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 antibody fixing part 10 which allows liquid to pass by a capillary phenomenon and which has an antibody fixation function. The antibody fixing part 10 includes a localized portion 11 which contains a fixing component M capable of bonding to antibodies and in which the fixing component M is localized. The localized portion 11 is arranged on the side of a surface 10US of the antibody fixing part 10, has flow paths inside, and has the fixing component M on the side of the surface 10US of the antibody fixing part 10 so as to be able to accumulate conjugates LC on the side of the surface 10US of the antibody fixing part 10.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 this test line, it is captured by the immobilized antibody fixed to the test line and develops color (coloration). This color development can be easily determined visually, and therefore 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 immobilized 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, and a method for producing the same. [Means for solving the problem]

[0007] The testing substrate of the present invention is a testing substrate that is provided with a flat antibody fixing portion having an antibody immobilization function that allows liquid to pass through by capillary action, and the antibody fixing portion contains a fixing compound that can bind to an antibody, and has a localized site where the fixing compound is localized, and the localized site is located on the surface side of the antibody fixing portion. The method for manufacturing a testing substrate of the present invention is a method for manufacturing a testing substrate, and includes the steps of applying an antibody fixing liquid in a strip shape to the surface of a sheet member containing a fibrous member and through which liquid can pass by capillary action, and drying the sheet member to form an antibody fixing portion, and cutting the sheet member into strips along a direction perpendicular to the antibody fixing portion formed in the strip shape, wherein the antibody fixing liquid contains a fixing compound that can bind to an antibody. [Effects of the Invention]

[0008] The testing substrate of the present invention can exhibit antibody fixation properties and flow path performance, and since no fixation compound is placed on the surface side of the antibody fixation section, the conjugate can be accumulated on the surface side of the antibody fixation section. 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 viewed from the liquid advance direction. [Figure 2] FIG. 2(B) is a schematic explanatory diagram of the antibody fixing portion 10 of the testing substrate 1 of this embodiment, in which fibers 14, granular members 15, and a binder member 16 are used in the forming member 13. FIG. 2(A) 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. 1 is a schematic explanatory diagram showing a state in which a testing substrate 1 of this embodiment is provided with an antibody fixing section 10, a flow path section 20, an absorption section 30, and a base section 40. [Figure 4] FIG. 2 is a schematic explanatory view of the inspection situation of the inspection substrate 1 of the present embodiment. [Figure 5] FIG. 1 is a schematic explanatory diagram of the inspection status of a conventional product. [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. [Figure 14] FIG. 10 is a diagram showing experimental results. [Figure 15] FIG. 10 is a diagram showing experimental results. [Figure 16] FIG. 10 is a diagram showing experimental results. [Figure 17] FIG. 10 is a diagram showing experimental results. [Figure 18] FIG. 10 is a diagram showing experimental results. [Figure 19] FIG. 10 is a diagram showing experimental results. [Figure 20] 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 an antibody-fixed portion 10 . The antibody-fixed section 10 is a flat testing member with many mesh-like voids 10h formed inside. These voids 10h form a mesh-like network, forming multiple fine flow paths for liquid to pass through within the antibody-fixed section 10. Therefore, when liquid is supplied to the antibody-fixed section 10, it can be passed through the mesh-like flow paths by capillary action. The antibody-fixing section 10 contains a fixing compound M. This fixing compound M is a compound that can bind to antibody A applied to the antibody-fixing section 10 (described later) and fix the antibody A within the antibody-fixing section 10, and is arranged in a fixed state on the inner surface of the void 10h, within the void 10h, or on the surface of the forming member 13 (for example, on the surface of 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 antibody-fixing section 10, making it difficult for the antibody A to move.

[0013] As shown in Figure 1(B) , in the cross-sectional view of antibody-fixing section 10, this fixing compound M is arranged so as to be localized on the surface 10US side of antibody-fixing section 10. The site where this fixing compound M is localized is localized site 11 of antibody-fixing section 10. In other words, localized site 11 is located on the surface 10US side, and is formed so that fixing compound M is significantly present compared to other sites of antibody-fixing section 10 (for example, back surface layer 12 located on the back surface 10BS side). The shape of the localized area 11 is not particularly limited as long as it is formed so that a larger amount of fixing compound M is present than in other areas. For example, it may be formed in a layer form over the entire surface of the antibody fixing area 10 as shown in Figure 1, or may be formed in a three-dimensional form (for example, in a block or block shape) in a partial area.

[0014] In this way, the antibody fixing portion 10 is a flat plate-like member having an antibody immobilization function that allows liquid to pass through by capillary action, and is formed so that the localized portion 11 containing a large amount of fixing compound M that binds antibody C is located on the surface 10US side.

[0015] The antibody A is not particularly limited as long as it can bind to the fixing compound M and capture, in the bound state, 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 such antibodies include immobilized antibody AI (sometimes referred to as a solid-phase antibody or a capture antibody) and 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 by coating or the like on the surface 10US of the antibody-fixing section 10 of the testing substrate 1 of this embodiment before the test, so that the antibody A binds to the fixing compound M at the localized site 11. In other words, at the time of the test, the antibody A is supported and fixed at the localized site 11 of the antibody-fixing section 10. As shown in FIG. 1, region 11a is a region where immobilized antibody AI of antibody A is applied, and region 11b is a region where control antibody AC of antibody A is applied.

[0016] Furthermore, there is no particular limitation on the method for supplying a liquid (hereinafter referred to as a test liquid) to the antibody-fixed section 10 of the testing substrate 1 of this embodiment. For example, the test liquid can be supplied to the antibody-fixed section 10 by contacting the edge of the antibody-fixed section 10 with the test liquid, adding the test liquid to the surface 10US near the edge, or contacting the back surface 10BS near the edge.

[0017] The test liquid is a liquid to be tested. The testing substrate 1 of this embodiment is a substrate used for testing as described above, and is designed to capture antigens in the test liquid at the antibody-fixed portion 10. Therefore, as long as the test liquid is a liquid that is expected to contain an antigen, the test liquid can be an undiluted solution, a diluted solution thereof, a filtered solution thereof, etc. For example, various liquids can be used as the test liquid or a raw material for the test liquid, such as body fluids such as blood, plasma, serum, urine, saliva, sweat, and tears, river water, seawater, groundwater, and aqueous solutions of soil and food. 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.

[0018] 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 antibody-fixing section 10 in which the forming member 13 contains, in addition to the fibers 14, granular members 15 and a binder member 16, 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. The liquid advance direction means the direction in which the test liquid moves when the test liquid is developed in the antibody-fixed part 10 of the testing substrate 1 of this embodiment, as shown in Fig. 1 (the direction of the arrow in Fig. 1). In this liquid advance direction, the side where the test liquid is supplied is called the upstream side, and the opposite side is called the downstream side.

[0019] As described above, when a test liquid is supplied to the antibody-fixed 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 advancement due to capillary action. When the spread test liquid reaches the region 11a of the antibody-fixed section 10 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. 4). Then, color develops in the application region 11a 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 antibody-fixed section 10 of 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.

[0020] 1 to 4, the fixing compound M is more locally located in the localized region 11 of the antibody-fixed region 10 than in other regions, and this localized region 11 is located on the surface side of the antibody-fixed region 10. In other words, in the region 11a where the immobilized antibody AI is applied, the fixing compound M is locally located in the localized region 11 on the surface 10US side, whereas below that (the back layer 12 on the back surface 10BS side in the thickness direction), no fixing compound M is present, or even if present, the amount is much less than that of the localized region 11. In other words, the antibody-fixed region 10 is formed so as to have the property of surface accumulation of the immobilized antibody AI. Therefore, when immobilized antibody AI is applied to this region 11a, it is supported within the localized region 11 on the surface 10US (the region surrounded by a thick dotted line in region 11a), and is substantially not supported or almost not supported below that (the region surrounded by a thin dotted line in the back layer 12) (see Figures 1(B) and 3). When the test liquid spread on this region 11a arrives, the antigen or conjugate LC can be accumulated at the localized region 11 on the surface 10US (see Figure 4(C)). Because the color development in this region 11a depends on the amount of antigen present, accumulating the antigen on the surface 11US of region 11a allows for a clear determination of the presence or absence of the antigen. Even when the amount of antigen in the test liquid is particularly small, it can be accumulated on the surface 10US of the antibody-fixed portion 10, preventing misinterpretation and ensuring the reliability of the analysis.

[0021] On the other hand, in the case of a conventional product (made of a porous polymer, for example, a nitrocellulose membrane) as shown in FIG. 5, antibody fixation is uniform across the cross section of the flow channel. Therefore, when the immobilized antibody AI is applied, the immobilized antibody AI is fixed uniformly over the entire application area (the entire area from the front to the back), as shown in Figure 5. In other words, when the same amount of immobilized antibody AI is applied to the antibody fixing portion 10 of the conventional product and the testing substrate 1 of this embodiment, the testing substrate 1 of this embodiment can accumulate the immobilized antibody AI at a higher density on the surface 10US side than the conventional product. In other words, the testing substrate 1 of this embodiment can improve the fixation of antibody A compared to the conventional product. As shown in Figures 4 and 5, when the test liquid is developed, in the area where the immobilized antibody AI is applied (area 11a in Figure 4), the antigen or conjugate LC is distributed and captured throughout the area in conventional products, whereas in the antibody fixing portion 10 of the testing substrate 1 of this embodiment, the antigen or conjugate LC can be accumulated and captured at a high density on the surface 10US side of the localized area 11. Therefore, the testing substrate 1 of this embodiment can improve the color intensity compared to conventional products, and therefore the state of the test result can be more easily grasped visually compared to conventional products. In particular, the testing substrate 1 of this embodiment has the advantage that it can perform the test appropriately even when the amount of antigen is such that visual judgment is difficult with conventional products.

[0022] In particular, the antibody-fixed section 10 of the testing substrate 1 of this embodiment contains a large number of fibrous members 14 (hereinafter simply referred to as fibers 14) as the forming member 13. The above-mentioned voids 10h are formed between the large number of fibers 14 (see FIGS. 1 and 2), and the voids 10h formed between the fibers 14 form flow paths through which the test liquid moves. In other words, the voids 10h formed between adjacent fibers 14 form a mesh-like network inside the antibody-fixed 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 antibody-fixing portion 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 channel through which the test solution moves. As a result, even if pressure or the like is applied during transportation or handling, the shape of the flow channel can be appropriately maintained, allowing the antigen or the like to appropriately accumulate at the localized site 11.

[0023] 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.

[0024] Therefore, by incorporating the fiber 14 as the forming member 13 in the antibody-fixed portion 10, the testing substrate 1 of this embodiment can improve the 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 antibody-fixed portion 10 improves the handleability in 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.

[0025] Next, the antibody-fixed portion 10 will be described in detail.

[0026] (localized site 11) As described above, the localized site 11 of the antibody fixing portion 10 is arranged in a localized state of the fixing compound M, and this fixing compound M is arranged in a state where it is supported on the surface of the forming member 13 of the localized site 11 or within the void 10h.

[0027] There are no particular limitations on the method for forming the localized site 11. For example, localized site 11 of a predetermined shape can be formed by applying an antibody fixing solution containing a predetermined amount of fixing compound M, which will be described later, to a predetermined region (e.g., a rectangular, circular, elliptical, or other shape in plan view) on the surface 10US of the antibody-fixed section 10, or by applying the solution to the entire surface 10US of the antibody-fixed section 10, a layer-like localized site 11 can be formed.

[0028] The localized site 11 may be located on the surface 10US side of the antibody-fixed site 10, and is preferably located so that the upper surface of the localized site 11 forms the surface 10US of the antibody-fixed site 10 (see Figures 1 and 2). The thickness of the localization site 11 (the distance from the top surface to the bottom surface of the localization site 11) is not particularly limited. For example, the bottom surface of the localization site 11 may be shallower than, approximately the same as, or slightly deeper than the center line passing through half the thickness of the antibody-fixed site 10. The center line of the antibody-fixed site 10 refers to a horizontal line that intersects with a line connecting the front surface 10US and the back surface 10BS in the thickness direction of the antibody-fixed site 10 when the antibody-fixed site 10 is placed horizontally and passes through the midpoint of this line (see Figure 9). The center line is approximately parallel to the front surface 10US and the back surface 10B of the antibody-fixed site 10. For example, as shown in Figure 1(B), the localization site 11 can be formed in a layer so as to be located above the center line of the antibody-fixed site 10.

[0029] The proportion of fixing compound M in this localized region 11 can be expressed by the localization index of fixing compound M, and if this localization index is greater than 50 (>50), it indicates that fixing compound M is localized and accumulated in localized region 11 (i.e., the surface 10US side of the antibody fixing portion 10). Conversely, fixing compound M may or may not be present in other regions (e.g., the back layer 12) other than localized region 11 (see, for example, Figures 1 and 2), as long as the content of fixing compound M in localized region 11 is biased compared to these regions.

[0030] This localization index is calculated by the following formula based on the spectral analysis of the cross section of the antibody anchoring portion 10 at the localized site 11 of the antibody anchoring portion 10 shown in the Examples. Spectroscopic analysis can be performed using absorption spectroscopy, emission spectroscopy, fluorescence spectroscopy, mass spectroscopy, molecular spectroscopy, atomic spectroscopy, infrared absorption, ultraviolet-visible absorption, terahertz wave spectroscopy, X-ray spectroscopy, chemiluminescence, atomic emission, ion emission, secondary ion mass spectroscopy, laser-induced spectroscopy, radiometric analysis, gamma-ray spectroscopy, atomic force microscope, scanning probe, Raman spectroscopy, etc.

[0031] Localization index=∫f(x):P / {∫f(x):P + ∫f(x):Q} × 100

[0032] In the formula, ∫f(x):P is the value obtained by taking a definite integral of the difference between the maximum value of the signal intensity at a wavelength specific to the substance and the background, i.e., the signal intensity difference, in the range from the point passing through the surface 10US of the antibody-fixed portion 10 on the x-axis to the point passing through the center line of the antibody-fixed portion 10 (the midpoint between the point passing through the surface 10US and the point passing through the back surface 10BS) on a graph obtained by measuring the line of the spectrum of the cross-section of the antibody-fixed portion 10 in the thickness direction of the antibody-fixed portion 10 (from the surface 10US to the back surface 10BS (for example, from left to right in Figure 9)). Furthermore, ∫f(x):Q in the equation is a numerical value obtained by performing a definite integral on the signal intensity difference in a graph obtained by measuring the line of a spectrum obtained by measuring a cross section of the antibody-fixed portion 10 in the thickness direction of the antibody-fixed portion 10 (from the surface 10US to the back surface 10BS (for example, from left to right in Figure 9)) in the range from a point passing through the center line of the antibody-fixed portion 10 on the x-axis (the midpoint between the point passing through the surface 10US and the point passing through the back surface 10BS) to a point passing through the back surface 10BS (if a base portion 40 described below is provided, a point passing through the interface where the surface of the base portion 40 and the back surface 10BS of the antibody-fixed portion 10 meet).

[0033] For example, when methyl nitrobenzoate is used as the fixing compound M, the localization index can be determined by Raman spectroscopy as shown in the Examples. In this case, the signal intensity difference in the above formula is calculated by subtracting the signal around 1360 cm as the background on the Raman spectrum from the peak at 1320 to 1330 cm, which is the characteristic Raman shift (for example, 1330 cm). -1 Peak intensity of -1360cm -1 background intensity).

[0034] The localization index can also be expressed by the following formula based on a color development test (mapping method). As with the above-mentioned spectral analysis, if the localization index based on this mapping method shows a value greater than 50 (>50), it indicates that the fixing compound M is localized and accumulated at the localization site 11 (i.e., the surface side of the antibody fixing portion 10), as in the above-mentioned case.

[0035] For this mapping method, for example, a color development test using iodine (iodine mapping method), dyes, osmium, fluorescent dyes, radioisotopes, latex beads, quantum dots, etc. can be used.

[0036] The localization index using the mapping method will be explained below. When the antibody fixing section 10 having fixing compound M carried in the localized region 11 is exposed to a color-developing reagent, the region carrying fixing compound M develops color, but the region not carrying fixing compound M does not change color. Utilizing this color change, the cross section of the discolored region (i.e., the region carrying fixing compound M) is observed with an optical microscope, and the image obtained by the observation is subjected to binary image processing to quantify the localization of fixing compound M in the cross section.

[0037] For example, in the case of a color development test using iodine on nitrocellulose, the threshold values ​​for the binarization process are set to Red: 22 to 80, Green: 17 to 65, and Blue: 29 to 88 in the RGB color system in the captured image, and these categories can be colored and quantified.

[0038] First, on the image of the cross-section micrograph, a center line perpendicular to the thickness direction is drawn, dividing the distance between the surface 10US (also called the coated surface when fixing compound M is applied to the surface 10US and fixed) and the back surface 10BS (the back surface 10BS on the side where fixing compound M is not applied is also called the non-coated surface) in the thickness direction of the antibody fixing portion 10. Next, two lines perpendicular to the center line are drawn, and the area is divided into sections enclosed by each line. The localized area 11 should be located between these two lines. This results in rectangular fraction images that are symmetrical about the center line (for example, fraction A image and fraction B image in FIG. 15). If the area of ​​the portion of the obtained fraction image that has been colored by binary image processing is I(A) (surface 10US side) and I(B) (back surface 10BS side), the localization index in the fractions in the range of fraction image A and fraction image B in the antibody fixation section 10 is calculated using the following formula. If the localization index is greater than 50, it indicates that the fixing compound M is localized in the localization site 11 (that is, the surface side of the antibody fixing portion 10).

[0039] Localization index (X fraction) = I(A) / {I(A) + I(B)} × 100

[0040] The localization index may be evaluated from values ​​obtained by dividing the localization site 11 into a plurality of fractions (for example, three fractions: X fraction, Y fraction, and Z fraction). For example, a line is drawn along the center line to divide the cross section into three equal parts. The image is then divided into six parts, A to F. Specifically, each of the resulting partial images is divided as follows: The six fractional images divided by each line are congruent rectangles. If the fractional images are A, B, C, D, E, and F, the areas of the fractional rectangles A, B, C, D, E, and F are (A), S(B), S(C), S(D), S(E), and S(F), respectively, as follows: S(A)=S(B)=S(C)=S(D)=S(E)=S(F). Next, let the areas of the colored parts of the fraction images A, B, C, D, E, and F be I(A), I(B), I(C), I(D), I(E), and I(F), respectively. Then, groups of fraction images symmetrical with respect to the center line are designated as fractions (X fraction, Y fraction, Z fraction). The localization index of fraction X can be calculated using the above formula. The localization index of other fractions (fraction Y, fraction Z) can be calculated by substituting the area of ​​each fraction in the same way as in the formula for fraction X above.

[0041] (Fixing Compound M) As described above, the fixing compound M has the function of binding to the antibody A applied to the antibody fixing portion 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.

[0042] 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, an amino group such as aniline, a carboxy group such as cyclohexanecarboxylic acid or benzoic acid, a phenyl group such as aniline, benzoic acid, or methyl 3-nitrobenzoate, a cyclohexyl group such as cyclohexanecarboxylic acid, an ester group such as methyl 3-nitrobenzoate, an alkoxy group such as anisole, a nitro group such as methyl 3-nitrobenzoate, a nitrate ester group such as nitrocellulose, a hydroxyl group such as nitrocellulose or cellulose, a sulfo group such as nitrobenzenesulfonic acid, an amide group such as acetanilide, an aldehyde group such as benzaldehyde, a ketone group such as acetophenone, an ether group such as polyether, a halogen group such as aryl halide, a nitrile group such as benzonitrile, a phosphate group such as diphenyl phosphate, a silicic acid group such as phenyl orthosilicate, and an acid anhydride group such as 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.

[0043] 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.

[0044] The present inventors have been the first to discover, 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 also has the function of being easily retained within the antibody fixing portion 10. Then, by fixing this fixing compound M within the antibody fixing portion 10, it has become possible to appropriately capture an antigen in the region of the antibody fixing portion 10 where antibody A has been fixed (localized site 11).

[0045] 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 is easily retained within the antibody-fixing section 10 (i.e., so that the bound antibody A is easily retained within the antibody-fixing 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.

[0046] Furthermore, the fixing compound M preferably has the property of being easily supported by the forming member 13 of the antibody-fixing 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 antibody-fixing 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 antibody-fixing section 10. 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 antibody-fixing section 10 (such as a state in which the color is difficult to visualize or a state in which color development corresponding to the amount of antigen is not obtained), thereby improving the sharpness of the color development line and thereby improving the analytical accuracy.

[0047] 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 embodiment, for example, if the forming member 13 contains fibers 14 made of hydrophilic cellulose, the fixing compound M can also be hydrophilic (for example, nitrocellulose or benzoic acid).In the latter embodiment, if the forming member 13 contains fibers 14 made of resin, the fixing compound M can also be hydrophobic (for example, methyl nitrobenzoate (NBM) or nitroaniline).

[0048] 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, polyvinyl alcohol, etc. can be used as the linking compound, and substances having surfactant properties can also be used as the linking compound.

[0049] 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.

[0050] (Forming member 13) The forming member 13 of the antibody fixing portion 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 specifically described below.

[0051] (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, filter paper (the material is not particularly limited) can be used as a sheet member when forming the antibody-fixed portion 10. 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.

[0052] 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, thereby appropriately preventing the fixing compound M from moving with the test liquid or becoming detached. Furthermore, since the contact area can be increased, it is possible to increase the content of the fixing compound M in the antibody fixing section 10, and therefore the amount of conjugate LC captured by the fixing compound M can be increased.

[0053] (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.

[0054] 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.

[0055] If a granular member 15 within this size range is used, the gap 10h (flow path) formed between adjacent fibers 14 can be narrowed by placing the granular member 15 between the fibers 14, and the flow path resistance can be increased, so that the pressure loss of the fluid moving through the flow path can be increased compared to when no granular member 15 is provided. As a result, the velocity of the test liquid can be slowed down. 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. Furthermore, the inclusion of granular members 15 can improve the smoothness of the surface 10US of the antibody fixing portion 10. By reducing the irregularities of the surface 10US and improving the smoothness, the fixing compound M can be applied evenly within a predetermined area, resulting in uniform and even fixing. In particular, the immobilized antibody AI or control antibody AC can be applied neatly when applied in a strip shape, allowing the line edges of the antibody line to be formed sharply. If the antibody line is sharp, the 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.

[0056] 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).

[0057] (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.

[0058] 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 a nanofiber membrane is an assembly of very fine fibers, it can have very low water permeability, so if it is formed on the back surface 10BS of the antibody fixing portion 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.

[0059] 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.

[0060] The antibody-fixing section 10 may be formed using a sheet member made of a 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.

[0061] The antibody-fixed portion 10 contains the above-mentioned forming member 13 (particularly the fiber 14), and therefore the mechanical strength can be improved. For example, the mechanical strength can be expressed by the change in thickness of the antibody-anchored 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 antibody-anchored section 10 when the press load is 23.1 MPa is 0.3 or more, preferably 0.4 or more, more preferably 0.5 or more, and even more preferably 0.6 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.

[0062] Since the thickness change of the antibody-fixed 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 antibody-fixed section 10, the test liquid can be appropriately passed through.

[0063] The mechanical strength of the antibody-fixed 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 antibody-fixed 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 antibody-fixed section 10 as described above, and a smaller liquid advance time change indicates that the shape of the voids 10h (flow path) is maintained better. 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 antibody-fixed section 10, when the press load is 23.1 MPa, the change in liquid penetration time 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 penetration time ratio is 3 or less, preferably 2 or less.

[0064] Since the antibody-fixed section 10 of the testing substrate 1 of this embodiment has the mechanical strength as described above, 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 antibody-fixed section 10. In particular, when the testing substrate 1 of this embodiment includes a flow path section 20 (described later) and this flow path section 20 and the antibody-fixed section 10 are integrally formed, it becomes easier to control the rate at which the test liquid advances.

[0065] (flow path section 20) 3 and 4, the testing substrate 1 of this embodiment may include a flow path section 20 connected to the antibody-fixed section 10 so as to come into contact with the antibody-fixed section 10. Specifically, the flow path section 20 has a test liquid supply region 20a at one end for supplying the test liquid, and when the test liquid is supplied here, it is moved toward the other end, so that the test liquid can be supplied to the antibody-fixed section 10 connected to the other end. 3, the flow path section 20 is connected to the antibody-fixed section 10 such that the region 11a of the antibody-fixed section 10 where the immobilized antibody AI is applied intersects with the direction of advance of the test liquid in the flow path section 20. For example, if the flow path section 20 is formed in a strip shape, the other end of the short axis of the flow path section 20 and one end of the antibody-fixed section 10 are connected along the long axis direction of the flow path section 20. In this case, the region 11a is connected so as to be approximately perpendicular to the long axis direction of the flow path section 20. In this way, in a plan view of the testing substrate 1 of this embodiment, the region 11a of the antibody-fixed section 10 can be arranged so as to intersect with the direction of advance of the test liquid, allowing the test liquid to appropriately contact the immobilized antibody AI.

[0066] As shown in FIG. 3, the flow path section 20 may have an area 20b for applying labeled antibody AL, which is antibody A that has the function of binding to an antigen and exhibiting color development when the antigen is captured by the immobilized antibody AI.

[0067] 3 and 4, the flow path section 20 is a porous member having numerous mesh-like voids (flow paths through which liquid passes) formed therein, and when a liquid is supplied, the liquid can pass through the mesh-like flow paths by capillary action. The flow path section 20 contains a fibrous member 24 (hereinafter referred to as fiber 24). The inclusion of fiber 24 makes it easier to maintain the shape of the flow path for the test liquid, similar to the antibody-fixed section 10, and therefore allows the test liquid to move appropriately to the antibody-fixed section 10.

[0068] As long as the flow path section 20 has the above-described structure, it may contain any material other than the fibers 24. For example, it may contain a material similar to the forming material 13 of the antibody-fixing section 10 described above (e.g., a granular material or a binder material). In this case, the structural effect, shape, material, etc. of the material are the same as those of the antibody-fixing section 10, and therefore the description thereof will be omitted.

[0069] In particular, if the flow path section 20 contains the same material as the forming material 13 of the antibody fixing section 10, the antibody fixing section 10 and the flow path section 20 can be integrally formed as the same sheet material, except for the antibody A and fixing compound M that are applied depending on the type of antigen, etc. Then, by fixing a fixing compound M to a predetermined area of ​​this sheet member by coating or the like, an antibody fixing section 10 having a localized site 11 containing the fixing compound M and a flow path section 20 continuously connected thereto can be integrally formed (see Figures 3 and 4). Therefore, by using the sheet member as described above, the testing substrate 1 of this embodiment, which is equipped with the antibody fixing section 10 and the flow path section 20, can be easily formed (for details, see the manufacturing method of the testing substrate 1 of this embodiment described later). Moreover, in this case, the difference in the forming materials between the two (the connecting surface between the flow path section 20 and the antibody-fixed section 10) can be eliminated, improving the continuity between the two. This allows the test liquid to move smoothly. This prevents the test liquid from stagnating at the connecting portion between the two, improving the analytical accuracy.

[0070] It goes without saying that the flow path section 20 may be made of a material different from that of the antibody-fixed section 10. In this case, the testing substrate 1 of this embodiment can be manufactured by forming both sections and connecting the antibody-fixed section 10 so that it intersects with the direction of advance of the test liquid in the flow path section 20. In this case, the connecting portion connecting the two sections is not particularly limited as long as it can move the test liquid, and for example, paper filter paper or the like can be used. Specifically, by placing water-soaked paper filter paper or the like between the connecting surfaces of the two sections and then drying it, the two sections are connected and the test liquid can pass between them.

[0071] In the above example, the antibody fixing section 10 and the flow path section 20 are connected on the same plane, but they may also be arranged so that the surface at the other end of the short axis side of the flow path section 20 is in contact with the back surface 10BS at one end of the antibody fixing section 10. Furthermore, the degree of intersection with the direction of advance of the test liquid is not particularly limited. For example, in a plan view (when the flow path section 20 and the antibody-fixed section 10 are viewed from the surface side), the two (flow path section 20 and the antibody-fixed section 10) may be connected so that the connecting surface is oblique to the direction of advance of the test liquid, or the connecting surface may be connected so that it meanders, or further so that the connecting surface is perpendicular or nearly perpendicular. In particular, when the connecting surface is formed so that it is perpendicular or nearly perpendicular, the timing at which the developed test liquid reaches the antibody-fixed section 10 can be made approximately the same, making it easier to understand the measurement of the target substance.

[0072] The shapes of the antibody fixing section 10 and the flow channel section 20 are not particularly limited. For example, the shape may be a strip or rectangle as shown in FIGS. 1 to 4, or may be a circle, ellipse, radial shape, or other various shapes. For example, in the case of a strip 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 antibody-fixed 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 solution 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.

[0073] (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. In the following description, a case where the antibody fixing section 10 and the flow path section 20 of the testing substrate 1 of this embodiment are integrally formed will be described as a representative example.

[0074] The method for producing the testing substrate 1 of this embodiment includes an antibody-fixed portion forming step and a cutting step.

[0075] In the antibody fixing section forming step, first, a sheet member is prepared. This sheet member is a sheet-like member formed from, for example, filter paper, glass fiber filter paper, nonwoven fabric, membrane, mesh, or the functional material described in WO 2015 / 152287. That is, the sheet member in this state is a sheet-like member corresponding to the flow path section 20.

[0076] Next, the above-mentioned fixing compound M is dissolved or dispersed in water, an organic solvent, or the like to prepare a solution (hereinafter referred to as an antibody fixing solution). This fixing compound M is the above-mentioned benzoic acid, methyl 3-nitrobenzoate, or the like, and may consist of one of these compounds or may contain two or more of them.

[0077] The antibody fixing solution may contain other components, such as a linking compound, a buffer solution, or a surfactant, in addition to the fixing compound M. For example, the linking compound has the function of improving the connectivity between the fixing compound M and the material constituting the sheet member when they have different properties (e.g., hydrophilicity and hydrophobicity). Furthermore, for example, nitrocellulose is a linking compound for methyl nitrobenzoate, and cellulose nanofiber is a linking compound for nitrocellulose.

[0078] Next, the prepared antibody fixing solution is placed in an application device (e.g., an immunochromatographic dispenser or an inkjet printer) and applied in a strip-like manner to the surface of a predetermined region of the sheet member. This region corresponds to the localized region 11, and the portion having this localized region 11 corresponds to the antibody fixing section 10. The remaining portion corresponds to the flow path section 20. After the antibody fixing solution is applied, the testing substrate 1 of this embodiment, which is integrally connected to the flow path section 20, can be formed on the sheet member by drying for a predetermined period of time.

[0079] Here, by applying the antibody fixer to the surface of a predetermined region of the sheet member, the fixer compound M can be arranged at a high concentration on the surface. Moreover, by using a fixer compound M that has a high affinity with the constituent member of the sheet member (a member corresponding to the above-mentioned forming member 13), the fixer compound M can be arranged in a state where it is firmly supported on the surface and in the vicinity of the surface. In other words, as the antibody fixer applied to the surface penetrates in the thickness direction, almost all of the fixer compound M is arranged in a fixed state in the portion corresponding to the localized site 11, including the surface.

[0080] After the antibody fixing solution is applied, the substrate is subjected to a drying process. In this drying process, the antibody fixing solution that has permeated the sheet member can be moved from the inside of the sheet member to the surface, where it can evaporate and dry. In other words, the fixing compound M that has permeated the inside of the sheet member can be moved toward the surface of the sheet member as the solvent in the antibody fixing solution evaporates. This phenomenon is called migration. In other words, by utilizing the natural drying that occurs when the antibody fixing solution is applied and the migration that occurs during the drying process, it is possible to form a portion on the surface side of the sheet member in the thickness direction where the content of fixing compound M is higher (a portion corresponding to localized portion 11).

[0081] Then, by applying the antibody fixing solution to the surface of a predetermined area of ​​the sheet member and drying it, the fixing compound M can be fixed so as to accumulate (ie, localize) on the surface side of the sheet member.

[0082] Next, the sheet member that has been coated with the antibody fixing solution in the predetermined area and dried as described above is subjected to a step of cutting into a predetermined size (cutting step). In this cutting step, the sheet member is cut in a direction substantially perpendicular to the band-shaped line on which the antibody fixing solution is applied. The cutting width is adjusted appropriately depending on the application. For example, when used in immunochromatography, the substrate is cut into strips. This results in the production of the testing substrate 1 of this embodiment, which includes a flow path section 20 and an antibody fixing section 10 connected continuously to the flow path section 20 in the order from one end of the short axis along the long axis (starting from the left in FIG. 4). As explained later in the method of use, the antibody fixing solution (fixing compound M) may be applied after cutting to a predetermined size.

[0083] As described above, the testing substrate 1 of this embodiment, in which the antibody fixing section 10 and the flow path section 20 are integrally formed, can be manufactured by a simple operation of simply applying the antibody fixing solution to a predetermined sheet member. The obtained testing substrate 1 of this embodiment has flow path performance and antibody fixing 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.

[0084] (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.

[0085] First, the testing substrate 1 of this embodiment is prepared in advance as follows according to the antigen to be tested.

[0086] (Application of immobilized antibody AI) As shown in Fig. 1, immobilized antibody AI is applied to the region of the antibody-fixing section 10 of the testing substrate 1 of this embodiment where the fixing compound M is fixed. When applying, the immobilized antibody AI is applied in a strip shape so as to intersect (e.g., approximately perpendicular to) the direction of the test liquid advance. In other words, as shown in Figs. 1 to 4, the immobilized antibody AI is applied in a line parallel to the minor axis direction of the flow path section 20. The region to which the immobilized antibody AI is applied is region 11a. 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 a fixing compound M carried on the forming member 13 of the antibody fixing portion 10, and is fixed to the localized portion 11 of the antibody fixing portion 10.

[0087] (Application of control antibody AC) 1 to 4, 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 antibody-immobilized portion 10. The region to which the control antibody AC is applied is the region 11b. 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 11b downstream of this line, where control antibodies AC that capture the labeled antibodies AL themselves have been fixed. In this region 11b, 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.

[0088] This region 11b is formed by applying the control antibody AC in a strip-like manner downstream (downstream in the direction of the test liquid advance) of region 11a where the immobilized antibody AI is applied, so that the strip-like application crosses (for example, is approximately perpendicular to) the direction of the test liquid advance. In other words, when viewed from the surface, the antibody-fixed section 10 is formed with two antibody lines that are approximately parallel and spaced a predetermined distance apart: an antibody line (also called a test line) where the immobilized antibody AI is applied in a strip-like manner, and an antibody line (also called a control line) where the control antibody AC is applied in a strip-like manner.

[0089] In addition, the antibody-fixed section 10 may be provided with a test line in which a band 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, so that the test line 2 is formed by immobilizing antibody AIb in a strip and approximately parallel to each other. In other words, when viewed from surface 10US, three antibody lines (two test lines 1 and 2 and one control line) are formed in antibody-fixed section 10, approximately perpendicular to the direction of liquid advance, 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.

[0090] (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. For example, as shown in Fig. 4, the labeled antibody AL can be applied to the flow path section 20 of the testing substrate 1 of this embodiment. This applied region is the region 20b. The region 20b is applied in a strip shape between the test liquid supply region 20a of the flow path section 20 and the antibody fixing section 10, so as to intersect (for example, approximately perpendicular to) the direction of advancement of the test liquid.

[0091] (Development of test solution) Next, as shown in Fig. 4(A), the test liquid is dropped and supplied to the test liquid supply region 20a of the flow path section 20 of the prepared testing substrate 1 of this embodiment. The supplied test liquid spreads toward the antibody fixing section 10 by capillary action, and when it reaches the region 20b, a conjugate LC is formed in which the antigen is combined with the labeled antibody AL by an antigen-antibody reaction (Fig. 4(B)). As shown in Figure 4(C), the formed conjugate LC continues to move in the liquid advance direction due to capillary action and reaches the antibody-fixed region 10. Upon arriving at the antibody-fixed region 10, the conjugate LC is captured by the immobilized antibody AI through an antigen-antibody reaction and remains in region 11a. It is then fixed to the forming member 13 via the fixing compound M. Meanwhile, other substances in the test solution, such as the solvent, pass through region 11a due to capillary action along the liquid advance direction. This means that only the conjugate LC remains in region 11a, where the immobilized antibody AI is applied. The solvent and free labeled antibody AL that have passed through region 11a then reach region 11b, located downstream, where the free labeled antibody AL is captured by the control antibody AC that has been fixed in region 11b. This means that only free labeled antibody AL remains in region 11b, where the control antibody AC is applied. As a result, in the antibody-fixed portion 10, color develops at the control line, making it possible to confirm that the test liquid has reliably moved, and in the region 11a, color develops (colors) at the antibody line if an antigen is present in the test liquid. As shown in Figures 1 to 4, in the antibody-fixed portion 10 of the testing substrate 1 of this embodiment, the fixing compound M is supported in an accumulated state at the localized portion 11 located on the surface 10US side, and therefore the conjugate LC can be accumulated at the localized portion 11, making it easy to visually determine whether or not the antigen is present in the test liquid even from the surface 10US of the antibody-fixed portion 10 (Figure 4(C)). 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).

[0092] As shown in Figure 5, the conventional product has a porous channel made of a polymer (nitrocellulose membrane) that allows antibody fixation uniformly 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 (Figure 5(C)).

[0093] Therefore, when the testing substrate 1 of this embodiment is compared with a conventional test kit, if the same amount of test liquid is spread, the testing substrate 1 of this embodiment can accumulate a larger amount of conjugate LC on the surface 10US side of the antibody-fixed portion 10 (the surface side of the localization site 11) than the conventional test kit. Moreover, as shown in Figures 4(C) and 5(C), the testing substrate 1 of this embodiment can achieve a higher accumulation density of conjugate LC at the localization site 11 of the antibody-fixed portion 10 than the conventional product. This phenomenon can be explained by the difference in the immobilization range of the immobilized antibody AI (specifically, the difference in the immobilization range of the immobilized antibody AI in the cross section of the region 11a where the immobilized antibody AI is applied). That is, in the case of the testing substrate 1 of this embodiment and the conventional product, the immobilization range of the immobilized antibody AI is localized site 11 in the former (testing substrate 1 of this embodiment), whereas in the latter it is the entire thickness direction from the front to the back (see Figures 4(C) and 5(C)). It is believed that this difference in the immobilization range of the immobilized antibody AI is due to the difference in color development between the two.

[0094] (Base part 40) 1 to 4, the testing substrate 1 of this embodiment may include a base section 40. This base section 40 is a member capable of arranging and holding the antibody-fixing section 10 and the flow path section 20 on its surface. This base section 40 is formed, for example, to be the same size as or slightly larger than the antibody-fixing section 10 and the flow path section 20 that it holds, and its shape is formed to be approximately similar to those of the antibody-fixing section 10 and the flow path section 20. If the testing substrate 1 of this embodiment includes the base portion 40, the antibody fixing portion 10 and the flow path portion 20 can be prevented from bending, thereby improving the ease of handling.

[0095] The material of the base portion 40 is not particularly limited, and examples thereof include paper and resin. In particular, the base portion 40 is preferably impermeable, and the base portion 40 can be formed using, for example, waterproof paper.

[0096] (Absorbing section 30) 3 and 4, the testing substrate 1 of this embodiment may be provided with an absorbing section 30 connected to the antibody-fixed section 10, which can absorb liquid, downstream of the antibody-fixed section 10 in the direction of advancement of the test liquid. This absorbing section 30 is designed to absorb the test liquid once the test liquid reaches the connecting section with the antibody-fixed section 10. This prevents the movement of the test liquid from stopping at the tip of the antibody-fixed section 10 when the test liquid has moved to the end of the connecting section with the antibody-fixed section 10, and therefore allows the added test liquid to reliably pass through the region 11a, enabling more accurate measurement.

[0097] The absorption section 30 is provided so as to be in contact with the antibody-fixing section 10, and there are no particular limitations on how it is arranged, so long as it can absorb the test liquid that has moved to the downstream end of the antibody-fixing section 10. For example, it may be connected horizontally to the downstream end of the antibody-fixing section 10, or it may be provided so that the top surface of the downstream end of the antibody-fixing section 10 and the bottom surface of the base end of the absorption section 30 are in contact.

[0098] The absorbent member 30 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 member 30. Examples of materials for the absorbent member 30 include filter paper, glass fiber filter paper, nonwoven fabric, and resin sponge members. In particular, when the absorption section 30 is formed from the same material as the antibody-fixing section 10 and the flow path section 20, for example, as shown in FIG. 3, if the absorption section 30 is formed to contain fibrous members 34 (hereinafter referred to as fibers 34) similar to those of the antibody-fixing section 10 and the flow path section 20, mesh-like voids (flow paths) can be formed between the fibers 34. In this case, the antibody-fixing section 10, the flow path section 20, and the absorption section 30 can be integrally formed. For example, as shown in FIGS. 3 and 4, 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 20, the antibody-fixing section 10, and the absorption section 30 are connected in this order from one end on the short axis side along the long axis direction. The absorbent section 30 may contain any material other than the fibers 34. For example, it may contain a material similar to the forming material 13 of the antibody-fixed section 10 described above (e.g., a granular material or a binder material). In this case, the structural effect, shape, material, etc. of the material are the same as those of the antibody-fixed section 10, and therefore the description thereof will be omitted. [Example]

[0099] 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.

[0100] Test substrates (samples A and B) of the present invention were prepared as follows.

[0101] (Preparation of Sample A) The flow path and antibody fixing part of sample A were made by applying a mixed liquid color consisting of PET fiber as the fiber, PET particles as the granular material, and a mixture of PVA and nanofiber as the binder material to the waterproof paper base using an applicator (Tester Sangyo Co., Ltd., PI-1210-S), and then drying. The dried product was cut to the specified size. After cutting to a specified size, an antibody fixing solution containing a fixing compound was applied to a specified area in a strip shape approximately perpendicular to the direction in which the test solution was advanced (advancing direction), allowing the fixing compound to be fixed, and then the sample was subjected to a drying process. In addition, a liquid containing labeled antibodies was applied in a stripe shape slightly downstream of the test liquid supply area, approximately perpendicular to the direction in which the test liquid was advanced (advancing direction), to fix the fixing compound, and then the substrate was subjected to a drying process. The area having the antibody fixing solution applied thereto is the antibody fixing section (corresponding to antibody fixing section 10 in this embodiment), and the area to which the antibody fixing solution is applied is the localized area (corresponding to localized area 11 of the testing substrate 1 in this embodiment).

[0102] Sample A was in the form of a strip, and was formed so as to have an area where the fixing compound was fixed, in a direction substantially perpendicular to the long axis. The size of Sample A was approximately 60 mm in length (length along the major axis) and approximately 5 mm in width (length along the minor axis) in a plan view. Sample A had a structure in which the channel section and antibody fixing section, which were integrally connected, were held by a base section, and its thickness (distance from the front surface to the back surface, including the base section) was approximately 350 μm (the thicknesses of the channel section and antibody fixing section were each approximately 150 μm (distance from the surface of the channel section and antibody fixing section to the top surface of the base section) + the thickness of the base section was approximately 200 μm (distance from the top surface of the base section to the back surface)). The film thickness was measured using a constant pressure thickness measuring instrument (model J-Type PG-02, manufactured by Teclock Corporation) and calculated from the average value of 10 points.

[0103] FIG. 6 shows SEM (scanning electron microscope) images of the cross section and surface of the antibody-fixed portion and base portion of Sample A.

[0104] (Preparation of Sample B) Sample B was formed using a sheet material (filter paper) made only of fibers. The filter paper used was made of cellulose fiber (manufactured by ADVANTEC, circular qualitative filter paper model numbers No. 1 and No. 131) and glass fiber (manufactured by ADVANTEC, GA-200). After cutting to a specified size, an antibody fixing solution (hereinafter sometimes referred to as an antibody fixing reagent) containing a fixing compound was applied to a specified area in a band shape approximately perpendicular to the direction in which the test liquid was advanced (advancing direction), and the fixing compound was fixed, and then the sample was subjected to a drying process. The area having the antibody fixing solution applied thereto is the antibody fixing section (corresponding to antibody fixing section 10 in this embodiment), and the area to which the antibody fixing solution is applied is the localized area (corresponding to localized area 11 of the testing substrate 1 in this embodiment).

[0105] Sample B was a strip, and was formed so as to have an area where the fixing compound was fixed, approximately perpendicular to the long axis. The size of Sample B was a strip of approximately 60 mm length (length in the long axis direction) and approximately 5 mm width (length in the short axis direction) in plan view. Sample B had a structure of a flow path section and an antibody-immobilized section, and its thickness (distance from the front surface to the back surface) was approximately 200 to 740 μm. Sample B was classified into three types depending on thickness and fiber material: Sample B-1 (fiber: cellulose fiber, thickness approximately 200 μm), Sample B-2 (fiber: cellulose fiber, thickness approximately 250 μm), and Sample B-3 (fiber: glass fiber, thickness approximately 740 μm). The film thickness was measured in the same manner as in Sample A.

[0106] As a comparative example, a nitrocellulose membrane, which is widely available commercially and is used in immunochromatography, was used. Other comparative examples were similar to Sample A or Sample B except that no fixative compound was applied.

[0107] Fixing compound application amount (mg / cm 2 ) is the fixation density (mg / cm) of the fixation compound in the antibody fixation area 2 ) and is expressed as the solid content. For example, the following coating amount of 1 mg / cm 2 This means that the fixing compound in the antibody fixing area is 1 mg / cm in solid content. 2 This means that it has become established in Japan.

[0108] The antibodies (immobilized antibodies, labeled antibodies, and control antibodies) were diluted 5-fold with pure water (Millipore ultrapure water production system, Merck, hereafter the same) and applied using a dispenser at a volume of 0.30 to 35 μL / cm. After application, the antibodies were left to dry for 12 hours before use. The labeled antibody may be mixed with the test solution before use.

[0109] 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.

[0110] 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.

[0111] 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 the area of ​​Sample A where the fixing compound had been applied (antibody-immobilized area). For the comparative example, the same sample as sample A was used except that no fixing compound was applied, 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. In the experiment, a mixture of methyl nitrobenzoate and nitrocellulose was used as the fixing compound.

[0112] The results are shown in Figure 7. In sample A, which had been fixed with the fixing compound, the color lines were clear and sharp, whereas in the comparative example, tailing (a phenomenon in which the color area stretches in the direction of the ink advance, causing bleeding) occurred. From this result, it was found that the color development could be improved by fixing the fixing compound. In other words, when the fixing compound is not provided, the antibody (immobilized antibody) does not firmly adhere to the forming member of the antibody fixing section and moves downstream together with the test liquid. fixed By fixing the adsorbent, the antibody (immobilized antibody) can be firmly fixed, and it was confirmed that the conjugate (complex of antigen in the test solution and labeled antibody) can be maintained in a captured state along the area where the antibody (immobilized antibody) is applied (antibody line). Furthermore, no color development was observed anywhere other than the area where the immobilized antibody was applied on this fixing compound (corresponding to area 11a 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 antibody fixing area.

[0113] Next, the following experiment was carried out on the type of fixing compound. The fixing compounds used are listed in Table 1 below.

[0114] [Table 1]

[0115] In the experiment, Sample A was used, in which the fixing compound was the substance shown in the table. An antibody fixative solution containing a fixative compound was applied to the designated area of ​​the antibody fixative. The test solution used was the same as that used in the HS described above, containing the antigen and the labeled antibody in water. In the experiment, the color development was evaluated 15 minutes and 30 minutes after the test liquid was allowed to advance (develop).

[0116] The experimental results are shown in Table 2 below and FIG. As shown in Table 2 and Figure 8, the color intensity after 15 minutes for the non-applied comparative example was 412.7±11.7 mABS, while for aniline, cyclohexanecarboxylic acid, benzoic acid, anisole, methyl nitrobenzoate, and nitrocellulose, the values ​​were 625.9±25.2 mABS, 805.3±34.3 mABS, 661.3±34.5 mABS, 564.6±35.0 mABS, 863.0±20.2 mABS, and 667.2±13.4 mABS, respectively, confirming improved color development. Furthermore, after the development time, the color intensity after 30 minutes was greater than that after 15 minutes. The accuracy of the color measurement was 10.0% or less for both the values ​​after 15 minutes and 30 minutes, which is within the standard accuracy of analytical evaluation. In the figures, each fixing compound is indicated by its abbreviation (for example, aniline is indicated by "AN"). In addition, a comparative example was used that was equivalent to sample A except that no fixing compound was applied. This is indicated by "non" in the tables and figures.

[0117] [Table 2]

[0118] Next, the surface accumulation of the fixing compound in the surface localized layer of the antibody fixing portion of the testing substrate of the present invention was evaluated. In the experiment, sample A was used. The fixing compound used was methyl nitrobenzoate.

[0119] The region of the antibody fixation section where the fixing compound was applied was cut using a cutter along the strip-shaped applied region to prepare a cross section (in other words, a cross section cut approximately perpendicular to the long axis of the strip-shaped sample A).The prepared cross section was then subjected to Raman spectroscopy analysis using a microscopic laser Raman spectroscopy analyzer, and a distribution image based on the Raman shift characteristic of the fixing compound, methyl nitrobenzoate, was obtained. The microscopic Raman spectroscopy used in the experiment detects interactions between molecular vibrations and electromagnetic waves, and is an effective analytical method for understanding the molecular structure of a local region, particularly functional groups and crystalline state. Analysis conditions: Raman spectroscopy (Nanophoton) and laser irradiation conditions (green laser: irradiation wavelength 532 nm, output 500 mW). The characteristic Raman shift of the fixative compound, methyl nitrobenzoate, is 1320–1330 cm based on the standard. -1 The distribution of the fixing compound (methyl nitrobenzoate) in the cross section was observed based on the Raman shift observed in the cross section.

[0120] The comparative example was similar to sample A except that no fixative compound was applied.

[0121] FIG. 9 shows an example of the observation results. The graph in Figure 9 shows the Raman intensity (1320-1330 cm, which is the characteristic Raman shift of methyl nitrobenzoate) when Raman spectroscopy was performed in the thickness direction (from left to right in the figure) from the front side to the back side of the cross section. -1 The peak at 1360 cm acts as a background on the Raman spectrum. -1 (subtracting nearby signals). As shown in Figure 9, it was confirmed that the fixing compound methyl nitrobenzoate was accumulated and localized in the surface layer (i.e., the surface localized layer, the layer located inward from the dotted line on the left side of the figure, hereinafter referred to as the localized layer). This was presumed to be because the fixing compound was accumulated in the layer near the surface by applying an antibody fixing solution containing the fixing compound to the surface of the antibody-fixed area and then drying the area near the surface.

[0122] The surface of sample A corresponds to the surface 10US of the antibody-fixed portion 10 in this embodiment, and the localized layer corresponds to the localized site 11 of the antibody-fixed portion 10.

[0123] Next, the accumulation state of the fixing compound in the localized layer of the antibody fixing portion was quantified. For quantification, a localization index based on Raman spectroscopy was used. This localization index was calculated by measuring the Raman shift intensity difference (1330 cm ) between the line passing through the point corresponding to the surface on the x-axis of the graph from the line passing through the point corresponding to the center line and the line passing through the point corresponding to the back surface on the graph (the point indicated by the dotted line on the left end of the horizontal axis (x-axis) of the graph (the point passing through the surface in the figure) corresponds to the surface of the antibody-immobilized area, and the point indicated by the dotted line on the right end of the x-axis (the point passing through the back surface in the figure) corresponds to the back surface of the antibody-immobilized area. The midpoint between these two points is the point passing through the center line in the figure). -1 -1360cm -1 ) We performed a definite integral with respect to the equation. The formula of the former definite integral is ∫f(x):P, and the formula of the latter definite integral is f(x):Q. The localization index of the fixing compound in the localization layer of the antibody fixing area can be expressed by the following formula, and if this value is greater than 50, it indicates that the fixing compound is localized and accumulated in the localization layer.

[0124] The localized layer refers to the layer extending from the center of the thickness of the antibody-immobilized portion to the back surface (the interface where the antibody-immobilized portion and the upper surface of the base portion meet in Sample A) to the surface, and in the graph of Figure 9, this is the area enclosed by the line passing through the point corresponding to the surface on the x-axis and the line passing through the point corresponding to the center line. This localized layer corresponds to localized region 11 in Figure 1 or Figure 4.

[0125] Localization index=∫f(x):P / {∫f(x):P + ∫f(x):Q} × 100

[0126] In the experiment, six samples A (samples A-1 to A-2) were prepared. For the comparative examples, five samples (Comparative Examples A to E) were prepared using conventional products.

[0127] First, as a comparative test, Raman measurements were carried out on the following (1) to (3). (1) Nitrocellulose membrane of Comparative Example 1 (2) A commercially available nitrocellulose reagent (Fujifilm Wako Pure Chemical Industries, Ltd. Collodion (10%)) is thinly spread into a glassy state and dried to form a film (collodion membrane). (3) Filter paper made from highly pure cellulose (Advantech 2A filter paper: Cellulose)

[0128] The experimental results are shown in Figure 10. As shown in Figure 10, the nitrocellulose membrane (1) exhibited a peak at 1268 cm -1 A characteristic Raman shift was observed around 1268 cm. The collodion film (2) also exhibited a similar Raman shift around 1268 cm. -1 On the other hand, a peak was observed at 1268 cm for the filter paper (3). -1 No Raman shift was observed. From the results of the Raman measurements (1) to (3) above, the 1268 cm observed from the nitrocellulose film and the collodion film -1 It can be seen that the Raman shift of is characteristic of nitrocellulose. So, this 1268cm -1 Based on the Raman shift of the specimens (1) and (2), cross sections were prepared in the same manner as in the above experiment (Figure 9), and Raman spectroscopic analysis was performed on these cross sections along the thickness direction from the front side to the back side. FIG. 11 shows an example of the observation results (an example of a nitrocellulose membrane). As shown in Figure 11, the cross section is uniformly distributed at 1268 cm -1 A Raman scattering spectrum of 0.01 mm was observed. This is significantly different from the Raman spectrum of Sample A, and it was confirmed that the antibody adhesion was uniform across the cross section of the channel because the entire channel was made of nitrocellulose. In other words, it was clear that the antibody adhesion was not localized on the surface of the channel.

[0129] Table 3 below shows the results of calculating the localization index for Sample A and the comparative example.

[0130] [Table 3]

[0131] As shown in the table, the average localization index for each sample in the localization layer of Sample A (Upper-Lower) coated with the fixing compound was significantly greater than 50, confirming that the fixing compound methyl nitrobenzoate was present in large amounts on the surface side (localization layer) where it was coated. On the other hand, the comparative sample (uncoated sample) that was not coated with the fixing compound methyl nitrobenzoate showed a localization index of 50 or less, indicating that it was not accumulated in a specific location but was distributed non-locally.

[0132] From the results of the Raman spectroscopy analysis described above, it is presumed that the testing substrate of the present invention can exhibit higher color development performance than the nitrocellulose membrane used in conventional products because the fixing compound is applied from the surface in the antibody fixing area, allowing the conjugate (complex of antigen and labeled antibody) that travels through the flow path to accumulate near the surface.

[0133] Next, the color intensity of the testing substrate of the present invention was confirmed. In the experiment, the color intensity was measured when the type and application amount (density) of fixing compound was changed using Sample A. The effect of mixing the fixing compound with a surfactant was also confirmed. Fixing compounds used were methyl nitrobenzoate (NBM), nitrocellulose, and a mixture of nitrocellulose and NBM. In immunochromatography, surfactants are used to improve the reactivity between antigens and antibodies and to suppress nonspecific adsorption (an increase in the blank value due to the immobilization of only the labeled antibody, not just the conjugate that is the bond between the antigen and the labeled antibody, at the antibody fixation site).

[0134] The comparative example was the same as sample A except that no fixing compound was applied (referred to as "uncoated" in the table).

[0135] The experimental results are shown in the table below and in FIG.

[0136] [Table 4]

[0137] When no fixing compound was applied, the color intensity was 394.4 mABS and 419.1 mABS at 15 and 30 minutes, respectively. However, when methyl nitrobenzoate was applied at 1 mg / cm as a fixing compound, the color intensity was 394.4 mABS and 419.1 mABS at 15 and 30 minutes, respectively. 2 When nitrocellulose was applied at 0.0008 mg / cm, the mABS increased to 653.7 mABS and 757.1 mABS. 2 and nitrocellulose (0.0008 mg / cm 2 ) and methyl nitrobenzoate (1 mg / cm 2 ) was also mixed and applied, and the color development was greatly improved. The presence of surfactants reduces the color intensity, while nitrocellulose (0.0008 mg / cm) as a fixing compound is effective. 2 ) and methyl nitrobenzoate (1 mg / cm 2 The mixture of the two compounds showed a color development of 283.8 mABS and 429.4 mABS compared to the mixture without the fixing compound, demonstrating that it exhibited higher color development than the uncoated product or the conventional product (nitrocellulose membrane). 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.

[0138] Next, the color intensity of Sample B was measured. The fixing compounds used were nitrocellulose and a mixture of nitrocellulose and methyl nitrobenzoate (NBM). Sample B used consisted of cellulose fiber (samples B-1 and B-2) and glass fiber (sample B-3).

[0139] For comparison of color intensity, the following samples were also measured in the same manner: a sample similar to Sample A except that no fixing compound was applied (Sample A uncoated in the table); a sample similar to Sample B-1 except that no fixing compound was applied (Sample B-1 uncoated in the table); a sample similar to Sample B-2 except that no fixing compound was applied (Sample B-2 uncoated in the table); and a sample similar to Sample B-3 except that no fixing compound was applied (Sample B-3 uncoated in the table).

[0140] The results of experiments using nitrocellulose as the fixative compound are shown in Table 5 below and in FIG. The results of an experiment using a mixture of nitrocellulose and methyl nitrobenzoate (NBM) as the fixing compound are shown in Table 6 below and FIG. It should be noted that the value after 30 minutes for sample B-3 in Table 5 could not be measured because the test piece was dried.

[0141] [Table 5]

[0142] From the results in Table 5, it was confirmed that the color development when nitrocellulose was applied as a fixing compound to cellulose fiber filter paper (samples B-1 and B-2) and glass fiber filter paper (sample B-3) was improved compared to the comparative example (unapplied).

[0143] [Table 6]

[0144] The results in Table 6 show that the color development of cellulose fiber filter paper (sample B-1) can be improved by using a mixture of nitrocellulose and methyl nitrobenzoate as the fixing compound.

[0145] Next, the surface accumulation of the fixing compound at the localized antibody fixing portion of the testing substrate of the present invention was evaluated using a color development test (mapping) method.

[0146] From the above experimental results, it was confirmed that the coating amount (fixed amount) of nitrocellulose can exhibit color development with a much smaller amount than that of methyl nitrobenzoate. However, it was also found that when the concentration is low, the measured values ​​in the Raman spectroscopic analysis vary. For example, when the coating amount (fixed amount) of nitrocellulose is 0.0008 mg / cm 2 While methyl nitrobenzoate can function at 1 mg / cm 2 It requires a certain degree. Therefore, when the amount of fixative compound applied (fixation amount) in such antibody fixation areas is low, it was found that the accumulation state of the fixative compound in the localized layer can be appropriately quantified by using a color development test (mapping) method.

[0147] In the experiment, sample B-1 was used. Nitrocellulose was used as the fixing compound. For fixation, a localization index based on a color development test (mapping) was used. The experiment used iodine, which develops a brown color in the presence of nitrocellulose.

[0148] The comparative example was similar to sample B-1 except that no fixative compound was applied.

[0149] First, a part (4 mm × 5 mm) of sample B-1 (4 mm × 60 mm) was coated with nitrocellulose at 0.0008 mg / cm as a fixing compound. 2The sample was placed in a petri dish together with 1 g of iodine powder, and the dish was covered and exposed for 72 hours. The amount of nitrocellulose applied is the solid content amount as described above.

[0150] 15 shows an optical microscope observation of the cross section of the antibody-immobilized area after iodine mapping. The distance between the dotted lines is 200 μm, which is the film thickness of sample B-1. As shown in FIG. 15, a large amount of color was observed on the side where nitrocellulose was applied (nitrocellulose-coated side) (photographed image).

[0151] Next, the cross-sectional images taken with this optical microscope were binarized to quantify the localization of nitrocellulose in the cross section.

[0152] From the obtained photographed images, the spatial portion (outside the dotted line in the figure) was deleted, and then binarized image processing was performed according to the following threshold values. The threshold value for the binarization process was calculated by quantifying the color development in the photographed image as Red: 22 to 80, Green: 17 to 65, and Blue: 29 to 88 in the RGB color system. Figure 16 shows a cross-sectional micrograph image superimposed on a binarized image, with the distance between both ends of the image being 200 μm, which is the film thickness of Sample B-1. The left side of Figure 16 is the comparative example, and the right side is Sample B-1. As shown in Figure 16, in the comparative example where nitrocellulose was not applied, there were almost no areas that developed color due to binary image processing, confirming that most of the colored areas in sample B-1 were due to the applied nitrocellulose.

[0153] Next, the binarized image is processed as follows. First, a center line perpendicular to the thickness direction was drawn on the micrograph image (photographed image) of the cross section, dividing the distance between the front (coated surface) and back (uncoated surface) in half in the thickness direction. Next, lines were drawn along this center line to divide the cross section into thirds. At this time, each of the resulting images (six divided images of fractions A to F in Figure 16) was divided as follows: The six fractional images divided by each line are congruent rectangles. If the fractional images are A, B, C, D, E, and F, the areas of the fractional rectangles A, B, C, D, E, and F are (A), S(B), S(C), S(D), S(E), and S(F), respectively, as follows: S(A)=S(B)=S(C)=S(D)=S(E)=S(F). Next, the areas of the colored portions of the fraction images A, B, C, D, E, and F that were colored by binary image processing are designated as I(A), I(B), I(C), I(D), I(E), and I(F), respectively. In Figure 16, the area indicated by sample B-1 corresponds to the colored portion. Then, if the groups of fraction images symmetrical with respect to the center line are designated as fractions (X fraction (fraction images A and B), Y fraction (fraction images C and D), and Z fraction (fraction images E and F)), the localization index for each fraction can be expressed by the following formula: If the localization index in each formula is greater than 50, it indicates that the fixing compound M is localized and accumulated on the surface side (i.e., the surface localization layer, the region corresponding to fraction images A, C, and E in Figure 16).

[0154] Localization index (X fraction) = I(A) / {I(A) + I(B)} × 100

[0155] Localization index (Y fraction) = I(C) / {I(C) + I(D)} × 100

[0156] Localization index (Z fraction) = I(E) / {I(E) + I(F)} × 100

[0157] The results of the localization index based on the iodine mapping method for sample B-1 are shown in the table below.

[0158] [Table 7]

[0159] As shown in Table 7, for all samples B-1, the localization index on the side coated with the fixing compound was 50 or higher, indicating significant localization of nitrocellulose on the nitrocellulose-coated surface. In other words, even when nitrocellulose was applied as a fixing compound to sample B-1 made from filter paper (a fibrous sheet material), it was confirmed that the antibody could be localized and fixed on the coated side (i.e., the localization layer) of the antibody-fixed area. While almost no color development was observed in the samples without nitrocellulose coating, color development and localization were observed in the coated samples. This result is consistent with the test results of Raman measurement in which methyl nitrobenzoate was applied as a fixing compound to sample A, and it was confirmed that the fixing compound accumulates on the side where it was applied. In the table, NC indicates that the coloring area on both the front and back sides was zero, so no calculation was made.

[0160] Next, the following experiment was carried out to examine the strength of the testing substrate of this embodiment. In the experiment, Sample A and Sample B-1 were used to confirm the effect of pressing the antibody-fixed portion of each sample in the thickness direction. A tabletop punching press (Tester Sangyo Co., Ltd.) was used for the pressing. In addition, a conventional (commercially available) nitrocellulose membrane was used as a comparative example.

[0161] FIG. 17 shows a cross-sectional image when a press pressure of 23.1 MPa was applied, and FIG. 18 shows a photograph of the surface. Cross-sectional images taken before and after pressing show that in Samples A and B-1, 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 Samples A and B-1, whereas in the Comparative Example, the structure is dense and almost entirely free 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 denting due to pressure, whereas Samples A and B-1 have a structure that is less susceptible to plastic deformation.

[0162] 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. 17, the thickness (B) of the base portion does not change, and therefore was removed from the thickness change equation for calculation. Thickness change (times) = P' / P

[0163] The change in the time of the advancement of the liquid was calculated using the following formula based on Table 8. 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.

[0164] The following table shows the thickness change when pressure is applied, as well as the time it took for the material to penetrate the liquid and the color intensity. Table 8 and Figure 20 also show 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, samples A and B-1 have 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 samples A and B-1, the thickness change due to pressure was greater than that of 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 thickness change of sample A was 0.844, while the comparative example showed a large compressive deformation of 0.273. The liquid advance time for samples A and B-1 was 95.3 sec / 40 mm and 53.7 sec / 40 mm without pressure, but was 103.0 sec / 40 mm and 49.3 sec / 40 mm, respectively, when 23.1 MPa was applied. In the comparative example, the values ​​were 111.3 sec / 40 mm and 749.7 sec / 40 mm, respectively. 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 23.1 MPa being 6.74 times (749.7 / 111.3), almost completely losing its function as a flow path, whereas samples A and B-1 showed a change in the liquid penetration time at an applied pressure of 23.1 MPa being 1.08 times (103.0 / 95.3) and 1.08 times (58.0 / 53.7), confirming that the test liquid was properly transported. Furthermore, with an applied pressure of 9.9 MPa, the change in the liquid penetration time at samples A and B-1 was 1.01 times and 0.92 times, respectively, while the comparative example showed a change of 4.09 times. Furthermore, in Samples A and B-1, the color development was maintained even after compression, whereas in the comparative example, the liquid progressed very slowly at pressures of 9.9 MPa or higher, and no color development was observed. Therefore, this experiment confirmed that the testing substrate of this embodiment is resistant to pressure.

[0165] [Table 8]

[0166] Next, the influence on the liquid adsorption characteristics of the test liquid when a fixing compound is fixed on the antibody fixing portion of the testing substrate of this embodiment was confirmed. The results are shown in Table 9 below. As shown in Table 9, applying a fixing compound to the surface of the antibody-fixed area slightly slowed the liquid wicking rate. When no fixing compound was applied to Sample A, the wicking rate was 78.0 sec / 40 mm. However, by applying the fixing compound and its application amount (fixation amount) to 10 mg / cm² of a 10% methyl nitrobenzoate solution, 20 mg / cm² of a 10% methyl nitrobenzoate solution, and 10 mg / cm² of 0.01% nitrocellulose, the wicking rates increased to 88.7, 124.3, and 113.7, respectively. The experimental results showed that applying a fixing compound created resistance to the advancement of the test liquid, slowing down the flow rate. In other words, it was confirmed that fixing the fixing compound improved the advancement characteristics. Therefore, it was confirmed that in 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 in the antibody fixing portion. The rate at which the test solution advances is an important factor in immunochromatography, which is based on an antigen-antibody reaction. Since an antigen-antibody reaction generally requires a certain amount of time, controlling the rate at which the test solution advances directly leads to improved measurement sensitivity and accuracy.

[0167] [Table 9] [Industrial Applicability]

[0168] The testing substrate of the present invention is suitable as a testing substrate used in tests such as immunoassays. [Explanation of symbols]

[0169] 1. Test substrate 10 Antibody fixing area 10h Void (flow path) 10US antibody fixation surface 10BS Back of antibody fixation area 11 Localized site 13 Forming member 14 Fibrous material of antibody fixation area 15 Granular materials 16 Binder material A antibody AC control antibody AI immobilized antibody AL-labeled antibody LC Conjugates 20 Flow path section 24 Fibrous material of flow path 30 Absorption section 40 Base

Claims

1. A substrate for testing, The plate-shaped antibody fixing section has an antibody fixing function and allows liquid to pass through by capillary action. The antibody fixing part is containing a fixative compound capable of binding to the antibody; The fixing compound has a localized site, The localized site is The antibody-fixed portion is disposed on the surface side of the antibody-fixed portion. A substrate for testing characterized by:

2. The antibody fixing part is Contains fibrous materials 2. The testing substrate according to claim 1.

3. The antibody fixing part is The antibody fixative solution containing the fixative compound is applied to a surface.

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

4. At the localized site, The fixative compound is formed so that its localization index is greater than 50 (>50).

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

5. The localization index is is a value calculated from the following formula based on a spectral analysis of the cross section of the antibody fixation part at the localized site:

5. The testing substrate according to claim 4. Localization index = ∫f(x):P / {∫f(x):P + ∫f(x):Q} × 100 (In the formula, ∫f(x):P is the value obtained by performing a definite integral on the signal intensity difference in the range from the point on the x-axis that passes through the surface of the antibody-immobilized section to the point that passes through the center line of the antibody-immobilized section (the midpoint between the point that passes through the surface and the point that passes through the back surface of the antibody-immobilized section) in a line measurement graph of the spectrum obtained by measuring the cross section of the antibody-immobilized section in the thickness direction of the antibody-immobilized section (from the front surface to the back surface). In the formula, ∫f(x):Q is the value obtained by performing a definite integral on the signal intensity difference in the range from the point passing through the center line of the antibody-immobilized section on the x-axis (the midpoint between the point passing through the surface and the point passing through the back surface of the antibody-immobilized section) to the point passing through the back surface of the antibody-immobilized section in a line measurement graph of the spectrum obtained by measuring the cross section of the antibody-immobilized section in the thickness direction of the antibody-immobilized section (from the front surface to the back surface).

6. The localization index is is a value calculated from the following formula based on a mapping method of the antibody fixation site at the localized site:

5. The testing substrate according to claim 4. Localization index = I(A) / {I(A) + I(B)} × 100 (I(A) and I(B) in the formula represent the areas of the colored portions obtained by binarizing the A and B fraction images obtained by taking a micrograph of the cross section of the antibody-immobilized area and then symmetrically taking the A and B fraction images about the center line of the antibody-immobilized area. A Fraction image A is a fraction image on the front side of the antibody-immobilized area, and fraction image B is a fraction image on the back side of the antibody-immobilized area.)

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

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

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

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

9. The antibody fixing part 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.

10. The antibody fixing part is and a granular member disposed between the fibrous members.

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

11. a base portion that holds the antibody-fixed portion; 3. The testing substrate according to claim 1 or 2.

12. a flow path connected to the antibody-fixed portion and through which a liquid can pass by capillary action; The flow path portion contains a fibrous member.

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

13. The antibody fixing section and the flow channel section are integrally formed.

13. The testing substrate according to claim 12.

14. 1. A method for manufacturing a substrate for testing, comprising: a step of applying an antibody fixing liquid in a stripe shape to a surface of a sheet member containing a fibrous member and through which a liquid can pass by capillary action, and drying the sheet member to form an antibody fixing portion; cutting the sheet member into strips along a direction perpendicular to the belt-shaped antibody-fixed portion, The antibody fixative solution Contains a fixing compound capable of binding to the antibody A method for manufacturing a substrate for testing, comprising:

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