each other or connecting the fibers 14 to the granular members 15, etc. Each component will be described in detail below.

[0029] (Fiber 14) First, the fibers 14 are not particularly limited as long as they are members whose aspect ratio (fiber length / fiber diameter) is greater than 1. In other words, the fiber F is not particularly limited in terms of fiber diameter or fiber length, and may be any member whose aspect ratio is greater than 1. For example, the fibers 14 may be of a size that is used for general filter paper, etc. In other words, the flow path portion 10 can be formed by mixing the fibers 14 of such a size with the fixing compound M using a paper machine or the like. Note that the material of the fibers 14 in this case is not particularly limited.

[0030] The fibers 14 may have a diameter of about 1 μm to 500 μm and a length of about 2 μm to 5 mm. The fibers 14 may also include nanofibers made of cellulose, resin, or the like, having an average diameter of about 1 nm to 100 nm and an average length of about 100 nm to 1 μm. The material of the fibers 14 is not particularly limited. For example, the material of the fibers F may be natural materials (natural fibers) whose main component is cellulose, such as pulp or cotton, synthetic materials (chemical fibers), such as rayon, polyethylene terephthalate (PET), polyethylene (PE), polypropylene (PP), or polyvinyl alcohol (PVA), materials made from calcium carbonate, silicon dioxide, such as glass fiber, metal materials, such as metal fiber, or carbon fiber. Note that the fibers 14 may include not only those made of a single fiber, but also fiber aggregates in which multiple thin fibers are bundled together.

[0031] In particular, when the fibers 14 contain nanofibers, the nanofibers can be arranged in a state in which they penetrate into gaps such as between the granular components 15, between the fibers 14, and between the fibers 14 and the granular components 15. The penetrated nanofibers are very thin fibers, and have a specific surface area that is much larger than that of ordinary fibers. Therefore, by disposing nanofibers in the gap, the contact area between the forming member 13 and the fixing compound M can be increased. As a result, when the fixing compound M is fixed by coating or the like, the fixing compound M can be more firmly immobilized on the forming member 13, and the fixing compound M can be appropriately prevented from moving with the test liquid or from detaching. Furthermore, since the contact area can be increased, it is possible to increase the content of the fixing compound M in the flow path section 10, and therefore the amount of conjugate LC captured by the fixing compound M can be increased.

[0032] (Granular material 15) The granular members 15 are members that are provided so as to be positioned between the fibers 14, and there are no particular limitations on their size or shape, as long as they can be provided in this manner. Examples include members having various shapes such as blocky members, spherical members, and particulate members. The material of the granular members 15 is also not particularly limited, and examples include synthetic resins such as polyethylene terephthalate (PET), polyethylene (PE), and polypropylene (PP), calcium carbonate, and silicon dioxide.

[0033] For example, particles having a particle size of 0.1 μm to 100 μm can be used for the granular members 15. This size is preferably 1 μm to 50 μm, and more preferably 10 μm to 25 μm.

[0034] If granular members 15 within this size range are used, they can be placed between the fibers 14 to narrow the gaps 10h (flow paths) formed between adjacent fibers 14 and increase the flow path resistance, thereby increasing the pressure loss of the fluid moving through the flow path compared to when no granular members 15 are provided, thereby slowing down the advancement speed of the test liquid. In other words, by incorporating granular material 15, it is possible to adjust the advancement speed of the test liquid. When the test liquid contains a small amount of antigen, the contact time with antibody A can be increased by moving the test liquid slowly, thereby enabling appropriate determination of the presence or absence of antigen. This makes it possible to adjust the advancement speed of the test liquid depending on the concentration of the antigen in the test liquid, etc.

[0035] Furthermore, the inclusion of granular members 15 can improve the smoothness of the surface 10US of the flow path section 10. By reducing the irregularities of the surface 10US and improving the smoothness, antibody A can be applied uniformly within a predetermined area, allowing it to be fixed uniformly and without unevenness. In particular, when applying immobilized antibody AI or control antibody AC in a strip-like shape, it can be applied neatly, allowing the edge of the antibody line to be formed sharply. If the antibody line is sharp, conjugate LC or labeled antibody AC can be uniformly accumulated along the antibody line edge, which has the advantage of making the colored antibody line more visible. Furthermore, when the binder material 16 is contained together with the particulate material 15, the effect of the binder is improved, and there is also an effect of improving the strength and smoothness.

[0036] The size of the particulate material is expressed as the average particle size, which is the value at which the volume-based cumulative distribution calculated in accordance with JIS-Z-8819-2 (2001) is 50% based on the particle size distribution measured by the laser diffraction / scattering method in accordance with JIS-Z-8815 (2013).

[0037] (binder member 16) The binder member 16 is not particularly limited as long as it can connect adjacent fibers 14 together. For example, the binder member 16 can be made of synthetic resins such as vinyl chloride, vinyl acetate, silicone resin, acrylic resin, polyethylene terephthalate (PET), polyethylene (PE), polypropylene (PP), and polyvinyl alcohol (PVA), or natural resins such as natural rubber and starch. For example, using a synthetic material such as PVA as the binder member 16 offers the advantage of chemical resistance. Furthermore, the above-described nanofibers may be used as the binder member 16 because they have the function of connecting adjacent fibers 14 by entangling with each other. The binder member may be one of the above-mentioned materials or a mixture of two or more materials. For example, if a synthetic resin such as polyvinyl alcohol (PVA) and nanofibers are used as the binder member 16, the strength can be further improved.

[0038] The binder member 16 may be made of nanofibers as described above. As described above, nanofibers are very thin fibers formed with an average fiber diameter of about 1 to 100 nm and an average fiber length of about 100 nm to 1 μm. If such fibers are entangled to form an aggregate, they can function as the binder member 16. For example, if the forming member 13 contains nanofibers, a nanofiber membrane can be formed, or a nanofiber layer can be formed that bundles a plurality of fibers 14. Furthermore, this nanofiber layer can be formed to bundle a plurality of fibers 14 and a plurality of granular members 15 arranged between them. Furthermore, since the nanofiber membrane is an aggregate of very fine fibers, it can have very low water permeability, so if it is formed on the back surface 10BS of the flow path section 10, it can also prevent leakage of the test solution. Furthermore, nanofibers are known to be lightweight, highly elastic, high strength, and low linear thermal expansion, so it can further improve strength.

[0039] Nanofibers can be prepared by mechanically or chemically micronizing raw material components (e.g., fibers). The method for micronizing raw material fibers is not particularly limited, and for example, low-pressure homogenizers, high-pressure homogenizers, grinders, cutter mills, jet mills, single-screw extruders, twin-screw extruders, ultrasonic mixers, etc. can be used to convert fibers into nanofibers. The fibers fed to the machine are not particularly limited, and examples include chemical fibers and pulp fibers. The raw material for pulp fibers is not particularly limited, and can be anything that contains cellulose as a main component, such as wood flour, plant residues, and papermaking residues.

[0040] The flow path section 10 may be formed using a sheet member of the forming member 13 containing, in addition to the above-described fibers 14, granular members 15, a binder member 16, and the like. Examples include a sheet-like functional material having polyethylene terephthalate (PET) fibers, a cellulose nanofiber membrane, and a cellulose nanofiber layer (the functional material described in International Publication No. 2015 / 152287), and a sheet member containing PET fibers, PET particle members, and a resin member. In the former functional material, the PET fibers correspond to the fibers 14, and the cellulose nanofiber membrane and cellulose nanofiber layer correspond to the binder member 16. In the latter sheet member, the PET fibers correspond to the fibers 14, the PET particle members correspond to the granular members 15, and the resin member corresponds to the binder member 16.

[0041] (Detection unit 12) Next, the detection unit 12 of the flow path unit 10 will be specifically described. As shown in Figures 1 and 2, the detection section 12 has an area 12a in the flow path section 10 where the immobilized antibody AI of antibody A is applied as described above, and this is the part where the applied immobilized antibody AI is fixed to the fixing compound M that is arranged in a supported state on the surface of the forming member 13 or within the void 10h.

[0042] This fixing compound M is arranged throughout the entire flow path section 10, but in particular in the detection section 12, the antibody A is fixed to the fixing compound M in the region 12a where the immobilized antibody AI of antibody A is applied, making it possible to detect the presence or absence of the antigen in this region 12a.

[0043] (Fixing Compound M) As described above, the fixing compound M has the function of binding to the antibody A applied to the flow path section 10 used in tests such as immunoassays. Examples of the antibody A that binds to the fixing compound M include the immobilized antibody AI (antibody A that specifically binds to an antigen or conjugate LC) in the above-mentioned immunochromatography, and the control antibody AC that captures the labeled antibody AL (antibody A that specifically binds to the labeled antibody AL). The conjugate LC is a complex in which the antigen is bound or linked to the labeled antibody AL.

[0044] The fixing compound M is not particularly limited as long as it has the function of binding to the above-mentioned predetermined antibody A. For example, the fixing compound M may be a compound having a predetermined functional group. For example, amino groups include aniline, hydroxyl groups include nitrocellulose, nitrophenol, and cellulose, carboxy groups include cyclohexylcarboxylic acid and nitrobenzoic acid, sulfo groups include sodium nitrobenzenesulfonate, phenyl groups include aniline, benzoic acid, and methyl nitrobenzoate, cyclohexyl groups include cyclohexanecarboxylic acid, ester groups include methyl nitrobenzoate, alkoxy groups include anisole, nitro groups include methyl nitrobenzoate, nitrate ester groups include nitrocellulose, amide groups include acetanilide, aldehyde groups include benzaldehyde, ketone groups include acetophenone, ether groups include polyethers, halogen groups include aryl halides, nitrile groups include benzonitrile, phosphate groups include diphenyl phosphate, silicic acid groups include phenyl orthosilicate, and acid anhydride groups include phthalic anhydride. The fixing compound M may have only one type of the above-mentioned functional group (active site), or may have two or more types. Examples of the fixing compound M include the following compounds. Examples of such compounds include nitro compounds such as methyl nitrobenzoate, nitric acid ester compounds such as nitrocellulose, aromatic compounds such as benzoic acid, amine compounds such as aniline, carboxy compounds such as nitrobenzoic acid, alkoxides such as anisole, polysaccharides such as nitrocellulose, and sulfuric acid compounds such as nitrobenzenesulfonic acid. The fixing compound M may consist of only one of these compounds, or may contain two or more of them.

[0045] The present inventors have first discovered, from among countless commonly available compounds, a fixing compound M that has the property of being able to bind to antibody A (antibody fixing function) and that has the function of being easily retained within the flow channel section 10. Then, by fixing this fixing compound M within the flow channel section 10, it has become possible to appropriately capture an antigen in the region of the flow channel section 10 where antibody A has been fixed (detection section 12).

[0046] It is preferable that the fixing compound M is poorly soluble in the solvent (e.g., an aqueous solvent) in the test liquid so that it can be easily retained within the flow path section 10 (i.e., so that the bound antibody A can easily remain within the flow path section 10). For example, when the solvent of the test liquid is aqueous, using a fixing compound M that is poorly soluble in water can prevent the fixed fixing compound M from being separated, thereby improving the sharpness of the colored line.

[0047] Furthermore, the fixing compound M preferably has the property of being easily supported by the forming member 13 of the flow path section 10. Specifically, if the fixing compound M has a high affinity for the forming member 13, it can be more appropriately immobilized and supported within the detection section 12 of the flow path section 10. This makes it possible to more appropriately prevent the fixing compound M from moving downstream along with the test liquid when the test liquid moves downstream through the detection section 12. In this case, it is possible to more appropriately prevent the occurrence of phenomena such as bleeding or lightening of the color development in the detection section 12 (such as a state in which the test liquid is difficult to visualize or a state in which coloring corresponding to the amount of antigen is not obtained), thereby improving the sharpness of the color development line and thereby improving the analytical accuracy.

[0048] In addition, the fixing compound M may have the following properties in relation to the forming member 13. For example, a fixing compound M having hydrophobic or hydrophilic properties, or both, is employed. Then, a forming member 13 having the same properties as the adopted fixing compound M is employed. Specifically, if the fixing compound M is hydrophilic, a forming member 13 having hydrophilic properties is also used. On the other hand, if the fixing compound M is hydrophobic, a forming member 13 having hydrophobic properties is also used. In this case, by using both compounds having similar properties, the bonding strength between them can be improved, and the fixing compound M can be firmly bonded to and fixed on the forming member 13. In the former case, if the forming member 13 contains fibers 14 made of hydrophilic cellulose, the fixing compound M can also be hydrophilic (for example, sodium nitrobenzenesulfonate or nitrophenol). In the latter case, if the forming member 13 contains fibers 14 made of resin, the fixing compound M can also be hydrophobic (for example, nitroaniline).

[0049] On the other hand, if the properties of the fixing compound M and the forming member 13 are different (for example, if one is hydrophobic and the other is hydrophilic), the bonding strength between them may be weak if left as is. For this reason, it is preferable to provide a linking compound between them to connect them. By providing a linking compound, the fixing compound M and the forming member 13 can be appropriately fixed via the linking compound. As an example of the linking compound, the forming member 13 may be a fiber 14 made of hydrophilic cellulose, and hydrophobic methyl nitrobenzoate (NBM) may be used as the fixing compound M. In such a case, nitrocellulose, which is also used as the fixing compound M, may be included as the linking compound. When nitrocellulose is used as the fixing compound M, cellulose nanofibers can be used as the linking compound, and substances having surfactant properties can also be used as the linking compound.

[0050] The time it takes for the test liquid to spread can be controlled by adding a fixing compound at a predetermined ratio. That is, the flow rate at which the test liquid spreads can be controlled by the fixing compound added to the flow path section 10. For example, the fixing compound M may be sodium nitrobenzenesulfonate (NS), nitrophenol (NP), nitroaniline (NA), or nitrobenzoic acid (NB). For example, when 3% by mass of sodium nitrobenzenesulfonate (NS) is contained as the fixing compound M, the liquid advance time ratio can be 0.9 or less, preferably 0.7 or less, and more preferably 0.5 or less. When 3% by mass of nitrophenol (NP) is contained as the fixing compound M, the liquid advance time ratio can be 0.9 or less, preferably 0.8 or less, and more preferably 0.6 or less. When 3% by mass of nitrobenzoic acid (NB) is contained, the liquid advance time ratio can be 0.9 or less, preferably 0.85 or less. On the other hand, when 3% by mass of nitroaniline (NA) is contained as the fixing compound M, the liquid advance time ratio can be 0.9 or more, preferably 1 or more.

[0051] The fixing compound M preferably has the property of easily binding to antibodies such as the immobilized antibody AI that binds to the antigen or the conjugate LC, but not of nonspecifically adsorbing to other substances. If the fixing compound M has nonspecific adsorption properties, it may react with test solutions that do not contain the antigen (blanks, control samples), which may lead to misinterpretation of the analytical evaluation.

[0052] The shape of the flow path portion 10 is not particularly limited. For example, the shape can be formed into various shapes such as a strip shape or a rectangle as shown in FIGS. 1 to 3, a circle, an ellipse, a radial shape, and the like. For example, in the case of a rectangular shape, a length in the major axis direction of 5 mm to 100 mm and a length in the minor axis direction of 2 to 50 mm in a plan view (as viewed from the surface 10US of the flow path portion 10) can be used, but the size and shape can be adjusted appropriately depending on the application. Also, in the case of a radial shape, the flow path can be branched to measure multiple items simultaneously. The thickness is not particularly limited. For example, the thickness can be formed to be 50 μm to 500 μm. The thickness is preferably 50 μm to 300 μm, and more preferably 50 μm to 250 μm. In particular, from the viewpoint of ease of handling, the thickness is preferably 100 μm to 200 μm.

[0053] (Method for manufacturing the testing substrate 1 of this embodiment) Next, a method for manufacturing the testing substrate 1 of this embodiment will be described.

[0054] The method for manufacturing the testing substrate 1 of this embodiment includes a flow path portion forming step for forming the flow path portion 10, and a cutting step. In the flow path forming step, first, a mixed liquid color is prepared by mixing the fixing compound M and the forming member. The forming member may contain, in addition to the fibers 14, a binder member 16, a granular member 15, etc. as needed. For example, a mixed liquid color can be prepared by adding the fixing compound M to an aqueous solvent or an organic solvent and stirring, and then adding forming members 13 such as the fibers 14 to this stirred liquid and further stirring it.

[0055] The fixing compound M to be mixed into the mixed liquid color is the above-mentioned benzoic acid, methyl nitrobenzoate, etc., and may consist of one of these compounds or may contain two or more of them. The mixed color liquid may also contain components such as a linking compound, a buffer solution, and a surfactant. For example, the linking compound has the function of improving the connectivity between the fixing compound M and the forming member 13 when they have different properties (e.g., hydrophilicity and hydrophobicity). For example, nitrocellulose is a linking compound for methyl nitrobenzoate, and cellulose nanofiber is a linking compound for nitrocellulose.

[0056] The mixed color liquid may be prepared while being heated. If necessary, a degassing treatment may be carried out. By carrying out the degassing treatment, the coated surface after coating can be made smooth.

[0057] The prepared mixed liquid color is used to form a sheet-like sheet member using a predetermined coating machine. This sheet member may be formed solely from the sheet member, or may include a sheet substrate capable of supporting the sheet member. For example, in the former case, the mixed liquid color is applied to a resin sheet substrate in sheet form and then dried. The sheet member can then be formed by peeling the sheet member from the sheet substrate. If the sheet substrate is resinous, the two can be easily peeled off. In the latter case, the mixed liquid color is applied to the surface of the sheet substrate corresponding to the base portion 30 described below, thereby forming a sheet member including the sheet substrate. The sheet substrate is preferably made of a material that is impermeable to the solvent of the mixed liquid, such as waterproof paper or a resin sheet.

[0058] After the mixed liquid color is applied to the sheet, the sheet member is supplied to a drying process. The drying method is not particularly limited. For example, natural drying or mechanical drying can be used. For mechanical drying, for example, a rotary dryer can be used. Use of a rotary dryer allows for high-speed drying and surface pressure application, thereby improving the bonding strength between the fibers 14 and the binder member 16. In this case, there is an advantage in that the strength (compression strength) of the flow path portion 10 to be formed can be improved.

[0059] Here, by subjecting the mixed liquid color applied to the surface of the sheet substrate to a drying process, the concentration of the fixing compound M can be arranged so that it is higher in the layer located on the surface side than in the layer corresponding to the back side (the layer located on the surface side of the sheet substrate). This phenomenon of moving the fixing compound M during the drying process is called migration, and the fixing compound M in the applied mixed liquid color can be moved toward the surface side as the solvent in the mixed liquid color evaporates. In other words, by utilizing migration during the drying process, it is possible to form the layer on the surface side so that the content of the fixing compound M is greater than that of the layer on the back side in the thickness direction of the sheet member.

[0060] Next, the dried sheet member is subjected to a step of cutting into a predetermined size and shape (cutting step). In this cutting step, the sheet member is cut into a predetermined size depending on the intended use. For example, when used in immunochromatography, the testing substrate 1 of this embodiment can be obtained by cutting it into strips. In this case, the testing substrate 1 of this embodiment can be obtained, which includes a flow path portion 10 having a main body portion 11 and a detection portion 12 continuously connected to the main body portion 11 in the order from one end of the short axis along the long axis (from the left in FIG. 3).

[0061] As described above, the testing substrate 1 of this embodiment having the flow path portion 10 can be manufactured by a simple operation of simply applying the mixed liquid color to a sheet. The obtained testing substrate 1 of this embodiment has flow path performance and antibody fixation ability without providing any other parts. Therefore, since the testing substrate 1 of this embodiment can be manufactured by a simple operation as described above, productivity can be improved compared to conventional products manufactured by assembling multiple parts. Moreover, manufacturing costs can be reduced. Furthermore, by forming the testing substrate 1 as an integral part, evaluation errors that occur when assembling parts can be eliminated, thereby improving quality.

[0062] (How to use) Next, a method of using the testing substrate 1 of this embodiment will be described. When measuring an antigen in a test liquid using the testing substrate 1 of this embodiment, it is used as follows. In the following, a case where an antigen in a test liquid is measured by immunochromatography will be described as a representative example, and the testing substrate 1 of this embodiment will be described as a representative example where it is formed in a strip shape.

[0063] First, the testing substrate 1 of this embodiment is prepared in advance as follows according to the antigen to be tested.

[0064] (Application of immobilized antibody AI) As shown in Fig. 1, immobilized antibodies AI are applied to a predetermined region in the detection section 12 of the flow path section 10 of the testing substrate 1 of this embodiment. When applying, the immobilized antibodies AI are applied in a strip shape that intersects (e.g., is approximately perpendicular to) the direction of the test liquid advancement. In other words, as shown in Figs. 1 to 3, the immobilized antibodies AI are applied in a line that is parallel to the minor axis direction of the flow path section 10. The region to which the immobilized antibodies AI are applied is the region 12a in the detection section 12. The immobilized antibody AI is capable of specifically binding to an antigen. The method for applying the immobilized antibody AI is not particularly limited, and for example, a dispenser or an inkjet printer can be used. The applied immobilized antibody AI then binds to and is fixed by a fixing compound M carried on the forming member 13 of the detection unit 12.

[0065] (Application of control antibody AC) 1 to 3, in order to confirm that the test liquid has reliably moved, a control antibody AC, which is an antibody A for capturing the labeled antibody AL, is applied to the detection unit 12. The region to which this control antibody AC is applied is the region 12b in the detection unit 12. During testing, labeled antibodies AL that do not form a complex with the antigen are not captured by the immobilized antibodies AI (corresponding to the test line) that capture the antigen, but instead migrate to and are captured in region 12b downstream of this line, where control antibodies AC that capture the labeled antibodies AL themselves have been fixed. In region 12b, color develops as the labeled antibodies AL are captured by the control antibodies AC. This makes it possible to determine whether the test liquid has moved appropriately.

[0066] This region 12b is formed by applying the control antibody AC in a strip-like manner downstream (downstream in the direction of the test liquid advance) of the region 12a where the immobilized antibody AI is applied, so that the strip-like application crosses (for example, is substantially perpendicular to) the direction of the test liquid advance. That is, as shown in Figures 1 to 3, when viewed from the surface, the detection unit 12 is formed with two antibody lines, a strip-like antibody line (also called a test line) where the immobilized antibody AI is applied and a strip-like antibody line (also called a control line) where the control antibody AC is applied, so that they are substantially parallel to each other with a predetermined distance between them.

[0067] The detection section 12 may be provided with a test line in which a strip of antibody A other than the immobilized antibody AI is applied. For example, if multiple antigens a and b are present in the test liquid, and antigen a (conjugate LC1) is captured by immobilized antibody AIa and antigen b (conjugate LC2) is captured by immobilized antibody AIb, test line 2 is formed between test line 1, which is a strip of immobilized antibody AIa, and control line, which is a strip of control antibody AC, and immobilized antibody AIb is applied in a strip, so as to be approximately parallel to each other. In other words, when viewed from surface 10US, detection unit 12 has three antibody lines (two test lines 1 and 2 and one control line) that are approximately perpendicular to the direction of liquid advance and are formed approximately parallel to each other at a specified interval. By using the testing substrate 1 of this embodiment, even if there are multiple antigens to be tested in the test liquid, multiple antibody lines can be easily formed in the desired positions in the same manner, thereby improving customizability.

[0068] (Labeled antibody AL application) When analyzing by immunochromatography, the labeled antibody AL described above is generally used. This labeled antibody AL is an antibody labeled with a labeling substance, such as a mouse anti-hα-subunit antibody. Examples of labeling substances include chromogenic substances and fluorescent substances. Examples of chromogenic substances include gold colloids, silver colloids, latex beads, and quantum dots. 3, for example, the labeled antibody AL can be applied to the main body 11 of the flow path 10 of the testing substrate 1 of this embodiment. This applied region is the region 11b of the main body 11. This region 11b is applied in a strip shape between the region 11a (test liquid supply region 11a) of the main body 11 of the flow path 10 that supplies the test liquid and the detection unit 12, so as to intersect (for example, approximately perpendicular) with the direction of advance of the test liquid.

[0069] (Development of test solution) Next, as shown in Fig. 3(A), the test liquid is dropped and supplied to the test liquid supply region 11a of the flow path portion 10 of the prepared testing substrate 1 of this embodiment. The supplied test liquid is spread toward the detection portion 12 by capillary action. Then, when the test liquid reaches the region 11b of the main body portion 11, a conjugate LC is formed in which the antigen is combined with the labeled antibody AL by an antigen-antibody reaction (Fig. 3(B)). As shown in Figure 3(C), the formed conjugate LC continues to move in the liquid advance direction due to capillary action and reaches the detection unit 12. Upon arriving at the detection unit 12, the conjugate LC is captured by the immobilized antibody AI through an antigen-antibody reaction and remains in region 12a, where it is fixed to the formation member 13 via the fixing compound M. Meanwhile, other substances in the test liquid, such as the solvent, pass through region 12a due to capillary action along the liquid advance direction. This means that only the conjugate LC remains in region 12a, where the immobilized antibody AI is applied. The solvent and free labeled antibody AL that have passed through region 12a then reach region 12b, located downstream, where the free labeled antibody AL is captured by the control antibody AC that has been fixed in region 12b. This means that only free labeled antibody AL remains in region 12b, where the control antibody AC is applied. Then, in the detection section 12, the control line develops color, making it possible to confirm that the test liquid has reliably moved, and in the region 12a, if an antigen is present in the test liquid, the antibody line develops color (coloration).

[0070] In particular, the fixable compound M in the detection unit 12 can be made to exist in a large amount in the layer on the surface 10US side due to the migration phenomenon described above, which makes it easier for the immobilized antibody AI to accumulate in this layer. This makes it easier for the conjugate LC to accumulate on the surface 10US side of the detection unit 12, making it easier to visually determine the presence or absence of the antigen in the test liquid even from the surface 10US of the detection unit 12 in the flow path unit 10. The conventional product has a porous channel made of a polymer (nitrocellulose membrane) that allows for uniform antibody fixation across the cross section of the channel. Therefore, the applied immobilized antibody AI is uniformly distributed within the porous channel, and in the conventional product, the conjugate LC is uniformly immobilized within the porous channel. In other words, when the same amount of test liquid is spread between the testing substrate 1 of this embodiment and a conventional test kit, the testing substrate 1 of this embodiment is more likely to accumulate a larger amount of conjugate LC on the surface 10US side of the detection unit 12 than the conventional test kit. Therefore, the use of the testing substrate 1 of this embodiment can achieve color development intensity equal to or greater than that of the conventional product.

[0071] The concentration of the target substance in the test solution can be appropriately quantified by measuring the intensity of color development resulting from the labeling substance using a measuring device (for example, measuring absorbance using an immunochromatography reader).

[0072] (Base part 30) 1 to 3, the testing substrate 1 of this embodiment may include a base portion 30. This base portion 30 is a member capable of disposing and holding the flow path portion 10 on its surface. This base portion 30 is formed, for example, so that its size is the same as or slightly larger than the flow path portion 10 it holds, and its shape is formed so as to be approximately similar to that of the flow path portion 10. If the testing substrate 1 of this embodiment includes the base portion 30, bending of the flow path portion 10 can be suppressed, and thus handling can be improved.

[0073] The material of the base 30 is not particularly limited, and examples thereof include paper and resin. In particular, the base 30 is preferably impermeable, and the base 30 can be made of, for example, waterproof paper.

[0074] (Absorbing section 20) 1 to 3, the testing substrate 1 of this embodiment may be provided with an absorbing section 20 capable of absorbing liquid, connected to the downstream side of the flow path section 10 in the direction of advancement of the test liquid. This absorbing section 20 is configured to absorb the test liquid once the test liquid reaches the connecting section of the flow path section 10 with the detection section 12. This prevents the movement of the test liquid from stopping at the tip of the detection section 12 when the test liquid has moved to the end of the connecting section of the detection section 12, and therefore allows the added test liquid to reliably pass through the region 12a, enabling more appropriate measurement.

[0075] The absorption unit 20 is provided so as to be in contact with the detection unit 12 of the flow path unit 10, and there is no particular limitation on the arrangement method thereof, as long as it can absorb the test liquid that has moved to the downstream end of the detection unit 12. For example, it may be connected horizontally to the downstream end of the detection unit 12, or it may be provided so that the top surface of the downstream end of the detection unit 12 and the bottom surface of the base end of the absorption unit 20 are in contact.

[0076] The absorbent part 20 is not particularly limited as long as it can absorb liquid. For example, a porous member having many mesh-like voids (flow paths through which liquid passes) formed therein can be used as the absorbent part 20, and examples of such materials include filter paper, glass fiber filter paper, nonwoven fabric, and resin sponge members. In particular, when the absorption section 20 is formed from the same material as the flow path section 10, the flow path section 10 and the absorption section 20 can be formed integrally. For example, as shown in Figures 1 to 3, when the testing substrate 1 of this embodiment is formed in a strip shape, the testing substrate 1 of this embodiment can be formed in which the flow path section 10 (main body section 11, detection section 12) and the absorption section 20 are connected in this order from one end on the short axis side along the long axis direction. [Example]

[0077] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0078] A testing substrate (Sample A) of the present invention was prepared as follows.

[0079] (Preparation of Sample A) Sample A had a flow path portion attached to the top surface of the base portion. The flow path portion was made by mixing PET fibers as the fibers, PET particles as the interfiber components, and PVA and nanofibers as the binder. This mixture was further mixed with a fixing compound to form a mixed liquid color. This was applied to the waterproof paper base portion using an applicator (Tester Sangyo Co., Ltd., PI-1210-S), and then dried to form an integral part of the waterproof paper. After drying, the flow path portion was cut into strips of a specified size.

[0080] Sample A, formed in a strip shape, had a length (length in the long axis direction) of approximately 60 mm and a width (length in the short axis direction) of approximately 5 mm in plan view. Sample A had a structure in which the flow path section was held by a base section, and its thickness (length from the front surface to the back surface, including the base section) was approximately 350 μm (thickness of the flow path section: approximately 150 μm (distance from the surface of the flow path section to the top surface of the base section) + thickness of the base section: approximately 100 μm (distance from the top surface of the base section to the back surface)).

[0081] The region in which the immobilized antibody is applied and fixed in a strip shape, approximately perpendicular to the liquid advance direction at a predetermined interval from the test liquid supply region of the flow path section, corresponds to the detection section in the testing substrate of this embodiment.

[0082] FIG. 4 shows SEM (scanning electron microscope) images of the cross section and surface of the flow channel portion of Sample A.

[0083] As a comparative example, a nitrocellulose membrane, which is widely available commercially and is used as a flow channel for immunochromatography, was used. Another comparative example was similar to sample A except that no fixing compound was applied.

[0084] Antibodies (immobilized antibodies, control antibodies) were diluted 5-fold with pure water and applied using a dispenser at a volume of 0.30 to 35 μL / cm. After application, the antibody was left to dry for 12 hours before use.

[0085] The half-strip method (HS) was used to measure the rate of advancement. This measurement method involves injecting 110 μL of 1% diluted nickel dye (nickel(II) phthalocyanine-tetrasulfonic acid tetrasodium salt, Sigma-Aldrich Japan Co., Ltd. 274909-5G) into a glass screw bottle, spreading it horizontally, inserting a sample vertically, and measuring the time it takes for the nickel dye to reach a depth of 30 mm to 40 mm. In measuring the rate of advancement, the 1% diluted nickel dye corresponds to the test solution.

[0086] To measure color intensity, mix and stir the following components in the specified volume ratio (developer solution: 749, blocking solution: 749, HCG: 2, gold colloid-labeled antibody: 113.75), in the following order: developing solution (ELISA Wash Buffer, Bethyl Laboratories E106), blocking solution (ELISA Blocking Buffer, Bethyl Laboratories E104), HCG antigen (human chorionic gonadotropin, a glycoprotein hormone, Sigma-Aldrich Japan, C1063-1VL), and colloidal gold antibody (Gold Anti-hCG beta antibody [M705159], Abcam, ab31206). The test strip is immersed in 110 μL of test solution using the half-strip method, and the color intensity is measured after 15 and 30 minutes using an immunochromato reader. In this case, the gold colloid-labeled antibody corresponds to the labeled antibody.

[0087] The film thickness was measured using a paper thickness meter TM600-L manufactured by Kumagai Riki Kogyo Co., Ltd.

[0088] First, the effect of the fixing compound on Sample A was confirmed. An antibody (immobilized antibody: Anti-h Alpha Subunit 6601 SPR-5, Medix Biochemica, 100066) was applied to a predetermined region of sample A. The portion having the region to which the immobilized antibody was applied corresponds to the detection section 12 of the flow path section 10 of the testing substrate 1 of this embodiment. Methyl nitrobenzoate was used as the fixing compound and was added to the mixed color liquid so that the solid content was 2% by mass. For the comparative example, the same sample as sample A was used except that the mixed liquid color did not contain a fixing compound, and an antibody (immobilized antibody) was applied to the same position. Then, the test liquid was supplied to the test liquid supplying areas of both plates and allowed to develop.

[0089] The results are shown in Figure 5. As shown in Figure 5, sample A, which contained a fixing compound, produced clear, sharp color lines, whereas the comparative example exhibited tailing (a phenomenon in which the colored area stretches in the direction of ink advance, causing bleeding). These results demonstrate that the inclusion of a fixative compound in the flow path section improved color development. In other words, without the fixative compound, the antibody (immobilized antibody) did not adhere strongly to the forming member and moved downstream with the test liquid, but by including the fixative compound, the antibody (immobilized antibody) could be firmly fixed, and the conjugate (complex of antigen and labeled antibody in the test liquid) could be maintained in a captured state along the area where the antibody (immobilized antibody) was applied (antibody line). Furthermore, no color development was observed anywhere other than the area where the immobilized antibody was applied on the flow path section (corresponding to area 12a of the testing substrate 1 in this embodiment), confirming that there was almost no nonspecific adsorption of the conjugate, antigen, or labeled antibody to the flow path section.

[0090] Table 1 below shows the measurement results of the color line bandwidth when the above experiment was performed three times. Figure 6 shows a graph of this table. As shown in the table, it can be seen that sample A has a narrower color line bandwidth than the comparative example.

[0091] [Table 1]

[0092] Next, the following experiment was carried out on the type of fixing compound. The fixative compounds used are listed in Table 2 below. The fixing compound was added to the mixed color liquid so that the solid content was 3% by mass.

[0093] [Table 2]

[0094] The test solution used was the same as that used in the HS described above, containing an antigen (human chorionic gonadotropin, a glycoprotein hormone; Sigma-Aldrich Japan, C1063-1VL) and a labeled antibody (gold colloid-labeled antibody, Gold Anti-hCG beta antibody [M705159]; Abcam, ab31206) in purified water (Millipore ultrapure water production system; Merck, hereafter the same). In the experiment, the color development was evaluated 15 minutes and 30 minutes after the test liquid was allowed to advance (develop).

[0095] The experimental results are shown in the table below and in FIG. As shown in Table 3 and FIG. 7, improved color-developing ability was confirmed for all of nitrobenzoic acid (NB), nitroaniline (NA), nitrophenol (NP), and sodium nitrobenzenesulfonate (NS). In the figure, each fixing compound is indicated by its abbreviation (for example, nitrophenol is indicated by "NP"). For the comparative example, a sample equivalent to Sample A was used except that no fixing compound was mixed in. This is indicated as "no addition" in the tables and figures.

[0096] [Table 3]

[0097] Next, the concentration of the fixing compound added to the mixed color liquid was confirmed. Nitrobenzoic acid was used as the fixing compound. The experimental results are shown in Table 4 below and in FIG. For the comparative example, a sample equivalent to Sample A was used except that no fixing compound was mixed in. This is indicated as "no addition" in the tables and figures. As shown in the experimental results, regardless of the concentration of the fixing compound added, the color development was improved compared to the comparative example, and the maximum color development was observed at 2% by mass. Furthermore, when the test solution did not contain an antigen, the color intensity of the sample to which 2 or 5% by mass of nitrobenzoic acid had been added after 30 minutes was 50 mABS or less, and color development was not confirmed even by visual inspection. On the other hand, when the test solution contained an antigen, the color intensity was enhanced as described above. From these results, it was found that the fixing compound enhances the color intensity by fixing the immobilized antibody that captures the antigen. In other words, it was found that these fixing compounds do not nonspecifically adsorb the antigen or labeled antibody, but fix only the immobilized antibody.

[0098] [Table 4]

[0099] Next, the following experiment was carried out to examine the strength of the testing substrate of this embodiment. In the experiment, the effect of pressing sample A in the thickness direction was confirmed. A tabletop punching press (Tester Sangyo Co., Ltd.) was used for the pressing. In addition, a commercially available nitrocellulose membrane was used as a comparative example.

[0100] FIG. 9 shows a cross-sectional image when a press pressure of 23.1 MPa was applied, and FIG. 10 shows a photograph of the surface. Cross-sectional images taken before and after pressing show that in Sample A, the voids between the fibers (voids to induce capillary action) necessary for the test liquid to penetrate remain even after pressure is applied, whereas in the Comparative Example, the film thickness changes significantly with pressing, and the voids have almost completely disappeared. Furthermore, images taken from the surface show that gaps remain between the fibers in Sample A, whereas in the Comparative Example, the structure is dense and almost completely devoid of voids. The reason for this is that the Comparative Example is primarily made of low-strength nitrocellulose, which makes it susceptible to plastic deformation such as peeling and dents due to pressure, whereas Sample A has a structure that is less susceptible to plastic deformation.

[0101] The thickness change was calculated using the following formula based on FIG. The change in thickness was calculated based on the change in thickness when a constant pressure was applied using a pressure press and the thickness was measured after the pressure was released. As can be seen from the cross-sectional observation image in FIG. 9, the thickness (B) of the base portion does not change, and therefore was removed from the equation for thickness change in the calculation. Thickness change (times) = P' / P

[0102] The change in the time of the advancement of the liquid was calculated using the following formula based on Table 5. The change in liquid penetration time was calculated based on the change in the liquid penetration time when a constant pressure was applied using a pressure press and the pressure was released. Fluid advance time change (times) = (fluid advance time after pressurization) / (fluid advance time before pressurization) The time required for the test solution to progress was measured using the HS described above.

[0103] The table below shows the thickness change when pressure is applied, as well as the time it took for the liquid to penetrate and the color intensity. Figure 12 also shows the thickness change (fold), color intensity, and time it took to penetrate the liquid. The liquid penetration time and color intensity were measured in an experiment to confirm the difference in plastic deformation before and after pressing. As shown in the table, sample A has a structure that makes plastic deformation less likely due to the inclusion of fibers, and it was confirmed that the liquid penetration time and color intensity were maintained before and after pressing compared to the comparative example. For sample A, the change in thickness due to pressure was smaller than for the comparative example, indicating that plastic deformation due to pressure is less likely to occur. For example, at a press load of 23.1 MPa, the change in thickness was 0.552, while the comparative example showed a large compressive deformation of 0.273. The liquid penetration time for Sample A was 64.7 sec / 40 mm without pressure, but was 129.0 sec / 40 mm when 23.1 MPa was applied, and for the Comparative Example it was 111.3 sec / 40 mm and 749.7 sec / 40 mm, respectively, meaning that the flow rate in the Comparative Example was so slow that it almost completely lost its function as a flow path. Furthermore, while Sample A maintained its color development even after compression, the liquid penetration in the Comparative Example was so slow that no color development was observed at 9.9 MPa or higher. Furthermore, with regard to the change in the liquid penetration time, the comparative example showed a very slow flow rate, with the change in the liquid penetration time at an applied pressure of 9.9 MPa being 4.09 times (456.0 / 111.3), almost completely losing its function as a flow path, whereas sample A showed a change in the liquid penetration time at an applied pressure of 9.9 MPa being 1.89 times (122.7 / 64.7), confirming that the test liquid was properly transported. Furthermore, the change in the liquid penetration time at an applied pressure of 23.1 MPa was 6.73 times for the comparative example and 1.99 times for sample A. From this experiment, it was confirmed that the testing substrate of this embodiment is resistant to pressure deformation.

[0104] [Table 5]

[0105] Next, the influence of the fixing compound on the flow path of the test liquid when the fixing compound was fixed in the flow path portion of the testing substrate of this embodiment was confirmed. The fixing compounds used were sodium nitrobenzene sulfonate (NS), nitrophenol (NP), nitroaniline (NA), and nitrobenzoic acid (NB).

[0106] The results are shown in Figure 13. As shown in Figure 13, it was confirmed that the permeation characteristics can be changed by fixing a fixing compound in the flow path section. When NS, NP, and NB were added, it was found that the permeation became faster (the permeation time became shorter) when 3 mass% was added. In addition, in the case of NA, a phenomenon in which the flow rate became slower was observed. Specifically, the change in the time course of the solution caused by the fixing compound is calculated by the following formula based on FIG. This change in liquid-advancement time is calculated as the ratio of the liquid-advancement time when 3% by mass of fixing compound is added to that when no fixing compound is added. Change in liquid advance time due to fixing compound (times) = (liquid advance time when 3% by mass of fixing compound is added) / (liquid advance time when no fixing compound is added)

[0107] In this case, the change in the liquid advance time was 0.44 times for NS, 0.58 times for NP, 1.08 times for NA, and 0.84 times for NB. In other words, it was confirmed that the flow rate when developing the test liquid can be controlled by the fixing compound contained in the flow path.

[0108] The above experimental results demonstrate that controlling the flow rate in immunochromatography is important for achieving an appropriate antigen-antibody reaction. For example, antigen-antibody reactions generally require a certain amount of time, and controlling the flow rate of the channel is directly linked to improving measurement sensitivity and accuracy. Increasing the flow rate enables more samples to be processed efficiently. Furthermore, slowing the flow rate allows reactions that would normally require a long time to be completed appropriately over a sufficient period of time. Therefore, it was confirmed that by using the testing substrate of this embodiment, the rate at which the test liquid advances can be controlled by adjusting the type and concentration of the fixing compound. [Industrial Applicability]

[0109] The test substrate of the present invention is suitable as a test substrate used in immunoassays. [Explanation of symbols]

[0110] 1. Test substrate 10 Flow path section 10h Void (flow path) 10US Flow path surface 10BS Back of flow path 11 Main body 12 Detector 13 Forming member 14 Fibrous materials 15 Granular materials 16 Binder material A antibody AC control antibody AI immobilized antibody AL-labeled antibody LC Conjugates 20 Absorption section 30 Base

Claims

1. A substrate for testing, The device is provided with a flat channel having an antibody immobilization function that allows liquid to pass through by capillary action, The flow path portion is The device has a main body and a detection unit, The flow path portion is A fibrous member and a fixing compound capable of binding to an antibody. A testing substrate characterized by:

2. The flow path portion is The thickness change is 0.4 or more, The thickness change is This is a value calculated based on the ratio of the thickness before pressing to the thickness after pressing (thickness after pressing / thickness before pressing) when pressed with a press load of 9.9 MPa.

2. The testing substrate according to claim 1.

3. The flow path portion is The change in time of the liquid advance is 3 or less, The change in the time of the advancement of the liquid is This is a value calculated based on the ratio of the liquid penetration time before pressing to the liquid penetration time after pressing (liquid penetration time after pressing / liquid penetration time before pressing) when pressurized with a press load of 9.9 MPa.

3. The testing substrate according to claim 1 or 2.

4. The fixing compound is Contains one or more functional groups selected from the group consisting of amino, carboxy, phenyl, nitro, sulfo, hydroxyl, alkoxy, cyclohexyl, ester, amide, aldehyde, ketone, ether, nitrile, halogen, phosphate, silicic acid, and acid anhydride groups.

3. The testing substrate according to claim 1 or 2.

5. The fixative compound is Contains one or more compounds selected from the group consisting of nitro compounds, aromatic compounds, amine compounds, carboxy compounds, sulfuric acid compounds, polysaccharides, alkoxides, and nitrate ester compounds.

3. The testing substrate according to claim 1 or 2.

6. The flow path portion is The fibrous members are made of resin, and a binder member capable of connecting the fibrous members to each other is included, The binder member is It has a nanofiber membrane and a nanofiber layer.

3. The testing substrate according to claim 1 or 2.

7. The flow path portion is Contains a binder material, The binder member is It has a nanofiber membrane and a nanofiber layer.

3. The testing substrate according to claim 1 or 2.

8. The flow path portion is and a granular member disposed between the fibrous members.

3. The testing substrate according to claim 1 or 2.

9. a base portion for holding the flow path portion; 3. The testing substrate according to claim 1 or 2.

10. 1. A method for manufacturing a substrate for testing, comprising: preparing a mixed color containing a forming member and a fixer compound; a step of applying the mixed liquid color in a sheet form to form a sheet member; a drying step of drying the sheet member obtained by coating; and cutting the dried sheet member into strips. The forming member contains a fibrous member. A method for manufacturing a substrate for testing, comprising:

Images