Immuno-chromatographic device and method for measuring sugar chain antigen in a test body

Abstract

Provided is an immunochromatographic device and a method for measuring a sugar chain antigen in a sample, which enables measurement with sufficient sensitivity by performing nitrous acid extraction treatment for a sufficient period of time. The test strip has a sample pad, a labeled body region, and a detection region, and has a neutralizing reagent region upstream of the labeled body region and a region in which a solid acid reagent or nitrite is impregnated upstream of the neutralizing reagent region, wherein (i) the immunochromatographic device has a relatively large sample addition port for holding an added sample solution of the sample and facilitating extraction of the sugar chain antigen by the nitrite and the solid acid reagent by supplying it to the region in which the solid acid reagent or nitrite is impregnated for a short period of time; and (ii) there is no gap between the sample addition port and the sample pad, so that the sample does not overflow from the addition port, and the sample addition port has a size that enables 10 to 200 μL of the sample solution of the sample to be supplied at one time.

Description

[0001] This application is a divisional application of patent application No. 201880018199.7, filed on March 14, 2018, entitled "Immuno-chromatographic device for extracting and measuring sugar chain antigen", the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to an immuno-chromatographic device for extracting and measuring sugar chain antigen, which is capable of nitrous acid extraction treatment of sugar chain antigen on an immuno-chromatographic test sheet. BACKGROUND

[0003] Many rapid diagnostic reagents based on the principle of immuno-chromatography are widely used as a means for rapidly and simply measuring viral or bacterial infectious diseases and determining a treatment plan.

[0004] In general, rapid diagnostic reagents based on the principle of immuno-chromatography are capable of rapidly and simply performing measurement by supplying a suspension of a test body to an immuno-chromatographic test sheet after suspending the test body in a test body suspension.

[0005] In order to detect microorganisms belonging to Streptococcus such as Group A beta hemolytic streptococcus (Group A beta hemolytic streptococcus) and oral streptococcus, it is necessary to extract sugar chain antigen and measure the sugar chain antigen.

[0006] For example, it has been reported that a method for performing nitrous acid extraction treatment on an immuno-chromatographic test sheet by previously containing sodium nitrite and a neutralizing reagent in the immuno-chromatographic test sheet, suspending a test body in an acidic solution such as acetic acid, and supplying the test body to the immuno-chromatographic test sheet (Patent Document 1).

[0007] In addition, there is a method for previously containing an acidic reagent and a neutralizing reagent in an immuno-chromatographic test sheet, suspending a test body in a nitrite salt, and supplying the test body to the immuno-chromatographic test sheet.

[0008] In immuno-chromatography, it is sometimes necessary to control the chromatography development speed in an immuno-chromatographic test sheet, and methods for controlling by the shape of the device, by the attachment position, by the raw material (material such as nonwoven fabric) used, by the reagent, and the like have been reported (Patent Documents 2 and 3).

[0009] PRIOR ART DOCUMENTS

[0010] PATENT DOCUMENTS

[0011] Patent Document 1: International Patent Publication No. WO2005 / 121794

[0012] Patent Document 2: Japanese Patent Application Publication No. 2005-331471

[0013] Patent Literature 3: Japanese Patent Application Laid-Open (JP A) No. 2005-331463 SUMMARY

[0014] PROBLEMS TO BE SOLVED BY THE INVENTION

[0015] In order to detect microorganisms belonging to Streptococcus such as Group A beta hemolytic streptococcus (Group A beta hemolytic streptococcus) and oral streptococcus, a sugar chain antigen is extracted and measured.

[0016] In order to extract a sugar chain antigen, nitrous acid is used. Since nitrous acid is unstable when used alone, in the case of nitrous acid extraction, the antigen is extracted by generating nitrous acid by mixing a nitrite solution and an acid solution (acetic acid, citric acid, and / or tartaric acid, etc.) on site.

[0017] In the case of detection using an immunochromatography method, either the nitrous acid solution or the acid solution is coated and dried, and incorporated into an immunochromatography test sheet. In this reagent form, the operation is simple since the mixing of the reagents is not required compared to the liquid reagent, but on the other hand, the following problem exists: if the sample is developed in a short time after mixing the reagents, the sensitivity is insufficient since the nitrous acid extraction treatment is not sufficiently performed as in the case of the conventional immunochromatography method.

[0018] In the case of the reagent form in which the liquid reagents are mixed for nitrous acid extraction, the sample is extracted for 1 to 2 minutes. Therefore, in order to obtain the same sensitivity as in the case of the liquid form, it is necessary to maintain the sample for 1 to 2 minutes upstream of the neutralization.

[0019] The object of the present application is to provide an immunochromatography method and an immunochromatography device in which a sugar chain antigen is extracted by nitrous acid extraction on an immunochromatography test sheet and measured, and in which a sufficient sensitivity can be achieved by performing the nitrous acid extraction treatment for a sufficient time.

[0020] MEANS FOR SOLVING THE PROBLEMS

[0021] The present inventors have conducted intensive research on the following technology: in an immunochromatography method for extracting and measuring a sugar chain antigen in which nitrous acid extraction treatment of the sugar chain antigen can be performed on an immunochromatography test sheet, the nitrous acid extraction treatment is performed for a sufficient time.

[0022] As a result, it was found that the above object can be achieved by increasing the addition port of the immunochromatography device to enable the addition of a large amount of a test sample, and by introducing a mechanism in which the test sample does not flow out of the addition port and the development speed is adjusted, and the present application was completed. That is, the present application is as described below.

[0023] [1] An immunochromatographic device comprising an immunochromatographic test strip and a container for storing the test strip, and having a sample application port on a sample pad of the test strip, the immunochromatographic test strip comprising: a sample pad to which a sample of a test body mixed with a nitrite or an acidic solution is applied, a labeled body region containing a labeled antibody labeled with an antibody to a sugar chain antigen, and a detection region in which an antibody-sugar chain antigen-labeled antibody complex is formed by solidifying the antibody to the sugar chain antigen, and the sugar chain antigen is measured, the immunochromatographic test strip having, upstream of the labeled body region, a region impregnated with a neutralizing reagent, and further, upstream of the region impregnated with the neutralizing reagent, a region impregnated with a solid acidic reagent in the case of using a test body mixed with a nitrite, and a region impregnated with a nitrite in the case of using a test body mixed with an acidic solution, the immunochromatographic test strip being used for extracting and measuring the sugar chain antigen in the test body, wherein

[0024] (i) the immunochromatographic device has a larger test body application port for holding an applied test body sample solution and promoting extraction of the sugar chain antigen by the nitrite and the solid acidic reagent by supplying the test body sample solution to the region impregnated with the solid acidic reagent or the nitrite in a short time.

[0025] (ii) there is no gap between the application port and the sample pad so that the sample does not overflow from the application port.

[0026] [2] The immunochromatographic device according to [1], wherein the test body application port of the immunochromatographic device has a size of 24 to 75 mm 2 .

[0027] [3] The immunochromatographic device according to [1] or [2], wherein the pad impregnated with the solid acidic reagent or the nitrite or the neutralizing reagent on the immunochromatographic test strip is composed of a higher hydrophobic raw material, thereby promoting extraction of the sugar chain antigen by the nitrite and the solid acidic reagent by delaying the development time of the test body sample solution.

[0028] [4] The immunochromatographic device according to [3], wherein the higher hydrophobic raw material is selected from the group consisting of polyethylene, polyester, polystyrene, polypropylene, rayon, and nylon.

[0029] [5] The immunochromatographic device according to [1] or [2], wherein the pad impregnated with the solid acidic reagent or the nitrite or the neutralizing reagent on the immunochromatographic test strip is composed of a higher hydrophilic raw material, thereby promoting extraction of the sugar chain antigen by the nitrite and the solid acidic reagent by delaying the development time of the test body sample solution.

[0030] [6] The immunochromatographic device according to [5], wherein the higher hydrophilic raw material is filter paper having a thickness.

[0031] [7] The immunochromatographic device according to any one of [1] to [6], wherein the elution time of the sample solution of the test body is adjusted by adding a surfactant to the pad impregnated with the solid acid reagent or the nitrite or the neutralizing reagent on the immunochromatographic test strip to promote the extraction of the sugar chain antigen by the nitrite and the solid acid reagent.

[0032] [8] The immunochromatographic device according to [7], wherein the surfactant is selected from the group consisting of polyoxyethylene octylphenyl ether, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl ether, sodium cholate, deoxycholic acid, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate.

[0033] [9] The immunochromatographic device according to any one of [1] to [8], wherein the elution time of the sample solution of the test body is adjusted by adding a high-molecular compound having a fixation action to the pad impregnated with the solid acid reagent or the nitrite or the neutralizing reagent on the immunochromatographic test strip to promote the extraction of the sugar chain antigen by the nitrite and the solid acid reagent.

[0034]

[10] The immunochromatographic device according to [9], wherein the high-molecular compound having a fixation action is PVA or PLL.

[0035]

[11] The immunochromatographic device according to any one of [1] to

[10] , wherein the region impregnated with the solid acid reagent or the nitrite is present on the sample pad or the pad coated with the labeled body region.

[0036]

[12] The immunochromatographic device according to any one of [1] to

[11] , wherein the solid acid reagent is selected from the group consisting of malonic acid, malic acid, maleic acid, citric acid, and tartaric acid.

[0037]

[13] The immunochromatographic device according to any one of [1] to

[12] , wherein the neutralizing reagent is tris-hydroxymethyl aminomethane or sodium hydroxide.

[0038]

[14] The immunochromatographic device according to any one of [1] to

[13] , wherein the sugar chain antigen is a sugar chain antigen of protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, or virus.

[0039]

[15] A method for measuring a sugar chain antigen in a test body using the immunochromatographic device according to any one of [1] to

[14] , the method for measuring a sugar chain antigen in a test body by an immunochromatographic method, the method comprising:

[0040] When the immunochromatographic apparatus has a region impregnated with a solid acidic reagent, the sample is mixed with a nitrite solution; when the immunochromatographic apparatus has a region impregnated with nitrite, the sample is mixed with an acidic solution and added to the sample pad of the immunochromatographic apparatus.

[0041] In areas impregnated with solid acidic reagents or nitrites, glycan antigens are extracted from the sample by the action of nitrite produced from the reaction of nitrite with the solid acidic reagent. In areas impregnated with neutralizing reagents, the acidic solution containing the glycan antigens is neutralized.

[0042] An antibody-glycan antigen-labeled antibody complex is formed in the detection area. The method promotes the extraction of glycan antigens by the above-described immunochromatography and measures the glycan antigens in the sample.

[0043] This specification contains the disclosure of Japanese Patent Application No. 2017-049163, which forms the basis of the priority claim of this application.

[0044] The effects of the invention

[0045] When using the immunochromatographic apparatus of the present invention, the development time of the sample solution on the immunochromatographic test strip after the sample has been added can be adjusted. As a result, the sample processing performed on the test strip by the sample processing reagent contained on the immunochromatographic test strip can be fully carried out. Attached Figure Description

[0046] Figure 1 A diagram illustrating the structure of an immunochromatographic test strip (one-piece test strip) having a solid acidic reagent region and a neutralizing reagent region.

[0047] Figure 2 A diagram illustrating the structure of an immunochromatographic test strip (two-piece test strip) having a solid acidic reagent region and a neutralizing reagent region.

[0048] Figure 3 A, Figure 3 B is a perspective view showing the appearance of the immunochromatographic apparatus. Figure 3 A is the device of the present invention with a larger opening 9. Figure 3 B is an existing device with a smaller port 9 added.

[0049] Figure 4 A, Figure 4 B is a top view showing the appearance of the immunochromatographic apparatus. Figure 4 A is the device of the present invention with a larger opening 9. Figure 4 B is an existing device with a smaller port 9 added. Detailed Implementation

[0050] The present application will be described in detail below.

[0051] The present application relates to an immunochromatography device capable of easily performing nitrous acid extraction treatment of sugar chain antigens on an immunochromatography test sheet, and capable of rapidly and accurately measuring a detected substance, i.e., a sugar chain antigen. The immunochromatography device includes an immunochromatography test sheet and a container for storing the immunochromatography test sheet, and the immunochromatography test sheet is incorporated into the immunochromatography device to be manufactured. The storage container is capable of preventing deterioration due to, for example, ultraviolet rays or moisture in the air. In addition, when a contaminated or infectious sample is used, the storage container is capable of preventing contamination or infection of an analyst who performs the analysis. For example, a box made of a resin of an appropriate size is used as the storage container, and the device of the present application is stored in the box. In addition, a film or the like (top laminate) made of a resin is used to cover the surface of the test sheet on which an antigen or an antibody is immobilized.

[0052] The immunochromatography test sheet includes a support having a detection region in which an antibody (antibody 1) that captures a detected substance (antigen or the like) is immobilized, a labeling body region having a mobile labeled antibody (antibody 2), a sample pad to which a sample is added, an absorption belt that absorbs a labeled substance solution after development, and a back liner sheet or the like that integrates these components.

[0053] Note that the number of detection regions and the types of labeled antibodies included in the labeling body region are not limited to one, and two or more antigens can be measured on the same test sheet by using antibodies corresponding to a plurality of detected substances.

[0054] The support is a material having a function of immobilizing an antibody for capturing a detected substance (antigen), and has a function of not interfering with the passage of a liquid in the horizontal direction. Preferably, it is a porous film (membrane) having a capillary action, and is a material capable of transporting a liquid and a component dispersed therein by absorption. The material constituting the support is not particularly limited, and cellulose, nitrocellulose, cellulose acetate, polyvinylidene fluoride (PVDF), glass fiber, nylon, polyketone, or the like can be used. Among them, a film or a membrane made of nitrocellulose is more preferable. The film on which an antibody is immobilized is referred to as an antibody immobilized membrane.

[0055] The labeling body region is composed of a porous base material including a labeled antibody, and the material of the base material can use a commonly used glass fiber or nonwoven fabric or the like. In order to impregnate a larger amount of a labeled antibody, the base material is preferably a pad having a thickness of 0.3 mm to 0.6 mm. The porous base material impregnated with a labeled antibody and dried is also referred to as a dried pad.

[0056] As a label for the labeling of the antibody, an enzyme such as alkaline phosphatase or horseradish peroxidase, a colloidal metal such as colloidal gold, silica particles, cellulose particles, colored polystyrene particles, and colored latex particles are used. When colored particles such as colloidal metal particles, colored polystyrene particles, or colored latex particles are used, the color of the particles changes after the particles are aggregated, and the color change is measured. The particles on which the antibody is immobilized are referred to as antibody-immobilized particles.

[0057] The detection region is a part of the support on which an antibody that captures a substance to be detected (antigen) is immobilized. In the detection region, at least one region on which an antibody that captures an antigen is immobilized is provided. The detection region can be included in the support, and the antibody can be immobilized on the support.

[0058] The sample pad is a site where a sample is applied, and is a porous material. The sample pad is a site that is located at the most upstream position of the immunochromatographic test strip. As the material, a filter paper, glass fiber, nonwoven fabric, or the like can be used. In order to use a large amount of sample in the immunoassay, a pad having a thickness of 0.3 mm to 1 mm is preferable. The sample can also include a sample prepared using a sample obtained by suspending a sample in another solution.

[0059] The absorption pad is a member for absorbing a component that is supplied to the support and does not participate in the reaction in the detection region. As the material, a filter paper, sponge, or the like having a high water retention property made of a conventional natural high molecular compound or a synthetic high molecular compound can be used. In order to promote the development of the sample, a material having a high water absorption property is preferable.

[0060] The backer sheet is a member for partially overlapping and fixing the above-described materials, i.e., the support, the sample pad, the labeled body region, the absorption pad, and the like. As long as the materials are arranged and fixed at the most appropriate intervals, a conventional backer sheet is preferable, although it is not necessarily required, in terms of the convenience in manufacturing or use.

[0061] The immunochromatographic test strip of the present application can further include a control display region (member). The control display region is a site that indicates that the test is accurately performed. For example, the control display region is present downstream of the detection region, and when a sample reaches the control display region from the detection region, a signal is emitted by coloring or the like. The control display region can also be solidified with a substance that binds to an antibody that binds to a labeled carrier, and can also be solidified with a reagent such as a pH indicator that changes color when a sample reaches. When the antibody that binds to the labeled carrier is a mouse monoclonal antibody, an anti-mouse IgG antibody can be used.

[0062] The size of the immunochromatographic test strip is not limited, and for example, the length in the longitudinal direction is several cm to several tens of cm, and the length in the lateral direction is several mm to several cm.

[0063] In the test piece of the above-described mode, the sample passes through a porous flow path formed by a series of connections of the sample pad, the marker region, the support, the detection region, the absorbent strip, and the like. Therefore, in the present mode, all of them are the sample moving regions. Based on the material quality or mode of each constituent material, it is also possible that the sample passes through the surface, not the inside, of the sample-impermeable material. Since the sample moving region defined in the present specification does not consider whether it is the inside or the surface of the material, the test piece of this mode is also included in the scope of the present specification.

[0064] When the saccharide chain antigen in the sample is measured using the immunochromatography test piece of the present application, it is necessary to first extract the saccharide chain antigen from the sample. The extraction of the saccharide chain antigen is performed by treating the sample containing the saccharide chain antigen with nitrous acid. The treatment of the sample containing the saccharide chain antigen with nitrous acid can be performed by mixing a nitrous acid salt such as sodium nitrite with an acid to generate nitrous acid. The extracted antigen binds to the antibody immobilized on the immunochromatography test piece through the antigen-antibody reaction. At this time, if nitrous acid remains in the reaction system, the reaction system becomes strongly acidic, and the antigen-antibody reaction is hindered. Therefore, it is necessary to neutralize the nitrous acid in the reaction system.

[0065] In the present application, in the method in which the saccharide chain antigen is extracted from the sample by mixing a nitrous acid salt with an acid to generate nitrous acid and the saccharide chain antigen binds to the antibody immobilized on the immunochromatography test piece after the neutralization of the nitrous acid, and the saccharide chain antigen is measured, as a method for extracting and measuring the saccharide chain antigen, the following methods can be listed. In any of the methods, the extraction and the neutralization of the saccharide chain antigen with nitrous acid are performed on the immunochromatography test piece. In order to perform the extraction of the saccharide chain antigen with nitrous acid on the immunochromatography test piece, it is only necessary to previously impregnate an acidic reagent or a nitrous acid salt on the immunochromatography test piece. In order to perform the neutralization on the immunochromatography test piece, it is only necessary to previously impregnate a neutralizing reagent on the immunochromatography test piece.

[0066] (A) The sample is mixed with an acidic solution in advance and added to the sample pad of the immunochromatography test piece in which a nitrous acid salt and a neutralizing reagent are impregnated. The mixture reaches the region containing the nitrous acid salt, and the nitrous acid salt reacts with the acid to generate nitrous acid, and the saccharide chain antigen in the sample is extracted. The extract of the saccharide chain antigen is neutralized in the region of the immunochromatography test piece in which the neutralizing reagent is impregnated, and the saccharide chain antigen binds to the antibody immobilized on the immunochromatography test piece, and the measurement can be performed. In this method, as the acidic solution to be used, acetic acid, hydrochloric acid, malonic acid, malic acid, maleic acid, citric acid, tartaric acid, and the like can be listed.

[0067] (B) The sample is mixed with a nitrite solution in advance, and the mixture is added to a sample pad of an immunochromatography test strip in which an acidic reagent and a neutralizing reagent are impregnated. When the mixture reaches a region containing the acidic reagent, the nitrite reacts with the acid to produce nitrous acid, and the sugar chain antigen in the sample is extracted. The extracted sugar chain antigen is neutralized in a region of the immunochromatography test strip in which the neutralizing reagent is impregnated, and the sugar chain antigen binds to the antibody immobilized on the immunochromatography test strip, so that the detection can be performed.

[0068] In the immunochromatography test strip of the present application used for the above-described (A) or (B), an acidic reagent or a nitrite and a neutralizing reagent are impregnated in the sample pad or between the sample pad and the marker region, which is more upstream than the marker region (upstream of the sample flow, the side of the sample pad). The immunochromatography test strip in which a nitrite and a neutralizing reagent are impregnated in the sample pad or between the sample pad and the marker region can be used for the above-described (A). The immunochromatography test strip in which an acidic reagent and a neutralizing reagent are impregnated in the sample pad or between the sample pad and the marker region can be used for the above-described (B). Note that, as the acidic reagent impregnated in the immunochromatography test strip, a solid acidic reagent is used. By these methods, the detected substance in the sample can be accurately and specifically measured without being affected by the amount of the sample supplied in the test.

[0069] The solid acidic reagent or the nitrite can be impregnated in the sample pad, or in another pad composed of a porous material such as a nonwoven fabric, other than the sample pad. The porous material impregnated with the solid acidic reagent or the porous material impregnated with the nitrite is disposed between the sample pad and the marker region, i.e., the upstream side of the marker region. The region impregnated with the solid acidic reagent or the nitrite can or can not be in contact with the sample pad or the marker region.

[0070] In the present application, the region impregnated with the reagent is also referred to as a pad impregnated with the reagent.

[0071] The neutralizing reagent is disposed more downstream than the region impregnated with the solid acidic reagent or the nitrite. The neutralizing reagent can be impregnated in the sample pad, in the support, or in another pad composed of a porous material such as a nonwoven fabric, other than the support. The porous material impregnated with the neutralizing reagent is disposed between the region impregnated with the solid acidic reagent or the nitrite and the marker region. That is, the region impregnated with the neutralizing reagent is present upstream of the marker region, and the region impregnated with the solid acidic reagent or the nitrite is present further upstream of the region impregnated with the neutralizing reagent. The region impregnated with the neutralizing reagent can or can not be in contact with the region impregnated with the solid acidic reagent or the nitrite or the marker region.

[0072] The region containing the solid acid reagent is referred to as a solid acid reagent region, the region containing the nitrite is referred to as a nitrite region, and the region containing the neutralizing reagent is referred to as a neutralizing reagent region or a basic reagent region.

[0073] In the immunochromatography test sheet having the solid acid reagent region or the nitrite region and the neutralizing reagent region, the sample pad, the solid acid reagent region or the nitrite region, the neutralizing reagent region, the marker region, the detection region, and the absorbent pad are sequentially provided on the support from the upstream, and the solid acid reagent region or the nitrite region can be provided on the sample pad. In addition, the sample pad, the solid acid reagent region or the nitrite region, the neutralizing reagent region, the marker region, the detection region, and the absorbent pad can be in contact with each other or can not be in contact with each other. Further, the solid acid reagent region or the nitrite region, the neutralizing reagent region, and the marker region do not necessarily contain the respective independent porous materials, and a plurality of or all of the regions can contain the same porous material.

[0074] The solid acid reagent used in the present application is in a solid state at normal temperature and does not volatilize at high temperature.

[0075] As the preferred solid acid reagent used in the present application, malonic acid, malic acid, maleic acid, citric acid, and tartaric acid can be exemplified.

[0076] In addition, as the preferred solid acid reagent used in the present application, a smaller amount is sufficient for extraction if a higher valence number acid such as citric acid is used. In addition, if the valence numbers are the same, a smaller acid dissociation constant is better, for example, maleic acid and tartaric acid.

[0077] In addition, as the preferred solid acid reagent used in the present application, it is preferable that the reagent is not colored on the immunochromatography test sheet, and specifically, the reagent is white in a dry state or is not easily colored by dry heat or oxidation.

[0078] As the nitrite used in the present application, sodium nitrite, potassium nitrite, and the like can be exemplified.

[0079] The amount of the solid acid reagent or the nitrite used in the present application, i.e., the amount impregnated in the immunochromatography test sheet, is not particularly limited, and generally, the amount is 0.01 μg to 1 mg, preferably 0.1 μg to 0.1 mg, per test sheet of the immunochromatography test sheet. It is understood that, preferably, the most appropriate amount that can achieve the effect is selected depending on the kind of the solid acid reagent or the nitrite used, the composition or the amount of the test body suspension, and the like.

[0080] In order to impregnate the solid acid reagent or the nitrite in the sample pad or the porous material, the solid acid reagent or the nitrite is once dissolved and dried.

[0081] The neutralizing agent used in the present application is solid at normal temperature and does not volatilize at high temperature.

[0082] As the preferred neutralizing agent used in the present application, Tris base (tris-hydroxymethyl aminomethane), sodium hydroxide, dipotassium hydrogen phosphate, trisodium citrate, and Good's buffer having a buffering property in the basic region can be exemplified.

[0083] The amount of the neutralizing agent used in the present application, i.e., the amount of the impregnation into the immunochromatography test piece, is not particularly limited, and generally, the average amount per test piece of the immunochromatography test piece is 0.01 μg to 1 mg, preferably 0.1 μg to 0.1 mg. It is understood that the most appropriate amount to obtain the effect is preferably selected depending on the kind of the neutralizing agent used, the composition or the amount of the addition of the test body suspension, and the like.

[0084] In order to impregnate the neutralizing agent into the sample pad or the porous material, the neutralizing agent is once dissolved, and the solution is applied to the sample pad or the porous material, and then dried.

[0085] Figure 1 and Figure 2 A diagram showing a preferred form of a typical immunochromatography test piece. Figure 1 and Figure 2 The immunochromatography test piece shown is an immunochromatography test piece impregnated with a solid acidic reagent and a neutralizing agent, but a nitrate salt can be impregnated instead of the solid acidic reagent, in which case, it is an immunochromatography test piece impregnated with a nitrate salt and a neutralizing agent. The immunochromatography test piece impregnated with a nitrate salt and a neutralizing agent can be appropriately designed and manufactured by those skilled in the art. Note that the immunochromatography test piece is not limited to Figure 1 and Figure 2 shown. Figure 1 and Figure 2 In the figure, 1 is a support, 2 is a marker region, 3 is a detection region, 4 is a sample pad, 7 is an absorbent tape, and 8 is a backer sheet. In addition, a top laminate can also be attached to the entire test piece.

[0086] Figure 1 A and Figure 2 A is a top view, Figure 1 B and Figure 2 B is a sectional view. In Figure 1 the example, a backer sheet 8 made of resin or the like has a support 1 in which one detection region 3 is formed, an absorbent tape 7, a marker region 2, a sample pad 4, and the like laminated thereon, respectively. In addition, as Figure 1As shown, one end of the absorbent band 7 overlaps with one end of the support 1, the other end of the support 1 overlaps with one end of the marker region 2, and the other end of the marker region 2 overlaps with one end of the sample pad 4. The upstream portion of the sample pad 4 is impregnated with a solid acidic reagent, and the downstream portion, slightly separated from the sample pad, is impregnated with a neutralizing reagent. The region impregnated with the solid acidic reagent is called the solid acidic reagent region 5, and the region impregnated with the neutralizing reagent is called the neutralizing reagent region 6. In this test piece, the sample pad serves as both the solid acidic reagent region 5 and the neutralizing reagent region 6. That is, the solid acidic reagent region and the neutralizing reagent region exist on the sample pad. In this test piece, since the solid acidic reagent region and the neutralizing reagent region are set as a single porous material (pad), it is sometimes also called a single-pad test piece. Figure 2 In the example, upstream of the labeled region 2, there are solid acidic reagent region 5 and / or neutralizing reagent region 6, which overlap each other, thereby forming a continuous transverse flow path. Figure 2 In the test piece shown, the solid acid reagent region 5 also serves as a sample pad. That is, the solid acid reagent region exists on the sample pad. In this test piece, because the solid acid reagent region and the neutralizing reagent region are set as two independent porous materials (pads), it is sometimes called a two-pad test piece. A sample pad may also exist upstream of the solid acid reagent region. Furthermore, in Figure 2 In the test piece shown, the solid acid reagent region 5 and the neutralizing reagent region 6 are impregnated with different porous materials, but they can also be like... Figure 1 The test piece sample pad has a solid acid reagent region 5 on the upstream part and a neutralizing reagent region 6 on the downstream part, just like the sample pad of the test piece.

[0087] The above-mentioned chromatography test pieces were stored in Figure 3 and Figure 4 The storage container described herein is used as an immunochromatographic apparatus. Figure 3 and Figure 4 )use. Figure 3 and Figure 4 The storage container consists of a lower container section and an upper lid section. For example... Figure 3 and Figure 4 As shown, the storage container has a sample addition port (also called an addition hole) 9 and a determination section 10. When the above-mentioned chromatography test strip is stored in the storage container, the sample addition port, which supplies the sample solution to the sample pad, is located at the top of the sample pad. When the sample is added to the sample addition port of the container, the sample is immersed in the sample pad. Furthermore, when the chromatography test strip is stored in the storage container, the determination section is located on the detection area, and the detection area can be observed through the hole of the determination section of the container.

[0088] The immunochromatography device of the present application enables nitrous acid extraction treatment to be performed for a sufficient time in an immunochromatography method for extracting and measuring sugar chain antigens, which is capable of performing sugar chain antigen nitrous acid extraction treatment on an immunochromatography test strip. Therefore, the following structural feature is provided. The following structural feature is a structural feature regarding a mechanism for adjusting the sample application speed.

[0089] (1) The addition port of the immunochromatography device of the present application is enlarged.

[0090] For example, the addition port of an existing immunochromatography device (Quick Navi (trademark) - Strep A, Denka Seiken Co., Ltd.) that stores an immunochromatography test strip having a longitudinal length of 5 to 9 cm, preferably 7 cm, and a lateral length of 0.3 to 0.7 cm, preferably 0.5 cm, is provided as an approximately rectangular addition port having a size of about 3.5 mm x about 5.5 mm (19.25 mm 2 ), and the addition port of the immunochromatography device of the present application that stores an immunochromatography test strip having the same size is provided as an approximately rectangular addition port having a size of about 3.5 mm x about 11 mm (38.5 mm 2 ). The size of the immunochromatography device and the immunochromatography test strip stored therein becomes approximately similar, and therefore, in an immunochromatography device having a lateral length of 3 cm and a longitudinal length of 9 cm, an immunochromatography device having an approximately rectangular addition port of 3 to 5 mm x 8 to 15 mm (24 to 75 mm 2 ) is an immunochromatography device having a large addition port, and is capable of exhibiting the effects of the immunochromatography device of the present application. The longer side of the addition port is a side parallel to the longer side of the chromatography test strip, i.e., a side in the flow direction of the sample solution. Sometimes, the length of the longer side of the addition port is referred to as the addition port length, and the length of the shorter side is referred to as the addition port width. The above example is a case of an approximately rectangular addition port, and the shape of the addition port can be triangular or circular. When the size of the addition port of the immunochromatography device of the present application is expressed in terms of area, it is 24 to 75 mm 2 , preferably 35 to 75 mm 2 .

[0091] In the existing immunochromatography method, when the sample is treated with a sample treatment reagent provided on the immunochromatography test strip, the sample solution and the region in which the sample treatment reagent is impregnated are in contact with each other over a small area, and therefore, the sample treatment ability is weak. Furthermore, since the sample treatment is performed while the immunochromatography test strip is gradually spread, a difference in the concentration of the sample treatment reagent occurs between the sample solution that is spread first and the sample solution that is spread later. Therefore, the sample treatment cannot be reliably performed.

[0092] Since the addition port of the immunochromatographic device of the present application is provided to be large, a large amount of sample solution can be supplied to the region of the immunochromatographic strip to which the sample treatment reagent is impregnated, i.e., the region to which the solid acid reagent is impregnated or the region to which the nitrous acid is impregnated, in one operation in a short time. As a result, extraction of the sugar chain antigen on the immunochromatographic test piece can be facilitated. The sample solution that can be supplied in one operation is 10 μL to 200 μL.

[0093] As a result, a difference in the concentration of the sample treatment reagent due to a time difference does not occur, and thus the sample treatment can be performed reliably and efficiently on the immunochromatographic strip.

[0094] (2) In the immunochromatographic device of the present application, the addition port and the sample pad thereunder are provided without a gap therebetween so that the sample does not flow out of the addition port.

[0095] The addition port of the immunochromatographic device of the present application is provided to be large, and a large amount of sample solution can be supplied in one operation. In addition, the addition port has a certain height (1 to 5 mm) in the outer contour, i.e., the edge portion, and the sample solution supplied can be temporarily stored in the addition port.

[0096] As a result, since a long time is required for the entire liquid of the sample solution to be absorbed by the immunochromatographic test piece, the sample solution sometimes flows out of the immunochromatographic test piece without being absorbed by the immunochromatographic test piece.

[0097] In the immunochromatographic device of the present application, the backer sheet supporting the sample pad can be configured in such a manner that, in order to prevent the sample from flowing out of the addition port, the sample pad is pressed toward the addition port by the backer sheet having a size equal to or larger than the outer contour of the addition port, and the force of the backer sheet acts so that there is no gap between the addition port and the sample pad, and thus the sample solution can be prevented from flowing out.

[0098] In addition, when there is a difference in the thickness of the sample pad, the thinner portion of the sample pad is likely to have a gap with the addition port, and the sample solution is likely to flow out.

[0099] In the present application, by designing the thinner portion of the sample pad to have a higher base of the container of the device and the thicker portion to have a lower base, even when there is a difference in the thickness of the pad, the device can be configured in such a manner that there is no gap between the addition port and the pad, and thus the sample solution can be prevented from flowing out.

[0100] As a result, extraction of the sugar chain antigen on the immunochromatographic test piece can be performed efficiently.

[0101] (3) Furthermore, the raw material (nonwoven fabric) used in the manufacture of the pad of the immunochromatography test strip, which is impregnated with a solid acidic reagent or a nitrite or a neutralizing reagent or the like, is selected from a raw material having a relatively high hydrophobicity, and the flow rate of the liquid, i.e., the time for the sample solution to spread on the immunochromatography test strip, can be adjusted to be relatively slow.

[0102] Examples of the raw material having a relatively high hydrophobicity include polyethylene, polyester, polystyrene, polypropylene, rayon, nylon, and the like.

[0103] Examples of the raw material of the porous material used in the region impregnated with the neutralizing reagent include a fabric having a weight of 10 to 400 g / m2and a thickness of 0.1 to 2.0 mm, and having a water absorption of 10 to 100 μl / cm2and a water absorption rate of 1.0 to 5.0 μl / sec per 1 cm / m2, and further having a water retention of 10 to 100 μl / cm2after a piece of 1 cm / m2is contacted with a film in a wet state and left for 5 minutes, and preferably having a spreading area of 20 mm2or more of the liquid after a piece of 1 cm / m2is contacted with a film in a wet state and left for 5 minutes. 2 2 2 2 2 2 2 In other words, the porous material has a relatively high water absorption, a relatively high water retention (liquid retention), and a relatively low liquid release, and the liquid release is sustained. Specifically, examples of the porous material include filter paper made of cotton fibers of cellulose or glass fiber filter paper made of glass fibers, and the like. As such filter paper, for example, No. 26-3 of Toyo Filter Paper Co., Ltd. can be used. Furthermore, the filter paper has a large capacity, and can sufficiently retain the acidic solution that reaches the test strip from the upstream side later than the sample. By using such a porous material, the neutralizing reagent that can sufficiently neutralize the sample containing the sugar chain antigen to which nitrous acid produced by the reaction of the nitrite and the acidic reagent is extracted can be impregnated. Furthermore, since the pad can absorb and retain a large amount of liquid, and the release is sustained, the liquid containing the nitrous acid remaining in the upstream side of the immunochromatography test strip can have sufficient neutralizing ability even when the liquid is spread after the determination time, and the acidic solution can be prevented from reaching the detection region in which the antibody is immobilized, and as a result, the non-specific reaction can be suppressed, and the sugar chain antigen can be detected without the non-specific reaction. On the other hand, the glass fiber filter paper having a relatively high water absorption, a relatively high water retention, and a relatively low liquid release, specifically, for example, GS-25 of Toyo Filter Paper Co., Ltd. has a relatively high water absorption, and can impregnate a larger amount of the neutralizing reagent, and has a relatively high water retention and a relatively low water release, and thus the acidic solution can be prevented from reaching the detection region in which the immobilization is performed. As a result, when the result is negative, the non-specific reaction can be suppressed, and when the result is positive, the development of the band after the determination time can be prevented.​​​​​​

[0104] The "gram weight" of the raw material for the porous material used in the impregnation and neutralization reagent area is 10–400 g / m³. 2 Among them, "gram weight" refers to the average weight per unit area (1m²) of fabric, etc. 2 The weight of the pad. The basis weight may vary depending on the amount or composition of the neutralizing agent impregnated in the pad. A basis weight of 30 g / m² is acceptable. 2 The following time-phase has a high porosity and is prone to breakage when impregnated with neutralizing reagents, making it difficult to process during immunochromatography. Therefore, a weight of 50 g / m³ is preferred. 2 The above applies when the weight is 300g / m³. 2 At the above values, the porosity is low. In some neutralizing reagent compositions, the sample cannot easily permeate the raw materials and cannot mix with the neutralizing reagent. Therefore, 300 g / m³ is preferred. 2 The optimal weight is 250–270 g / m³. 2 .

[0105] Furthermore, the thickness of the raw material of the porous material used in the region impregnated with the neutralizing reagent is preferably 0.1 to 2.0 mm, and the thickness can be appropriately changed according to the amount or composition of the neutralizing reagent impregnated in the pad. When the thickness is less than 0.4 mm, it is easy to break when impregnated with neutralizing reagent, and it is difficult to handle during the manufacture of immunochromatography. Therefore, the thickness is preferably 0.4 to 0.8 mm. Considering that the amount of impregnated neutralizing reagent should be easy to adjust and the ease of handling during the manufacture of immunochromatography, a thickness of 0.6 mm is more preferred.

[0106] The preferred water absorption is an average of 1 cm / m 2 The water absorption capacity is 10–100 μl / cm 2 The water absorption rate was 1.0–5.0 μl / sec. The methods for determining water absorption and rate were as follows: 200 μl of dyed solution (1% Tween 20 + Red 102) was prepared in one well of a 96-well EIA plate. A test piece with a 5×60 mm raw material attached to the backing sheet was placed in the solution, with the side immersed in the solution as the lower end of the test piece. The time required for the solution to reach the upper end of the test piece was measured. Immediately after reaching the upper end, the test piece was removed, and the amount of liquid remaining in the well was measured. The water absorption rate was calculated as 200 μl minus the amount of liquid remaining in the well, representing the average absorption rate per 1 cm of raw material. 2 The liquid volume allocated to the area is used to calculate the water absorption rate by dividing the water absorption rate by the time required to reach the top. For the porous material used in the region containing the neutralizing reagent, a material with high water absorption and a slow water absorption rate is preferred. Based on the concentration and water content of the solid acidic reagent, when the water absorption rate is 30 μl / cm²... 2In some cases, the neutralizing reagent required for the immunochromatographic test strip cannot be completely impregnated. Therefore, it is preferable that the amount of water absorption be 30 μl / cm 2 The water absorption rate of the raw material of the porous material used in the neutralizing reagent-impregnated region affects the extraction time of the sugar chain antigen in the sample and the neutralization time of the test solution after extraction. The extraction time of the sugar chain antigen can be adjusted to some extent by adjusting the raw material or composition of the solid acid reagent, but it is not completely possible. Therefore, the water absorption rate of the raw material used in the neutralizing reagent-impregnated region is an important factor for the sufficient extraction and neutralization of the sugar chain antigen. Specifically, the water absorption rate is preferably 1.0 to 5.0 μl / sec, and more preferably the water absorption rate is 2.0 μl / sec or less.

[0107] The "water retention" is obtained by the following method: a 1 cm / m 2 piece of the raw material of the porous material used in the neutralizing reagent-impregnated region is placed on a film, 70 μl of a solution (1% Tween 20 + red No. 102) is added thereto, and the weight before and after 5 minutes of standing is measured. The amount of the solution based on the composition of the added solution (the amount of the surfactant or the protein) varies, and the water retention amount is preferably 10 to 100 μl / cm 2 , more preferably 15 μl / cm 2 or more when a 1% Tween 20 solution is used for the test.

[0108] The "release" is measured by the following method: a 1 cm / m 2 piece of the raw material of the porous material used in the neutralizing reagent-impregnated region is placed on a film, 70 μl of a solution (1% Tween 20 + red No. 102) is added thereto, and the area of the solution spread on the film after 5 minutes of standing is measured. The area based on the composition of the added solution (the amount of the surfactant or the protein) varies, and the area is 30 mm 2 or more when a 1% Tween 20 solution is used for the test, and more preferably the release is 20 mm 2 or more.

[0109] That is, the present application includes the following inventions.

[0110] [1] An immunochromatography test sheet for extracting and measuring a sugar chain antigen in a sample, comprising: a sample pad to which a sample of a test body mixed with nitrite or an acidic solution is added; a labeled body area containing a labeled antibody obtained by labeling an antibody against the sugar chain antigen; and a detection area in which an antibody-sugar chain antigen-labeled antibody complex is formed and the sugar chain antigen is measured, the immunochromatography test sheet having, upstream of the labeled body area, a neutralizing agent-impregnated area, and further, upstream of the neutralizing agent-impregnated area, in the case of using a test body mixed with nitrite, a solid acidic reagent-impregnated area, and in the case of using a test body mixed with an acidic solution, a nitrite-impregnated area, wherein

[0111] The raw material of the neutralizing agent-impregnated area is filter paper or glass fiber filter paper having three characteristics of higher absorbency, higher water retention, and lower or sustained release,

[0112] The higher absorbency and higher water retention of the neutralizing agent-impregnated area allow an acidic solution containing the sugar chain antigen to be sufficiently neutralized, and the lower release or sustained release of the neutralizing agent-impregnated area inhibits the remaining acidic solution from reaching the detection area or inhibits a non-specific reaction by the test solution being continuously developed in the detection area after being sufficiently neutralized.

[0113] [2] An immunochromatography test sheet for extracting and measuring a sugar chain antigen in a sample, comprising: a sample pad to which a sample of a test body mixed with nitrite or an acidic solution is added; a labeled body area containing a labeled antibody obtained by labeling an antibody against the sugar chain antigen; and a detection area in which an antibody-sugar chain antigen-labeled antibody complex is formed and the sugar chain antigen is measured, the immunochromatography test sheet having, upstream of the labeled body area, a neutralizing agent-impregnated area, and further, upstream of the neutralizing agent-impregnated area, in the case of using a test body mixed with nitrite, a solid acidic reagent-impregnated area, and in the case of using a test body mixed with an acidic solution, a nitrite-impregnated area, wherein

[0114] The raw material of the neutralizing agent-impregnated area is a fabric having a grammage of 10 to 400 g / m 2 and a thickness of 0.1 to 2.0 mm.

[0115] [3] The immunochromatography test sheet according to [1] or [2], wherein the average water absorption per 1 cm / m 2 of the fabric is 10 to 100 μl / cm 2and the water absorption speed is 1.0 to 5.0 μl / sec, and the water retention amount when a 1 cm / m 2 of the fragment is contacted with the detection region in a wet state and left for 5 minutes is 10 to 100 μl / cm 2 of the fragment is contacted with the membrane in a wet state and left for 5 minutes is 20 mm 2 of the fragment is contacted with the membrane in a wet state and left for 5 minutes is 20 mm 2 of the fragment is contacted with the membrane in a wet state and left for 5 minutes is 20 mm

[0116] [4] The immunochromatography test piece according to any one of [1] to [3], wherein the higher water absorption and the higher water retention of the region impregnated with the neutralizing reagent sufficiently neutralize the acidic solution containing the above sugar chain antigen, and the sustained force of the release of the region impregnated with the neutralizing reagent allows the remaining acidic solution to reach the detection region while still having sufficient neutralizing ability, thereby suppressing a non-specific reaction.

[0117] [5] The immunochromatography test piece according to any one of [1] to [4], wherein the higher water absorption and the higher water retention of the region impregnated with the neutralizing reagent sufficiently neutralize the acidic solution containing the above sugar chain antigen, and the lower release of the region impregnated with the neutralizing reagent suppresses the remaining acidic solution from reaching the detection region, thereby suppressing a non-specific reaction when negative and preventing a band from developing after the determination time when positive.

[0118] [6] The immunochromatography test piece according to any one of [1] to [5], wherein the region impregnated with the solid acidic reagent or the nitrite is present on the sample pad or the pad coated with the labeled body region.

[0119] [7] The immunochromatography test piece according to any one of [1] to [6], wherein the solid acidic reagent is selected from the group consisting of malonic acid, malic acid, maleic acid, citric acid, and tartaric acid.

[0120] [8] The immunochromatography test piece according to any one of [1] to [7], wherein the neutralizing reagent is tris-hydroxymethyl aminomethane or sodium hydroxide.

[0121] [9] The immunochromatography test piece according to any one of [1] to [8], wherein the sugar chain antigen is a sugar chain antigen of protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, or virus.

[0122]

[10] A method for measuring a sugar chain antigen in a test body, using the immunochromatography test piece according to any one of [1] to [9], measuring a sugar chain antigen in a test body by an immunochromatography method, the method comprising:

[0123] When the immunochromatography test piece has a region impregnated with a solid acidic reagent, the test body is mixed with a nitrous acid solution, and when the immunochromatography test piece has a region impregnated with a nitrite salt, the test body is mixed with an acidic solution, and added to the sample pad of the above-described immunochromatography test piece,

[0124] In the region impregnated with a solid acidic reagent or the region impregnated with a nitrite salt, the sugar chain antigen is extracted from the test body by the action of nitrous acid generated by the reaction of the nitrite salt with the solid acidic reagent, and in the region impregnated with a neutralizing reagent, the acidic solution containing the sugar chain antigen is neutralized,

[0125] In the detection region, the antibody-sugar chain antigen-labeled antibody complex is formed, and by the higher water absorbency and higher water retention of the region impregnated with a neutralizing reagent, the acidic solution containing the sugar chain antigen is sufficiently neutralized by the above-described immunochromatography, the remaining acidic solution is inhibited from reaching the detection region by the lower release or sustained release of the region impregnated with a neutralizing reagent, or the non-specific reaction is inhibited by continuously adding the test solution after sufficient neutralization to the detection region, and the sugar chain antigen in the test body is measured.

[0126] As a result, the extraction of the sugar chain antigen on the immunochromatography test piece can be facilitated.

[0127] Further, by adding a surfactant such as Triton X-100, Triton X-114, polyoxyethylene octylphenyl ether, Tween 20, Tween 80, polyoxyethylene sorbitan fatty acid ester, Brij-35, Brij-58, polyoxyethylene alkyl ether, sodium cholate, deoxycholic acid, an anionic surfactant such as sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate to the reagent impregnated in the pad having a higher hydrophobicity, the hydrophobicity of the reagent can be controlled, and the time for the test body to be added can be designed to be any time. Preferably, by adding the surfactant, the test body can be treated for a sufficient time, and the test sample solution can be suitably spread. That is, the test body can be sufficiently treated with the solid acidic reagent or the nitrite salt to extract the sugar chain antigen, and can be spread in the downstream in the presence of the pad impregnated with the neutralizing reagent.

[0128] Further, the control can be performed without the pad having a higher hydrophobicity and the surfactant, and as a raw material used for the manufacture of the pad for the test body treatment reagent such as the solid acidic reagent or the nitrite salt or the neutralizing reagent of the immunochromatography test piece, a raw material having a higher hydrophilicity is selected, and the flow rate of the liquid, that is, the time for the test sample solution to be spread on the immunochromatography test piece is adjusted to be longer. As a raw material having a higher hydrophilicity, for example, filter paper having a thickness of 210 to 580 μm can be exemplified.

[0129] Further, in order to easily keep the sample solution on the pad, a surfactant can not be used, and other high-molecular compounds having immobilization action (e.g., PVA (polyvinyl alcohol) or PLL (poly-L-lysine) can be used. This method also enables the extraction of sugar chain antigens on the immunochromatography test sheet to be facilitated.

[0130] In the immunochromatography device having at least one configuration of any one of the above (1), (2), and (3), the time for the added sample solution of the test body to spread on the immunochromatography test sheet can be made longer, and the spreading to the pad (neutralizing reagent region) in which the neutralizing reagent is impregnated downstream can be delayed.

[0131] Further, the immunochromatography device having at least one configuration of any one of the above (1), (2), and (3) can be utilized not only as a device for performing nitrite extraction processing of sugar chain antigens on the immunochromatography test sheet, but also as an immunochromatography device for processing the test body on the test sheet by the reagent impregnated on the test sheet.

[0132] The present application can be used not only for nitrite extraction, but also for cases where the analyte to be detected as the object of detection is incubated on the test device of the immunochromatography reagent by pH or a compound.

[0133] The present application is based on the immunochromatography test sheet having Figure 1 The present application is based on the immunochromatography test sheet having Figure 4 The use method of the device of the present application is described. The following use method is a method for mixing the test body with a nitrite solution, and performing measurement using the immunochromatography method in which a solid acid reagent and a neutralizing reagent are impregnated, and a method for mixing the test body with an acid solution, and performing measurement using the immunochromatography test method in which a nitrite salt and a neutralizing reagent are impregnated can be performed by referring to the following description of the use method.

[0134] Measurement is started by mixing the test body or a sample prepared using the test body with a nitrite salt solution, suspending the test body in the nitrite salt solution, and supplying it to the test body addition port of the device. At this time, 5 to 100 μL of the test body is mixed with 0.01 to 2 mL of a 0.1 M to 8 M nitrite salt, and 5 to 200 μL is supplied to the addition port. As the nitrite salt, sodium nitrite, potassium nitrite, and the like can be exemplified.

[0135] The sample containing the detected substance, i.e., the sugar chain antigen, supplied to the addition port 9 moves to the sample pad, spreads to the solid acid reagent region 5 and the neutralizing reagent region 6 on the sample pad 4 by capillary action, and further spreads to the labeled body region 2, the support 1, and the absorption band 7 in the horizontal direction. In the solid acid reagent region 5, the nitrite mixed with the sample reacts with the solid acid reagent on the solid acid reagent region 5 to produce free nitrous acid, and the sugar chain antigen is extracted from the sample by the action of the nitrous acid. The extracted sugar chain antigen moves with the acid spreading solution to the neutralizing reagent region 6 and spreads in the neutralizing reagent region 6, in which the pH of the acid spreading solution containing the sugar chain antigen is adjusted to the neutral region. As a result, the sugar chain antigen further moves and spreads downstream under the neutral condition. In the labeled body region 2, the labeled antibody is released into the liquid and spreads to the support 1 while the sample spreads. When the sugar chain antigen is present in the sample, the sugar chain antigen is specifically captured by the capture antibody in the detection region 3 of the support 1, and the sugar chain antigen further forms a complex with the labeled antibody by specific reaction. Thus, the sandwich of the antibody of the sugar chain antigen is achieved in the detection region 3, and the labeled antibody-sugar chain antigen complex can be measured in the detection region 3. The detection region can be observed by the judging section of the immunochromatography device.

[0136] According to the method using the immunochromatography test piece of the present application, since the extraction of the sugar chain antigen in the sample is performed on the immunochromatography test piece, it is not necessary to extract the sugar chain antigen in the sample in advance before measurement using the immunochromatography test piece, and the sugar chain antigen in the sample can be measured in one step.

[0137] The immunochromatography test piece of the present application, which incorporates the pad containing the solid acid reagent or the nitrite salt after drying, can efficiently perform extraction on the reagent.

[0138] The area of the sample addition port can be substantially equal to the area of the pad containing the solid acid reagent (solid acid reagent region). In this case, more sample can be instantaneously brought into contact with the solid acid reagent. Further, by using a nonwoven fabric having high hydrophobicity for the member subjected to coating and impregnation, the sample does not immediately spread to the downstream pad impregnated with the neutralizing reagent (neutralizing reagent region) after the sample is added. In this case, the sample is held in the sample addition portion for 1 to 2 minutes, and the antigen extraction can be sufficiently performed.

[0139] Further, in the present application, when the solid acid reagent region or the nitrite salt region is in contact with the neutralizing reagent region, a sheet through which liquid cannot pass is provided between the solid acid reagent region or the nitrite salt region and the neutralizing reagent region. By providing the sheet, the following three effects can be achieved.

[0140] (A) It is possible to inhibit the movement of the reagents in the two regions adjacent to each other during the storage of the test piece. As a result, it is possible to prevent the reaction of the solid-state acidic reagent or nitrite with the neutralizing reagent. As a result, it is possible to improve the stability of the reagents.

[0141] (B) Furthermore, when the specimen mixed with the nitrite or the acidic reagent is added, it is possible to prevent the specimen from moving to the neutralizing reagent region immediately after reaching the solid-state acidic reagent region or the nitrite region, so that the extraction of the sugar chain antigen is not sufficient. That is, the speed of the liquid containing the specimen moving from the solid-state acidic reagent region or the nitrite region to the neutralizing reagent region is reduced, and the residence time of the liquid containing the specimen in the solid-state acidic reagent region or the nitrite region is extended, as a result, the sugar chain antigen can be sufficiently extracted, and the measurement sensitivity is improved.

[0142] (C) Furthermore, when the specimen mixed with the nitrite or the acidic reagent is added, it is possible to prevent the specimen from moving to the neutralizing reagent region immediately after reaching the solid-state acidic reagent region or the nitrite region, and the liquid containing the specimen after the movement is backflowed to the solid-state acidic reagent region or the nitrite region, so that the activity of the solid-state acidic reagent or the nitrite is reduced, and the extraction efficiency is reduced. That is, the backflow of the liquid containing the specimen to the solid-state acidic reagent region or the nitrite region after reaching the neutralizing reagent region is temporarily prevented, the reduction in the activity of the solid-state acidic reagent or the nitrite is prevented, the extraction process of the nitrite to the sugar chain antigen is favorably performed, and the measurement sensitivity is improved.

[0143] The raw material of the sheet provided between the solid-state acidic reagent region or the nitrite region and the neutralizing reagent region is not limited as long as it is a raw material that does not allow the passage of liquid, and for example, a sheet made of resin such as a PET (polyethylene terephthalate) sheet, a polyethylene sheet, or the like can be used. The sheet made of resin is also referred to as a film made of resin.

[0144] The sheet can also be provided so that the solid-state acidic reagent region or the nitrite region and the neutralizing reagent region are partially in contact and overlap. At this time, the length of the overlapping portion of the solid-state acidic reagent region or the nitrite region and the neutralizing reagent region is preferably as short as possible, for example, 5 mm or less, preferably 3 mm or less, and further preferably 2 mm or less.

[0145] Furthermore, the sheet can also be provided so that the solid-state acidic reagent region or the nitrite region and the neutralizing reagent region are not in contact, and is in a shape in which a pad impregnated with an acidic reagent or a nitrite is covered on a PET sheet. At this time, the neutralizing region is completely covered with the sheet, and it is sufficient to provide the solid-state acidic reagent region or the nitrite region on the sheet. At this time, the liquid in the solid-state acidic reagent region or the nitrite region does not directly move to the neutralizing region, but flows on the sheet and then reaches the neutralizing reagent region.

[0146] The sheet can be provided only between the solid acid reagent region or the nitrite region and the neutralizing reagent region, for example. On the other hand, when the sheet made of resin is attached as a top laminated sheet in a manner to cover the upper side of the immunochromatography test sheet, the top laminated sheet can be attached in a manner to cover the neutralizing reagent region, the marker region, the support, the detection region, and the absorbent band except for the sample pad when the solid acid reagent region or the nitrite region serves as the sample pad, provided that the solid acid reagent region or the nitrite region is attached to the upper portion of the top laminated sheet attached to the upper portion of the neutralizing reagent portion. In Figure 2 and Figure 3 The structure of such an immunochromatography test sheet is shown in

[0147] That is, the present application includes the following inventions.

[0148] [1] An immunochromatography test sheet comprising: a sample pad to which a specimen mixed with nitrite or an acidic solution is added; a marker region including a marker antibody in which an antibody to a sugar chain antigen is labeled; and a detection region in which the antibody to the sugar chain antigen is solidified, an antibody-sugar chain antigen-marker antibody complex is formed in the detection region, and the sugar chain antigen is measured, the immunochromatography test sheet having, upstream of the marker region, a region in which a neutralizing reagent is impregnated, and further, upstream of the region in which the neutralizing reagent is impregnated, a region in which a solid acid reagent is impregnated in the case of using a specimen mixed with nitrite, or a region in which nitrite is impregnated in the case of using a specimen mixed with an acidic solution, the immunochromatography test sheet being used for extracting and measuring the sugar chain antigen in the specimen, wherein

[0149] A sheet made of resin is interposed between the region in which the solid acid reagent or the nitrite is impregnated and the region in which the neutralizing reagent is impregnated to inhibit movement of the reagents or movement of the specimen solution between the regions.

[0150] [2] The immunochromatography test sheet according to [1], wherein the region in which the solid acid reagent or the nitrite is impregnated is present on the sample pad.

[0151] [3] The immunochromatography test sheet according to [1] or [2], wherein the sheet made of resin is interposed in a manner to partially contact the region in which the solid acid reagent or the nitrite is impregnated and the region in which the neutralizing reagent is impregnated.

[0152] [4] The immunochromatography test sheet according to [1] or [2], wherein the sheet made of resin is interposed in a manner to not contact the region in which the solid acid reagent or the nitrite is impregnated and the region in which the neutralizing reagent is impregnated.

[0153] [5] The immunochromatography test strip according to any one of [2] to [4], wherein a region impregnated with a solid acid reagent or a nitrite salt is present on the most upstream sample pad on the immunochromatography test strip, a region other than the sample pad is covered with a sheet made of a resin, and a sample pad impregnated with a solid acid reagent or a nitrite salt is present on the upper portion of the sheet made of a resin on the upper portion of the region impregnated with a neutralizing reagent.

[0154] [6] The immunochromatography test strip according to any one of [1] to [5], wherein the solid acid reagent is selected from the group consisting of malonic acid, malic acid, maleic acid, citric acid, and tartaric acid.

[0155] [7] The immunochromatography test strip according to any one of [1] to [6], wherein the neutralizing reagent is tris-hydroxymethyl aminomethane or sodium hydroxide.

[0156] [8] The immunochromatography test strip according to any one of [1] to [7], wherein the sugar chain antigen is a sugar chain antigen of a protozoan, a fungus, a bacterium, a mycoplasma, a rickettsia, a chlamydia, or a virus.

[0157] [9] A method for measuring a sugar chain antigen in a test body, using the immunochromatography test strip according to any one of [1] to [8], measuring a sugar chain antigen in a test body by an immunochromatographic method, the method for measuring a sugar chain antigen in a test body comprising:

[0158] when the immunochromatography device has a region impregnated with a solid acid reagent, mixing the test body with a nitrous acid solution, when the immunochromatography device has a region impregnated with a nitrite salt, mixing the test body with an acid solution, and adding to a sample pad of the immunochromatography device,

[0159] in the region impregnated with a solid acid reagent or the region impregnated with a nitrite salt, extracting a sugar chain antigen from the test body by the action of nitrous acid produced by the reaction of the nitrite salt with the solid acid reagent, and in the region impregnated with a neutralizing reagent, neutralizing an acid solution containing the sugar chain antigen,

[0160] in the detection region, forming an antibody-sugar chain antigen-labeled antibody complex, controlling the development speed or direction of the test body on the immunochromatography test strip by the above-described immunochromatographic method, controlling the treatment using the acid reagent, the nitrite salt, and the neutralizing reagent, and measuring the sugar chain antigen in the test body.

[0161] In the method of the present application, a biological sample as a test body is not particularly limited, and examples include a body fluid such as serum, plasma, blood, urine, feces, saliva, tissue fluid, spinal fluid, swab fluid, or the like, or a dilution thereof.

[0162] In the method using the immunochromatographic device of the present application, the detected substance as the measurement object is a sugar chain antigen that can be measured by analysis using an immunoassay, i.e., an antigen-antibody reaction. As the antigen, a sugar chain antigen, i.e., a polysaccharide, etc., present in the cell wall of bacteria, which is extracted by nitrous acid extraction treatment, can be exemplified. Protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, viruses, etc., containing these substances can also be measured. By the method using the immunochromatographic test piece of the present application, it is possible to confirm whether or not a biological sample of a test subject contains a sugar chain antigen derived from protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, viruses, etc., and when the sugar chain antigen is contained, it is possible to judge that the test subject has an infectious disease caused by protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, viruses, etc. For example, it is possible to detect the presence or absence of infection with Group A β-hemolytic streptococcus (Streptococcus pyogenes), Escherichia coli, Legionella, Campylobacter, etc.

[0163] The present application will be specifically described by the following examples, but the present application is not limited to these examples.

[0164] In the following examples, % means w / v % unless otherwise specified.

[0165] Example 1: Study of the size of the addition port

[0166] 1. Immobilization of anti-Streptococcus pyogenes (Group A β-hemolytic streptococcus) antibody on a nitrocellulose membrane (support)

[0167] A liquid of anti-Streptococcus pyogenes antibody diluted to 1.0 mg / mL with purified water and an anti-rabbit IgG antibody were prepared, and the anti-Streptococcus pyogenes antibody was applied linearly on the sample pad side of the nitrocellulose membrane with a PET (polyethylene terephthalate) film as a back liner, and the anti-rabbit IgG antibody was applied linearly on the absorbent tape side. Thereafter, the nitrocellulose membrane was dried at 45°C for 30 minutes to obtain an anti-Streptococcus pyogenes antibody immobilized membrane. In this example, this membrane was referred to as an "antibody immobilized membrane".

[0168] 2. Immobilization of anti-Streptococcus pyogenes antibody on colored polystyrene particles

[0169] The anti-Streptococcus pyogenes antibody diluted with pure water to 1.0 mg / mL was added with colored polystyrene particles to 0.1%, and after stirring, carbodiimide was added to 1%, and further stirred. The supernatant was removed by centrifugation, and resuspended in 50 mM Tris (pH 9.0) and 3% BSA to obtain a 0.04% suspension of colored polystyrene particles to which the anti-Streptococcus pyogenes antibody was bound. In this example, the particles are referred to as "antibody-immobilized particles".

[0170] 3. Coating and drying of colored polystyrene particles to which the anti-Streptococcus pyogenes antibody was bound

[0171] The antibody-immobilized particle suspension prepared in 2 was coated on a nonwoven fabric in a prescribed amount, and dried at 45°C for 30 minutes. In this example, the resulting nonwoven fabric is referred to as a "drying pad".

[0172] 4. Preparation of a neutralizing reagent (alkaline reagent) pad

[0173] As the neutralizing reagent (alkaline reagent), 3 M Trizma (trade name) base (Tris base) and 1.5% Triton X100 were coated on filter paper (Toyo Filter Paper Co., Ltd., No. 26-3) at 30 μL / cm.

[0174] 5. Preparation of a solid acidic reagent pad

[0175] As the solid acidic reagent, 1.0 M tartaric acid and 0.5% Triton X100 were coated on a nonwoven fabric (Unitika Ltd., Elves) at 13 μL / cm. Immediately after coating, the nonwoven fabric was dried at 45°C for 1 hour to obtain a nonwoven fabric impregnated with the solid acidic reagent.

[0176] 6. Preparation of an immunochromatographic test sheet for detection of Streptococcus pyogenes

[0177] The antibody-immobilized membrane produced in 1, the dry pad produced in 3, the neutralizing reagent (alkaline reagent) pad produced in 4, and the solid-state acidic reagent pad produced in 5 were attached to other components (backing sheet, absorbent tape) and cut into a width of 5 mm to produce Streptococcus pyogenes test pieces. In this example, the test pieces in which filter papers impregnated with solid-state acidic reagent and neutralizing reagent (alkaline reagent) were used as sample pads are referred to as "immunochromatographic test pieces of the present application". Note that in the immunochromatographic test pieces, there were, in order from the upstream of the flow of the test body, a nonwoven fabric impregnated with solid-state acidic reagent, a nonwoven fabric impregnated with neutralizing reagent (alkaline reagent), a dry pad (marker region), an antibody-immobilized membrane (detection region), and an absorbent tape.

[0178] 7. An immunochromatographic device

[0179] The immunochromatographic test pieces of the present application were respectively loaded into an immunochromatographic device (conventional device) having an addition port length of 5 mm and an immunochromatographic device (device of the present application in which the addition port was enlarged) having an addition port length of 10 mm, and the following test was performed.

[0180] 8. Test body

[0181] Streptococcus pyogenes was cultured, and the culture solution was adjusted to a bacterial count of 1.0 x 10 7 CFU / mL using physiological saline.

[0182] In addition, as a negative test body, physiological saline was used.

[0183] 9. Measurement

[0184] Twenty μL of the test body was suspended in 180 μL of sodium nitrite solution (2.0 M NaNO3, 1% Tween 20), and 75 μL of the suspension was added to the addition port of the immunochromatographic device of the present application. In addition, as a conventional method, the test body was suspended in a nitrite extract solution obtained by mixing sodium nitrite and hydrochloric acid, and 50 μL of the test body suspension neutralized with a tris-hydroxymethyl aminomethane solution was added to the addition port of the immunochromatographic device (neither acidic reagent nor neutralizing reagent was immobilized) of the conventional method. After 5 minutes, the presence or absence and the degree of deposition of colored polystyrene particles on the prescribed position where the anti-Streptococcus pyogenes antibody was immobilized were visually determined. The degree of linear deposition was sequentially recorded as +++, ++, + from strong to weak, recorded as ± when it was difficult to determine, and recorded as - when deposition was not confirmed.

[0185] 10. Results

[0186] [Table 1]

[0187]

[0188] As shown in Table 1, the determination sensitivity of the immunochromatographic device to which the longer 10 mm length of the addition port was added was higher than that of the immunochromatographic device to which the 5 mm length of the addition port was added. It is considered that this is due to the increase in the extraction efficiency of the nitrous acid-treated antigen.

[0189] Example 2: Study of the coating amount of the surfactant

[0190] The test results (Table 2) were compared under the conditions that the concentration of the surfactant Triton X100 of the solid acid reagent in the production of the nonwoven fabric impregnated with the solid acid reagent of Example 1-5 was 0.5% and 2.0%.

[0191] [Table 2]

[0192] R2 additive 1 min 2 min 3 min 4 min 5 min 0.5% Tx 100 - - + ++ ++ 2.0% Tx 100 - - ± + +

[0193] As shown in Table 2, the determination sensitivity was higher when the concentration of the surfactant was lower. It is considered that this is due to the increase in the extraction efficiency of the nitrous acid-treated antigen.

[0194] 1... support (including a detection region); 2... marker region; 3... detection region; 4... sample pad; 5... solid acid reagent region; 6... neutralization reagent region; 7... absorption band; 8... backer sheet; 9... addition port of the device; 10... determination portion of the device.

[0195] The immunochromatographic device of the present application can be used to detect the infection of group A β hemolytic streptococcus with high sensitivity.

[0196] All publications, patents, and patent applications cited in this specification are directly incorporated by reference into this specification.

Claims

1. An immunochromatographic apparatus, characterized in that, The immunochromatographic test strip includes an immunochromatographic test strip and a container for storing the test strip, and has a sample addition port on the sample pad of the test strip. The immunochromatographic test strip is used to extract and measure glycan antigens in a sample. The immunochromatographic test strip includes: Sample pads are used to add samples prepared by mixing with nitrite or acidic solutions. The marker region contains a labeled antibody obtained by labeling the glycan antigen with an antibody; and In the detection area, the antibody against the glycan antigen is immobilized, forming an antibody-glycan antigen-labeled antibody complex, and the glycan antigen is then measured. The immunochromatographic test strip has a region impregnated with a neutralizing reagent upstream of the labeled region, a region impregnated with a solid acidic reagent upstream of this region when using a sample prepared with nitrite, and a region impregnated with nitrite when using a sample prepared with an acidic solution. (i) The immunochromatographic apparatus has a large sample addition port for holding the added sample solution and for promoting the extraction of glycan antigens by nitrite and solid acid reagent by supplying the sample solution to the area impregnated with the solid acid reagent or nitrite for a short time; (ii) There is no gap between the filling port and the sample pad, so that the sample does not overflow from the filling port. The immunochromatographic device has a sample addition port that is sized to supply 10 μL to 200 μL of sample solution at a time.

2. The immunochromatographic apparatus according to claim 1, characterized in that, The pad on the immunochromatographic test strip, which is impregnated with a solid acidic reagent, nitrite, or neutralizing reagent, is made of a highly hydrophobic raw material. This delays the development time of the sample solution, thereby promoting the extraction of glycan antigens by nitrite and solid acidic reagent.

3. The immunochromatographic apparatus according to claim 2, characterized in that, The highly hydrophobic raw materials are selected from the group consisting of polyethylene, polyester, polystyrene, polypropylene, rayon, and nylon.

4. The immunochromatographic apparatus according to claim 1, characterized in that, The pad on the immunochromatographic test strip, which is impregnated with a solid acidic reagent, nitrite, or neutralizing reagent, is made of a highly hydrophilic raw material. This delays the development time of the sample solution, thereby promoting the extraction of glycan antigens by nitrite and solid acidic reagent.

5. The immunochromatographic apparatus according to claim 4, characterized in that, The raw material with high hydrophilicity is filter paper with thickness.

6. The immunochromatographic apparatus according to claim 1, characterized in that, The extraction of glycan antigens by nitrite and solid acid reagents is promoted by adjusting the development time of the sample solution by adding surfactants to the pads impregnated with solid acid reagents, nitrites, or neutralizing agents on the immunochromatographic test strips.

7. The immunochromatographic apparatus according to claim 6, characterized in that, The surfactant is selected from the group consisting of polyoxyethylene octylphenyl ether, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl ether, sodium cholate, deoxycholic acid, sodium dodecyl sulfate, and sodium dodecylbenzene sulfonate.

8. The immunochromatographic apparatus according to claim 1, characterized in that, By adding a polymeric compound with immobilization properties to the pad impregnated with solid acidic reagents, nitrites, or neutralizing agents on the immunochromatographic test strip, the development time of the sample solution can be adjusted to promote the extraction of glycan antigens by nitrites and solid acidic reagents.

9. The immunochromatographic apparatus according to claim 8, characterized in that, The polymeric compound with immobilization function is polyvinyl alcohol or poly-L-lysine.

10. The immunochromatographic apparatus according to claim 1, characterized in that, Areas impregnated with solid acidic reagents or nitrites may be present on the sample pad or on the pad with a labeled area.

11. The immunochromatographic apparatus according to claim 1, characterized in that, The solid acidic reagent is selected from the group consisting of malonic acid, malic acid, maleic acid, citric acid and tartaric acid.

12. The immunochromatographic apparatus according to claim 1, characterized in that, The neutralizing agent is tris(hydroxymethyl)aminomethane or sodium hydroxide.

13. The immunochromatographic apparatus according to claim 1, characterized in that, The glycan antigen is a glycan antigen of protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, or virus.

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