Immunochromatographic device for extracting and determining sugar chain antigens

Abstract

The present invention provides a method and an immunochromatographic device for extracting and measuring sugar chain antigens by nitrous acid extraction on an immunochromatographic test piece, which can perform measurements with sufficient sensitivity by performing nitrous acid extraction treatment for a sufficient time. An immunochromatographic device includes an immunochromatographic test piece for extracting and measuring sugar chain antigens in a specimen and a container for storing the test piece, the immunochromatographic test piece including: a sample pad for adding a specimen mixed with nitrite or an acidic solution; a marker region including a labeled antibody, the labeled antibody being labeled with an antibody against the sugar chain antigen; and a detection region having an antibody against the sugar chain antigen solid-phased thereon, the immunochromatographic test piece forming an antibody-sugar chain antigen-labeled antibody complex in the detection region to measure the sugar chain antigen, an area impregnated with a neutralizing agent is provided upstream of the marker region, and in the area impregnated with the neutralizing agent, Upstream, when a specimen mixed with nitrite is used, there is an area impregnated with a solid acidic reagent, and when a specimen mixed with an acidic solution is used, there is an area impregnated with nitrite. The upper part of the sample pad of the test piece has a sample dropping port. In the immunochromatographic device, (i) there is a wide sample dropping port, and the sample dropping port is used to promote the extraction of sugar chain antigens using nitrite and solid acidic reagent by maintaining the added specimen sample solution and supplying the specimen sample solution to the area impregnated with the solid acidic reagent or nitrite in a short time; (ii) there is no gap between the dropping port and the sample pad so that the sample does not overflow from the dropping port.

Description

Technical Field

[0001] The present invention relates to an immunochromatographic device for extracting and measuring sugar chain antigens, which can perform nitrous acid extraction treatment of sugar chain antigens on an immunochromatographic test piece. Background Art

[0002] Most rapid diagnostic tests based on immunochromatography are able to quickly and easily detect viral or bacterial infections and are therefore widely used as a means of determining treatment strategies.

[0003] A common rapid diagnostic reagent based on the immunochromatography method suspends a specimen in a specimen suspension and then supplies the suspension to an immunochromatographic test strip, thereby enabling rapid and easy measurement.

[0004] In order to detect microorganisms belonging to the genus Streptococcus, such as group A β-hemolytic streptococci (group A β-hemolytic streptococci) and oral streptococci, it is necessary to extract and measure sugar chain antigens.

[0005] For example, a method has been reported in which sodium nitrite and a neutralizing agent are preliminarily contained in an immunochromatographic test strip, a specimen is suspended in an acidic solution such as acetic acid and then supplied to the immunochromatographic test strip, and nitrous acid extraction is performed on the immunochromatographic test strip only by this operation (Patent Document 1).

[0006] There is also a method in which an acidic reagent and a neutralizing reagent are contained in advance in an immunochromatographic test piece, and a specimen is suspended in nitrite and then supplied to the immunochromatographic test piece.

[0007] In immunochromatography, it is sometimes necessary to control the chromatographic development speed on the immunochromatographic test piece, and there are reports of immunochromatography methods such as control by device shape, control by bonding position, control by the raw materials used (materials such as non-woven fabrics), and control by the use of reagents (Patent Documents 2 and 3).

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: International Publication No. WO2005 / 121794

[0011] Patent Document 2: Japanese Patent Application Publication No. 2005-331471

[0012] Patent Document 3: Japanese Patent Application Publication No. 2005-331463 Summary of the invention

[0013] In order to detect microorganisms belonging to the genus Streptococcus, such as group A β-hemolytic streptococci (group A β-hemolytic streptococci) and oral streptococci, sugar chain antigens are extracted and measured.

[0014] Nitrous acid is required to extract sugar chain antigens. Since nitrous acid is unstable when used alone, it is necessary to mix a nitrite solution with an acid solution (acetic acid, citric acid, tartaric acid, etc.) to generate nitrous acid for antigen extraction.

[0015] In the case of detecting group A β-hemolytic streptococci (group A β-hemolytic streptococci) and the like by immunochromatography, either a nitrous acid solution or an acid solution is applied and dried, and then assembled on an immunochromatographic test piece. In such a reagent form, compared with a liquid reagent, the reagent mixing operation is not required and the operation is convenient, but on the other hand, there is a technical problem that after the reagents are mixed, if the sample is developed in a short time as in the conventional immunochromatography, the nitrous acid extraction treatment cannot be fully performed, resulting in insufficient sensitivity.

[0016] In the reagent format of mixing a liquid reagent for nitrous acid extraction, the sample is usually extracted for 1 to 2 minutes. Therefore, in order to show the same sensitivity as the liquid format, the sample must be kept upstream of neutralization on the immunochromatographic test piece for 1 to 2 minutes.

[0017] In contrast, some methods use a pad made of a hydrophobic material containing nitrous acid or acid to fully extract, but if the sample develops too slowly, there is a technical problem that the sample cannot be fully developed on the test piece within a 5-minute reaction time.

[0018] In order to solve these technical problems, some methods use a method such as using a raw material with a certain thickness compared to the usual immunochromatographic test piece in the gasket, but the following technical problems still exist: Due to the thickness and softness of the raw material, a gap is generated on the dropping port for adding the specimen, and the sample that should be extracted for 1 to 2 minutes overflows from the dropping port into the interior of the container and spreads along the side of the test piece, resulting in the reaction being unable to proceed normally (the acidic sample is not neutralized and spreads directly on the membrane, resulting in a non-specific reaction).

[0019] An object of the present invention is to provide a method and an immunochromatographic device for extracting and measuring sugar chain antigens by nitrous acid extraction on an immunochromatographic test piece, wherein the nitrous acid extraction treatment is performed for a sufficient time so that measurement can be performed with sufficient sensitivity.

[0020] The present inventors have conducted intensive studies on a technique for performing a nitrous acid extraction treatment for a sufficient time in an immunochromatography method for extracting and measuring a sugar chain antigen, wherein the nitrous acid extraction treatment of the sugar chain antigen can be performed on an immunochromatographic test piece.

[0021] Therefore, the inventors have found that the above-mentioned object can be achieved by adjusting the development time by inclining the support body portion consisting of the protrusions supporting the immunochromatographic test piece in the immunochromatographic device, thereby completing the present invention. That is, the present invention is as follows.

[0022] [1] An immunochromatographic device comprising an immunochromatographic test piece for extracting and measuring a sugar chain antigen in a specimen and a container for storing the test piece,

[0023] The immunochromatographic test piece includes: a sample pad for adding a sample mixed with nitrite or an acidic solution; a labeling region containing a labeled antibody, wherein the labeled antibody is labeled with an antibody against a sugar chain antigen; and a detection region in which an antibody against the sugar chain antigen is solid-phased, wherein the immunochromatographic test piece forms a complex of an antibody-sugar chain antigen-labeled antibody in the detection region to measure the sugar chain antigen, wherein the immunochromatographic test piece has an area impregnated with a neutralizing reagent upstream of the labeling region, and wherein, upstream of the area impregnated with the neutralizing reagent, there is an area impregnated with a solid acidic reagent when a sample mixed with nitrite is used, and an area impregnated with nitrite when a sample mixed with an acidic solution is used.

[0024] The container includes an upper cover portion and a lower container portion, the upper cover portion has an upper support body, the upper support body includes a protrusion supporting the upper side of the immunochromatographic test piece, and the lower container portion has a lower support body, the lower support body includes a protrusion supporting the lower side of the immunochromatographic test piece,

[0025] The upper part of the sample pad of the test piece has a sample dropping port.

[0026] In the immunochromatographic device, the protrusions forming the upper support and / or the lower support for supporting the immunochromatographic test piece are inclined.

[0027] [2] The immunochromatographic device according to [1], wherein the protrusions forming the upper support and the lower support for supporting the immunochromatographic test piece are inclined.

[0028] [3] The immunochromatographic device according to [1] or [2], characterized in that

[0029] The inclination of the protrusion of the lower support of the immunochromatographic test strip is downward in the downstream direction of the immunochromatographic test strip.

[0030] [4] The immunochromatographic device according to any one of [1] to [3], characterized in that

[0031] The inclination of the protrusion of the upper support of the immunochromatographic test strip is upward in the downstream direction of the immunochromatographic test strip.

[0032] [5] The immunochromatographic device according to any one of [1] to [4], wherein

[0033] The sugar chain antigen is a sugar chain antigen of a protozoa, fungus, bacteria, mycoplasma, rickettsia, chlamydia or virus.

[0034] [6] A method for measuring sugar chain antigens in a sample, using any one of the immunochromatographic devices of [1] to [5] to measure sugar chain antigens in the sample by immunochromatography, the method comprising:

[0035] When the immunochromatographic device has a region impregnated with a solid acidic reagent, the sample is mixed with a nitrous acid solution; when the immunochromatographic device has a region impregnated with a nitrite, the sample is mixed with an acidic solution and added to a sample pad of the immunochromatographic device.

[0036] In the area impregnated with the solid acidic reagent or the area impregnated with the nitrite, the sugar chain antigen is extracted from the specimen by the action of nitrous acid generated by the reaction of the nitrite with the solid acidic reagent, and in the area impregnated with the neutralizing reagent, the acidic solution containing the sugar chain antigen is neutralized.

[0037] In the detection area, a complex of antibody-sugar chain antigen-labeled antibody is formed, which promotes the extraction of sugar chain antigen and determines the sugar chain antigen in the specimen by immunochromatography.

[0038] This specification includes the disclosure of Japanese Patent Application No. 2019-184047 which is the basis of priority of this application.

[0039] When the immunochromatographic device of the present invention is used, the development time of the sample solution on the immunochromatographic test strip after the sample is added can be adjusted to an appropriate time. As a result, the sample processing on the test strip using the sample processing reagent contained in the immunochromatographic test strip can be fully performed. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a diagram schematically showing the structure of an immunochromatographic test piece (single pad test piece) having a solid acidic reagent region and a neutralizing reagent region.

[0041] Figure 2 This is a diagram schematically showing the structure of an immunochromatographic test piece (double pad test piece) having a solid acidic reagent region and a neutralizing reagent region.

[0042] Figure 3 It is a perspective view showing the appearance of a storage container of the immunochromatographic device.

[0043] Figure 4 is a cross-sectional view of a storage container of an immunochromatographic device.

[0044] Figure 5 It is a perspective view showing a cover portion of a storage container of the immunochromatographic device, an immunochromatographic test piece, and a container portion of the storage container.

[0045] Figure 6 It is a cross-sectional view showing a cover portion of a storage container of the immunochromatographic device, an immunochromatographic test piece, and a container portion of the storage container.

[0046] Figure 7 It is a perspective view of the cover portion of the storage container of the immunochromatographic device.

[0047] Figure 8 It is a top view of the cover portion and the container portion of the storage container of the immunochromatographic device. DETAILED DESCRIPTION

[0048] Hereinafter, the present invention will be described in detail.

[0049] The present invention relates to an immunochromatographic device, which is simplified so that nitrous acid extraction of sugar chain antigens can be performed on an immunochromatographic test piece, and the sugar chain antigens as a substance to be detected can be measured quickly and accurately. The immunochromatographic device includes an immunochromatographic test piece and a container for storing the same, and can be made by assembling the immunochromatographic test piece in the immunochromatographic device. Through the storage container, it is possible to prevent, for example, ultraviolet rays and moisture in the air from causing degradation. In addition, when using a contaminating or infectious specimen sample, the storage container can be used to prevent the person conducting the measurement from being contaminated or infected. For example, as long as a resin shell of an appropriate size is used as a storage container, and the immunochromatographic test piece is stored in the shell. In addition, the surface of the test piece immobilized with an antigen or antibody can also be covered with a resin film (top laminate).

[0050] The immunochromatographic test piece comprises: a support body having a detection area on which an antibody (antibody 1) for capturing a substance to be detected (antigen, etc.) is fixed; a labeling area having a movable labeled antibody (antibody 2); a sample pad for adding a specimen; an absorption band for absorbing the specimen liquid after development; and a backing plate for bonding the above components together.

[0051] It should be noted that the number of detection regions and the type of labeled antibody contained in the label region are not limited to one, and two or more antigens can be measured on the same test strip using antibodies corresponding to a plurality of detection substances.

[0052] The support is a material having the performance of immobilizing antibodies for capturing the detected substance (antigen), and has the performance of not hindering the passage of liquid in the horizontal direction. It is preferably a porous film (diaphragm) with capillary action, and its material can transport liquid and components dispersed in the liquid by absorption. The material constituting the support is not particularly limited, and examples thereof include: cellulose, nitrocellulose, cellulose acetate, polyvinylidene fluoride (PVDF), glass fiber, nylon, polyketone, etc. Among them, nitrocellulose is more preferably used to make a film or a diaphragm. The diaphragm immobilized with antibodies is called an antibody immobilized diaphragm.

[0053] The labeling region is composed of a porous substrate containing the labeled antibody, and the material of the substrate can be commonly used glass fiber (Fiberglass), non-woven fabrics, etc. In order to impregnate a large amount of labeled antibodies, the substrate is preferably in a pad shape with a thickness of about 0.3mm to 0.6mm. The porous substrate impregnated with the labeled antibody and dried is also called a dry pad.

[0054] The labeling of the labeled antibody is often carried out using enzymes such as alkaline phosphatase and horseradish peroxidase, metal colloids such as gold colloids, silica particles, cellulose particles, colored polystyrene particles, and colored latex particles. When colored particles such as metal colloid particles, colored polystyrene particles, and colored latex particles are used, coloration is generated due to aggregation of these labeling agents, so the coloration is measured. The particles on which the antibody is immobilized are called antibody-immobilized particles.

[0055] The detection region refers to a partial region of the support body on which an antibody for capturing the substance to be detected (antigen) is immobilized. The detection region is provided with at least one region on which an antibody for capturing the antigen is immobilized. The detection region only needs to be included in the support body, and the antibody only needs to be immobilized on the support body.

[0056] The sample pad is a part for adding specimens, and a porous material is used. The sample pad is located at the most upstream part of the immunochromatographic test piece. The material can use commonly used filter paper, glass fiber, non-woven fabric, etc. In order to use a large number of specimens for immunoassays, it is preferably in a pad shape with a thickness of about 0.3mm to 1mm. The specimen also includes samples prepared using specimens, such as samples obtained by suspending the specimen in other solutions.

[0057] The absorption band is a component for absorbing components supplied to the support but not reacting in the detection area. The material of the absorption band can be filter paper, sponge, etc. with high water retention made of common natural polymer compounds, synthetic polymer compounds, etc., but in order to promote the development of the specimen, a material with high water absorption is preferred.

[0058] The backing plate is a component to which all the aforementioned materials, i.e., the support body, sample pad, labeling body area, absorption band, etc., are partially overlapped and fixed. As long as these materials are arranged and fixed at the optimal intervals, the backing plate is not essential, but it is usually preferred to use a backing plate from the convenience of manufacturing or use.

[0059] The immunochromatographic test piece of the present invention may further include a control display area (component). The control display area is a portion that indicates that the test has been correctly performed. For example, the control display area is present downstream of the detection area, and when the specimen sample passes through the detection area and reaches the control display area, a signal is emitted by coloring, etc. The control display area may be pre-solidified with a substance that binds to an antibody bound to a labeled carrier, or may be pre-solidified with a pH indicator or other reagent that changes color after the specimen arrives. In the case where the antibody bound to the labeled carrier is a mouse monoclonal antibody, an anti-mouse IgG antibody may be used.

[0060] The size of the immunochromatographic test piece is not limited, and is, for example, about several centimeters to several dozen centimeters in length and several millimeters to several centimeters in width.

[0061] In the test piece of the above scheme, the specimen passes through a porous flow channel formed by a series of connections of the sample pad, the marker area, the support body, the detection area, the absorption band, etc. Therefore, in this scheme, all of the above are specimen movement areas. Depending on the material and form of each constituent material, in some schemes, the specimen may not penetrate into the material but pass through the surface, but the specimen movement area defined in this specification does not distinguish between the inside or the surface of the material, so the test piece of this scheme is also included in the scope of this specification.

[0062] When the sugar chain antigen in the specimen is measured using the immunochromatographic test piece of the present invention, it is first necessary to extract the sugar chain antigen in the specimen. The extraction of the sugar chain antigen is performed by treating the specimen containing the sugar chain antigen with nitrous acid. Nitrous acid can be produced by mixing a nitrite such as sodium nitrite with an acid, and the specimen containing the sugar chain antigen can be treated with the nitrous acid produced in this way. The extracted antigen is bound to the antibody immobilized on the immunochromatographic test piece through an antigen-antibody reaction, but if there is residual acid in the reaction system at this time, the reaction system becomes acidic, thereby inhibiting the antigen-antibody reaction. Therefore, it is necessary to neutralize the acid in the reaction system.

[0063] In the present invention, nitrite and acid are mixed to generate nitrous acid, and the sugar chain antigen in the specimen is extracted by the nitrous acid, and the nitrous acid is neutralized, and then the sugar chain antigen is bound to the antibody solid-phased on the immunochromatographic test piece, and the sugar chain antigen is measured. In this method, the following methods can be cited as methods for extracting and measuring sugar chain antigens. In all methods, the extraction and neutralization of sugar chain antigens using nitrous acid are performed on the immunochromatographic test piece. In order to extract sugar chain antigens using nitrous acid on the immunochromatographic test piece, the immunochromatographic test piece can be impregnated with an acidic reagent or nitrite in advance. In order to neutralize on the immunochromatographic test piece, the immunochromatographic test piece can be impregnated with a neutralizing reagent in advance.

[0064] (A) The specimen and the acidic solution are mixed in advance and added to the sample pad of the immunochromatographic test piece impregnated with nitrite and a neutralizing agent. When the mixed solution reaches the area containing nitrite, the nitrite reacts with the acid to produce nitrous acid, thereby extracting the sugar chain antigen in the specimen. The sugar chain antigen extract is neutralized in the area impregnated with the neutralizing agent on the immunochromatographic test piece, and the sugar chain antigen can bind to the antibody solid-phased on the immunochromatographic test piece and be detected. In this method, the acidic solution used includes: acetic acid, hydrochloric acid, malonic acid, malic acid, maleic acid, citric acid, tartaric acid, etc.

[0065] (B) The specimen and nitrite solution are mixed in advance and added to the sample pad of the immunochromatographic test piece impregnated with an acidic reagent and a neutralizing reagent. When the mixed solution reaches the area containing the acidic reagent, the nitrite reacts with the acid to produce nitrous acid, which extracts the sugar chain antigen in the specimen. The sugar chain antigen extract is neutralized in the area impregnated with the neutralizing reagent on the immunochromatographic test piece, and the sugar chain antigen can be bound to the antibody immobilized on the immunochromatographic test piece and detected.

[0066] In the immunochromatographic test piece of the present invention for carrying out the method of (A) or (B) above, an acidic reagent or nitrate and a neutralizing reagent are impregnated upstream of the marker region (upstream of the sample flow, on the side where the sample pad exists), i.e., in the sample pad or between the sample pad and the marker region. An immunochromatographic test piece impregnated with nitrite and a neutralizing reagent in the sample pad or between the sample pad and the marker region can be used for the method (A) above. In addition, an immunochromatographic test piece impregnated with an acidic reagent and a neutralizing reagent in the sample pad or between the sample pad and the marker region can be used for the method (B) above. It should be noted that a solid acidic reagent is used as an acidic reagent impregnated in the immunochromatographic test piece. By these methods, the substance to be detected in the test sample can be accurately and specifically detected without relying on the amount used for the test sample test.

[0067] The solid acidic reagent or nitrite can be impregnated in the sample pad, or in a gasket made of a porous material such as a non-woven fabric that is different from the sample pad, and the obtained solid acidic reagent impregnated porous material or nitrite impregnated porous material is arranged between the sample pad and the marker area, that is, on the upstream side of the marker area. Here, the area impregnated with the solid acidic reagent or nitrite may or may not be in contact with the sample pad or the marker area. In the present invention, the area impregnated with the reagent is also referred to as a gasket impregnated with the reagent.

[0068] The neutralizing agent is arranged downstream of the region impregnated with a solid acidic agent or nitrite. The neutralizing agent can be impregnated in a sample pad, or in a support, or in a pad made of a porous material such as a non-woven fabric different from the support, and the porous material impregnated with the neutralizing agent is arranged between the region impregnated with a solid acidic agent or nitrite and the marker region. That is, there is a region impregnated with a neutralizing agent upstream of the marker region, and there is a region impregnated with a solid acidic agent or nitrite upstream of the region impregnated with the neutralizing agent. Here, the region impregnated with the neutralizing agent may or may not contact the region impregnated with a solid acidic agent or nitrite or the marker region.

[0069] The region impregnated with the solid acidic reagent is referred to as a solid acidic reagent region, the region impregnated with the nitrite is referred to as a nitrite region, and the region impregnated with the neutralizing reagent is referred to as a neutralizing reagent region or an alkaline reagent region.

[0070] As an immunochromatographic test piece with a solid acidic reagent region or a nitrite region and a neutralizing reagent region, it has a sample pad, a solid acidic reagent region or a nitrite region, a neutralizing reagent region, a label region, a detection region and an absorption band on a support body from upstream, and the solid acidic reagent region or the nitrite region can also be located on the sample pad. In addition, in the sample pad, the solid acidic reagent region or the nitrite region, the neutralizing reagent region, the label region, the detection region and the absorption band, adjacent regions can be in contact with each other or not. And, the solid acidic reagent region or the nitrite region, the neutralizing reagent region, the label region need not necessarily be impregnated in a single porous material, and multiple or all regions can also be impregnated in the same porous material.

[0071] The solid acidic reagent used in the present invention is solid at room temperature and does not volatilize at high temperature.

[0072] Preferred solid acidic reagents used in the present invention include malonic acid, malic acid, maleic acid, citric acid, and tartaric acid.

[0073] In addition, as the preferred solid acid reagent used in the present invention, if use the more acid of valence number such as citric acid, then can be extracted by smaller amount. In addition, if the valence number is the same, then the efficiency of the acid with smaller acid dissociation constant such as maleic acid, tartaric acid is better.

[0074] Furthermore, as a preferred solid acidic reagent used in the present invention, a reagent that does not stain the immunochromatographic test piece is preferred, and specifically a reagent that is white in a dry state or is not easily colored by drying heat or oxidation is preferred.

[0075] Examples of the nitrite used in the present invention include sodium nitrite and potassium nitrite.

[0076] The amount of the solid acidic reagent or nitrite used in the present invention, i.e., the amount impregnated in the immunochromatographic test piece, is not particularly limited, and is generally about 0.01 μg to 1 mg per immunochromatographic test piece, preferably about 0.1 μg to 0.1 mg. It is most preferred to select the best amount that can achieve the effect according to the type of solid acidic reagent or nitrite used, the composition of the specimen suspension, and the amount added.

[0077] The solid acidic reagent or nitrite is dissolved once, applied and dried, so that the solid acidic reagent or nitrite is impregnated into the sample pad or the porous material.

[0078] The neutralizing agent used in the present invention is solid at room temperature and does not volatilize at high temperatures.

[0079] Preferred neutralizing agents used in the present invention include Tris base (trishydroxymethylaminomethane), sodium hydroxide, dipotassium hydrogen phosphate, trisodium citrate, and Good's buffer having a buffering capacity in the alkaline region.

[0080] The amount of the neutralizing agent used in the present invention, i.e., the amount impregnated in the immunochromatographic test piece, is not particularly limited, and is generally about 0.01 μg to 1 mg per immunochromatographic test piece, preferably about 0.1 μg to 0.1 mg. It is most preferred to select the optimal amount that can achieve the effect according to the type of neutralizing agent used, the composition of the specimen suspension, the amount added, etc.

[0081] In order to impregnate the sample pad or the porous material with the neutralizing reagent, the neutralizing reagent may be dissolved once, the solution may be applied to the sample pad or the porous material, and then dried.

[0082] Figure 1 and Figure 2 1 is a diagram showing a preferred form of a typical immunochromatographic test piece. Figure 1 and Figure 2The immunochromatographic test piece shown is an immunochromatographic test piece impregnated with a solid acidic reagent and a neutralizing reagent, but it can also be impregnated with nitrate instead of the solid acidic reagent. In this case, an immunochromatographic test piece impregnated with nitrate and a neutralizing reagent is formed. Those skilled in the art can appropriately design and manufacture immunochromatographic test pieces impregnated with nitrate and a neutralizing reagent. It should be noted that the immunochromatographic test piece is not limited to Figure 1 and Figure 2 Test piece shown. Figure 1 and Figure 2 1 represents a support, 2 represents a label region, 3 represents a detection region, 4 represents a sample pad, 7 represents an absorption band, and 8 represents a backing plate. In addition, a top laminate may be attached to the entire test piece.

[0083] Figure 1 A and Figure 2 A is the top view. Figure 1 B and Figure 2 B is a cut-away cross-sectional view. Figure 1 In the example of FIG. 1 , a support 1 having a detection region 3, an absorption band 7, a label region 2, a sample pad 4, etc. are stacked on a backing plate 8 made of resin or the like. Figure 1 As shown, one end of the absorption 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 after a certain interval, the downstream portion of the sample pad is impregnated with a neutralizing reagent. The region impregnated with the solid acidic reagent is referred to as the solid acidic reagent region 5, and the region impregnated with the neutralizing reagent is referred to as the neutralizing reagent region 6. In this test piece, the sample pad 4 serves as both the solid acidic reagent region 5 and the neutralizing reagent region 6. That is, there are a solid acidic reagent region and a neutralizing reagent region on the sample pad. In this test piece, since the solid acidic reagent region and the neutralizing reagent region are in the form of a piece of porous material (gasket), it is sometimes referred to as a single pad test piece. Figure 2 In the example of FIG. 1 , there is a solid acidic reagent region 5 and / or a neutralizing reagent region 6 upstream of the label region 2, and these regions overlap to form a continuous lateral flow channel. Figure 2 In the test piece shown, the solid acid reagent area 5 also serves as a sample pad. That is, there is a solid acid reagent area on the sample pad. In this test piece, the solid acid reagent area and the neutralizing reagent area are provided in the form of two separate porous materials (pads), which is sometimes called a double pad test piece. There may be a sample pad further upstream of the solid acid reagent area. In addition, although Figure 2 In the test piece shown, the solid acid reagent area 5 and the neutralizing reagent area 6 are impregnated with different porous materials, but it is also possible to Figure 1Like the sample pad of the test piece, a solid acid reagent area 5 is provided in the upstream portion on the same porous material (gasket), and a neutralizing reagent area 6 is provided in the downstream portion.

[0084] The above-mentioned immunochromatographic test piece is stored in a storage container and is used as an immunochromatographic device. That is, the state in which the immunochromatographic test piece is assembled in the storage container is called an immunochromatographic device. Usually, the above-mentioned storage container includes a lower container part and an upper cover part. Here, the immunochromatographic test piece is stored in the lower container part, and when the immunochromatographic test is performed, the upper part of the lower container part is covered by the upper cover part. Sometimes the lower container part and the upper cover part are respectively referred to as the lower part and the upper part of the storage container. After the above-mentioned immunochromatographic test piece is stored in the storage container, the specimen dropping port that can supply the specimen sample solution to the sample pad is located at the upper part of the sample pad, and after adding the specimen to the specimen dropping port of the container, the specimen is immersed in the sample pad. Here, the dropping port is sometimes also referred to as the adding port. In addition, after the immunochromatographic test piece is stored in the storage container, the determination part is located above the detection area, and the detection area can be observed through the opening of the determination part of the container.

[0085] After the upper cover portion covers the lower container portion, the immunochromatographic test piece contained in the lower container portion is sandwiched by the protrusions provided on the upper cover portion and the lower container portion of the storage container and supported so as to be immobile. Therefore, the upper cover portion provided on the storage container and the protrusions provided on the lower container portion are respectively referred to as an upper support body including the protrusions and a lower support body.

[0086] The upper support of the upper cover part is arranged in a manner that the edge of the opening is formed on the back side of the side where the sample is dripped at the dripping port, and the upper support of the upper cover part is formed by a protrusion having an inclination. The upper support of the upper cover part supports the upper side of the sample pad of the immunochromatographic test piece. The upper support has an inclination, and when the upper support of the upper cover part presses the upper side of the sample pad of the immunochromatographic test piece from above, the inclination makes the height of the protrusion smaller toward the downstream part of the immunochromatographic test piece, that is, the inclination is upward along the downstream direction of the immunochromatographic test piece. After the upper cover part covers the lower container part, the upper side of the sample pad of the immunochromatographic test piece is pressed from above and fixed so that it cannot move, and it is made to fit closely with the sample pad, so that the sample solution does not leak to the part other than the sample pad. In addition, when the upper support of the upper cover part squeezes the sample pad, the pressing gradually weakens from upstream to downstream. Sometimes the support is referred to as a support composed of inclined protrusions.

[0087] On the other hand, the upper support body of the cover portion above the storage container of the conventional immunochromatographic device has no inclination but has a flat shape.

[0088] The lower support of the lower container part is formed by a protrusion with an inclination, which is arranged in the part corresponding to the sample dripping port in the lower container part. Here, the part corresponding to the sample dripping port in the lower container part refers to the part overlapping with the opening of the sample dripping port after the upper cover part is covered on the lower container part. After covering the upper cover part on the lower container part, the lower support of the lower container part supports the lower side of the sample pad of the immunochromatographic test piece. The lower support of the lower container part presses the lower side of the sample pad of the immunochromatographic test piece from below to fix it and prevent it from moving. The lower support is arranged in the part corresponding to the sample dripping port as a protrusion with an inclination. The lower support has an inclination, and when pressing the sample pad of the immunochromatographic test piece, the inclination makes the height of the protrusion smaller toward the downstream part of the immunochromatographic test piece, that is, the inclination is downward along the downstream direction of the immunochromatographic test piece, and the pressing of the sample pad gradually weakens from upstream to downstream. This support body is sometimes referred to as a support body composed of inclined protrusions.

[0089] On the other hand, the lower support body of the container below the storage container of the conventional immunochromatographic device does not have an inclination but has a flat shape.

[0090] The cover portion having a support body including a protrusion can be referred to as an upper support body having an inclined cover portion, wherein the protrusion presses and supports the upper side of the immunochromatographic test piece, and the test piece has an upward inclination along the downstream direction of the immunochromatographic test piece; the container portion having a support body composed of protrusions can be referred to as a lower support body having an inclined container portion, wherein the protrusion presses and supports the lower side of the immunochromatographic test piece, and the test piece has a downward inclination along the downstream direction of the immunochromatographic test piece; the cover portion not having a support body composed of inclined protrusions can be referred to as an existing cover portion; and the container portion not having a support body composed of inclined protrusions can be referred to as an existing container portion. In this case, the combination of the upper cover portion and the lower container portion of the storage container of the immunochromatographic device of the present invention has the following combinations A to C.

[0091] A: The upper support has an inclined cover part and an existing container part

[0092] B: The upper support has an inclined cover portion, and the lower support has an inclined container portion

[0093] C: The existing cover part and the lower support body have an inclined container part

[0094] In the immunochromatographic device of the present invention, the support body composed of inclined protrusions may be present in both the upper cover portion and the lower container portion of the storage container of the immunochromatographic device as in B above, or may be present only on one side as in A and C above. Preferably, it is present in both portions as in B above.

[0095] In this way, there is an inclined portion on the support body portion of the upper cover portion and / or the lower container portion that contacts the sample pad of the immunochromatographic test piece, and the inclined portion includes a protrusion whose height gradually decreases toward the downstream side of the immunochromatographic test piece, so that when the upper support body and the lower support body clamp and squeeze the sample pad of the immunochromatographic test piece, the upper support body and the lower support body are in close contact with the immunochromatographic test piece, and the pressing of the downstream part is less than the pressing of the upstream part. Therefore, the dripped liquid specimen will not leak from the immunochromatographic test piece, and the flow path of the liquid on the downstream side of the sample pad of the immunochromatographic test piece will not be strongly squeezed by the support body, so the liquid specimen is not easy to flow. In addition, the upstream part of the sample pad exists at a higher position than the downstream part, and the liquid specimen is easy to flow by gravity. After the specimen is easy to flow, the start time of the specimen development can be adjusted to an appropriate speed, so that the performance of the highest sensitivity can be exerted within the set judgment time.

[0096] The immunochromatographic device of the present invention has a support body in a storage container of the immunochromatographic test piece, and the support body includes the above-mentioned inclined protrusions, so that the development of the sample can be adjusted to an appropriate speed in the immunochromatographic method for extracting and measuring sugar chain antigens by nitrous acid extraction treatment of sugar chain antigens on the immunochromatographic test piece. In addition, the immunochromatographic device of the present invention may also have a structural feature related to the structure for adjusting the development speed of the specimen sample as follows.

[0097] (1) The immunochromatographic device of the present invention can be provided with a relatively wide dropping port.

[0098] For example, the dropping port of a conventional immunochromatographic device (Quick Navi (trademark) -Strep A, Denka Seiken) storing an immunochromatographic test piece having a length of 5 to 9 cm, preferably 7 cm, and a width of 0.3 to 0.7 cm, preferably 0.5 cm, is set to have a size of about 3.5 mm × about 5.5 mm (19.25 mm 2 ) size, and the dropping port of the immunochromatographic device of the present invention for storing immunochromatographic test pieces of the same size is set to have a size of about 3.5 mm × about 11 mm (38.5 mm 2The immunochromatographic device and the immunochromatographic test piece stored therein are of roughly similar size. Therefore, the immunochromatographic device of 3 cm in width and 9 cm in length has a roughly rectangular 3 to 5 mm × 8 to 15 mm (24 to 75 mm) 2 ) is an immunochromatographic device with a wider dropping port, which can play the role of the immunochromatographic device of the present invention. The long side of the dropping port is a side parallel to the long side of the chromatographic test piece, that is, a side along the flow direction of the specimen sample solution. Sometimes the length of the long side of the dropping port is called the dropping port length, and the length of the short side is called the dropping port width. The above example is the case of a roughly rectangular dropping port. The shape of the dropping port can be triangular or circular. When the size of the dropping port of the immunochromatographic device of the present invention is expressed by area, the area is 24 to 75 mm 2 , preferably 35 to 75 mm 2 .

[0099] For example, the immunochromatographic device of the present invention stores an immunochromatographic test piece with a length of 5 to 9 cm and a width of 0.3 to 0.7 cm, and has a size of 3 to 5 mm (dropping port width) × 8 to 15 mm (dropping port length) (24 to 75 mm 2 ) is a roughly rectangular drop-feeding port. The drop-feeding port length is preferably 10 mm or more, and more preferably 10 mm. A device having a drop-feeding port length of less than 8 mm (eg, 5 mm) is a device having a small width of an existing drop-feeding port.

[0100] In the conventional immunochromatography method, when the sample is processed using the sample processing reagent provided on the immunochromatographic test piece, the area where the sample solution contacts the region impregnated with the sample processing reagent is small, so the sample processing capability is weak, and since the sample is processed while the immunochromatographic test piece is gradually developed, a difference in concentration of the sample processing reagent occurs between the sample solution developed first and the sample solution developed later. Therefore, the sample cannot be processed reliably.

[0101] Since the dropping port of the immunochromatographic device of the present invention is set wide, a large amount of sample solution can be supplied at one time in a short time to the area impregnated with the sample processing reagent on the immunochromatographic device, that is, the area impregnated with the solid acidic reagent or the area impregnated with nitrous acid. As a result, the extraction of sugar chain antigens on the immunochromatographic test piece can be promoted. The sample solution that can be supplied at one time is 10 μL to 200 μL.

[0102] As a result, there is no concentration difference of the sample processing reagent due to time difference, so the sample processing on the immunochromatography can be accelerated and performed reliably and efficiently.

[0103] (2) The immunochromatographic device of the present invention may be configured to have no gap between the dropping port and the sample pad located therebelow so that the sample does not overflow from the dropping port.

[0104] The immunochromatographic device of the present invention has a wider droplet opening to supply a large amount of sample solution at one time. In addition, the edge portion of the outer contour of the droplet opening has a certain height (1-5 mm), and the supplied sample material solution is temporarily stored in the droplet opening.

[0105] As a result, since it takes a certain amount of time for all the specimen sample solution to be absorbed by the immunochromatographic test piece, there is a possibility that the sample solution is not absorbed by the immunochromatographic test piece but overflows from the immunochromatographic test piece.

[0106] The immunochromatographic device of the present invention can be constructed such that a sample pad is supported by a backing plate having an outer contour dimension equal to or greater than that of a dropping port, so that the sample does not overflow from the dropping port from the backing plate supporting the sample pad. Thus, a force is exerted through the backing plate to press the sample pad toward the dropping port, thereby eliminating a gap between the dropping port and the sample pad, thereby preventing leakage of the sample solution.

[0107] In addition, when there is a difference in thickness of the sample pad, a gap is easily formed between the thinner portion of the sample pad and the dropping port, and the sample solution is easily leaked.

[0108] In the present invention, the base of the container of the device is designed to be higher where the sample pad is thinner and lower where the sample pad is thicker. Therefore, even if there is a difference in the thickness of the gasket, there is no gap between the dripping port and the gasket, thereby preventing leakage of the sample solution.

[0109] As a result, sugar chain antigens can be efficiently extracted on the immunochromatographic test strip.

[0110] (3) In addition, a highly hydrophobic raw material can be selected as the raw material (non-woven fabric) for manufacturing the gasket for the immunochromatographic test piece. The gasket is impregnated with specimen processing reagents such as solid acid reagents, nitrites and neutralizing reagents. This can reduce the flow rate of the liquid, that is, slow down the time it takes for the specimen sample solution to develop on the immunochromatographic test piece.

[0111] In this case, examples of the material having high hydrophobicity include polyethylene, polyester, polystyrene, polypropylene, rayon, and nylon.

[0112] As the raw material of the porous material used in the region impregnated with the neutralizing agent, there can be mentioned materials having a basis weight of 10 to 400 g / m 2 The fabric has a thickness of 0.1 to 2.0 mm, and the fabric is a porous material, each 1 cm / m 2 The water absorption capacity is 10~100μl / cm2 , and the water absorption rate is 1.0~5.0μl / sec, and 1cm / m 2 The water retention of the fragments in wet state in contact with the membrane and left to stand for 5 minutes is 10-100 μl / cm 2 , preferably 1cm / m 2 The fragments are in contact with the membrane in a wet state and left to stand for 5 minutes. The diffusion area of ​​the liquid is 20 mm 2 The following, that is, the porous material has three characteristics: high water absorption, high water retention (liquid retention), and low or continuous release of liquid. Specifically, filter paper made of cellulose cotton fiber and glass filter paper made of glass fiber can be cited. As such filter paper, for example, No. 26-3 of Toyo Filter Paper Co., Ltd. can be cited. In addition, the volume of the filter paper used is large, and it can fully retain the acidic solution that arrives from the upstream later than the specimen. By using such a porous material, it is possible to impregnate the required amount of neutralizing reagent, and the required amount of neutralizing reagent can fully neutralize the specimen after the sugar chain antigen is extracted by nitrous acid produced by the reaction of nitrite and acidic reagent. In addition, the gasket can absorb and retain a large amount of liquid, and the release can be continuous. Therefore, even if the liquid containing nitrous acid remaining in the upstream of the immunochromatographic test piece is unfolded after the judgment time, it has sufficient neutralization ability, so that the acidic solution can be inhibited from reaching the detection area where the antibody is solid-phased, and then the non-specific reaction is inhibited, so that the sugar chain antigen can be detected without producing non-specific reactions. On the other hand, as a glass filter paper with high water absorption, high water retention and low water release, specifically, GS-25 of Toyo Roshi Co., Ltd. can be cited. Due to its high water absorption, it can be impregnated with more neutralizing reagents, and due to its high water retention and low water release, it prevents the acidic solution from reaching the detection area after solidification. As a result, non-specific reactions can be suppressed in negative cases, and color development of lines after the judgment time can be prevented in positive cases.

[0113] The raw material weight of the porous material used in the area impregnated with the neutralizing agent is 10 to 400 g / m 2 Here, "weight" refers to the weight per unit area (1m 2 ) weight. The gram weight can be appropriately changed according to the amount and composition of the neutralizing agent impregnated in the gasket. If the gram weight is 30g / m 2 If the porosity is lower than 50 g / m, the film will be easily torn after being impregnated with the neutralizing reagent, and will be difficult to operate during the manufacture of immunochromatography. Therefore, the preferred weight is 50 g / m 2 Above, the weight is 300g / m 2 In the above case, the porosity is low. Although it varies depending on the composition of the neutralizing agent, the specimen will not penetrate into the raw material smoothly and cannot be mixed with the neutralizing agent. Therefore, 300 g / m 2The most preferred weight is 250 to 270 g / m 2 .

[0114] In addition, the "thickness" of the raw material of the porous material used in the area impregnated with the neutralizing agent can be 0.1 to 2.0 mm, and the thickness can be appropriately changed according to the amount and composition of the neutralizing agent impregnated in the gasket. When the thickness is less than 0.4 mm, it is easy to tear after impregnation with the neutralizing agent, and it is difficult to operate when manufacturing the immunochromatogram. Therefore, the preferred thickness is 0.4 to 0.8 mm. Considering that the amount of the impregnated neutralizing agent is easy to adjust and easy to handle when manufacturing the immunochromatogram, it is more preferably about 0.6 mm.

[0115] As for "water absorption", it is preferred that per 1cm / m 2 The water absorption capacity is 10~100μl / cm 2 , and the water absorption rate is 1.0~5.0μl / sec. Regarding the water absorption amount and water absorption rate, prepare 200μl of colored solution (1% Tween20+Red No. 102) in a groove of a 96-well EIA plate, and put a test piece into it. The test piece is made of 5×60mm raw material pasted on a backing plate. The side immersed in the solution is the lower end of the test piece. The time required for the solution to reach the upper end of the test piece is measured, and the test piece is taken out immediately after the solution reaches the upper end, and the amount of liquid remaining in the groove is measured. The water absorption amount is obtained by subtracting the amount of liquid remaining in the groove from 200μl, and dividing it by the amount of raw material per 1cm 2 The water absorption rate is calculated by the water absorption amount / time required to reach the top. The raw material of the porous material used in the area impregnated with the neutralizing agent is preferably a raw material with a large water absorption amount and a slow water absorption rate. Although it varies depending on the concentration and water content of the solid acidic agent, when the water absorption amount is 30μl / cm 2 In the following cases, the immunochromatographic test piece may not be impregnated with all the necessary neutralizing reagents. Therefore, specifically, the water absorption is preferably 30 μl / cm 2 The above. The water absorption rate of the raw material of the porous material used in the area impregnated with the neutralizing reagent will affect the time for extracting the sugar chain antigen in the specimen and the time for the test solution after extraction to be neutralized. Although the time for extracting the sugar chain antigen can be adjusted to a certain extent by the raw material or composition of the solid acid reagent, it is not completely adjusted. Therefore, the water absorption rate of the raw material used in the area impregnated with the neutralizing reagent is a very important factor for fully extracting the sugar chain antigen and neutralizing. Specifically, the water absorption rate is preferably 1.0 to 5.0 μl / sec, and the more preferred water absorption rate is 2.0 μl / sec or less.

[0116] "Water retention" is measured by using a porous material with an area impregnated with a neutralizing agent of 1 cm / m 2The fragments are placed on a membrane, 70 μl of a solution (1% Tween 20 + Red No. 102) is added thereto, and the weight before and after standing is measured. The amount of liquid for water retention varies depending on the composition of the solution added (surfactant and protein amount). It is preferred that the water retention amount be 10 to 100 μl / cm when tested with a 1% Tween 20 solution. 2 The more preferred water retention capacity is 15 μl / cm 2 above.

[0117] "Releasability" is achieved by using a porous material with an area of ​​1cm / m of the raw material impregnated with the neutralizing agent. 2 The fragments are placed on a membrane, 70 μl of a solution (1% Tween 20 + Red No. 102) is added thereto, and the area of ​​the liquid diffused on the membrane after standing for 5 minutes is measured. The area varies depending on the composition of the solution added (surfactant and protein amount), and preferably the area is 30 mm when using a 1% Tween 20 solution test. 2 Below, the more preferred release is 20mm 2 the following.

[0118] For example, the raw material weight of the porous material used in the area impregnated with the neutralizing agent is 50 to 300 g / m 2 Or 250~270g / m 2 , thickness is 0.4~0.8mm, every 1cm / m 2 The water absorption capacity is 30~100μl / cm 2 , the water absorption rate is 1.0~2.0μl / sec, making 1cm / m 2 The water retention of the fragments in the wet state after contacting the detection area and standing for 5 minutes is 15-100μl / cm 2 , so that 1cm / m 2 The liquid diffusion area of ​​the fragments in wet state in contact with the membrane and left to stand for 5 minutes is 20mm 2 The raw material having this characteristic has a higher ability as follows, and therefore has an effect of suppressing non-specific reactions compared to other raw materials: "The acidic solution containing the sugar chain antigen is fully neutralized by the high water absorption and high water retention of the area impregnated with the neutralizing agent, and the acidic solution is suppressed from reaching the detection area by the low release or continuous release of the area impregnated with the neutralizing agent, or the fully neutralized test solution is allowed to continue to spread to the detection area."

[0119] The following is a description of the method of using the device of the present invention. The following method of use refers to a method of mixing a sample with a nitrous acid solution and measuring it using an immunochromatographic test method containing a solid acidic reagent and a neutralizing reagent, but a method of mixing a sample with an acidic solution and measuring it using an immunochromatographic test method containing a nitrite and a neutralizing reagent can also be performed with reference to the following description of the method of use.

[0120] The specimen or a sample prepared using the specimen is brought into contact with the nitrite solution and mixed, the specimen is suspended in the nitrite solution, and added to the specimen drop port of the device to start the measurement. At this time, 5 to 100 μL of the specimen and 0.01 to 2 mL of 0.1M to 8M nitrite are mixed, and 5 to 200 μL are supplied to the drop port. Examples of nitrite include sodium nitrite and potassium nitrite.

[0121] The substance to be tested supplied to the dripping port, i.e., the specimen containing the sugar chain antigen, moves to the sample pad, and is developed to the solid acid reagent area 5 and the neutralizing reagent area 6 on the sample pad 4 by capillary action, and is developed in sequence to the labeling body area 2, the support body 1, and the absorption band 7 in the horizontal direction. In the solid acid reagent area 5, the nitrite mixed in the specimen reacts with the solid acid reagent on the solid acid reagent area 5 to produce free nitrous acid, and the sugar chain antigen is extracted from the specimen by the action of the nitrous acid. The extracted sugar chain antigen moves to the neutralizing reagent area 6 together with the acidic developing solution, and the pH of the acidic developing solution containing the sugar chain antigen is neutralized in the neutralizing reagent area 6 and adjusted to a neutral range. As a result, the sugar chain antigen moves further downstream under neutral conditions. In the labeling body area 2, while the specimen sample is developed, the labeled antibody is released into the liquid and developed toward the support body 1. When sugar chain antigens are present in the specimen sample, the sugar chain antigens are specifically captured by the capture antibody in the detection region 3 of the support 1, and the sugar chain antigens also react specifically with the labeled antibody to form a complex. Thus, a sandwich of the antibodies sandwiching the sugar chain antigens is constructed in the detection region 3, so that the labeled antibody-sugar chain antigen complex can be measured in the detection region 3. The detection region can be observed by the determination unit of the immunochromatographic device.

[0122] According to the method using the immunochromatographic test strip of the present invention, since the sugar chain antigens in the specimen are extracted on the immunochromatographic test strip, there is no need to pre-extract the sugar chain antigens in the specimen before measuring using the immunochromatographic test strip, thereby enabling the sugar chain antigens in the specimen to be measured in one step.

[0123] According to the present invention, in an immunochromatographic test piece incorporating a spacer containing a dried solid acidic reagent or nitrite, efficient extraction can be performed on the reagent.

[0124] The area of ​​the specimen drop port can also be made substantially the same as the gasket containing the solid acid reagent (solid acid reagent area). In this way, more samples can be instantly contacted with the solid acid reagent. In addition, by using a highly hydrophobic non-woven fabric as a component for coating and impregnation, it is possible to prevent the sample from spreading to the gasket (neutralizing reagent area) impregnated with the neutralizing reagent downstream immediately after addition. In this way, the sample is kept in the specimen addition section for 1 to 2 minutes, so that antigen extraction can be fully performed.

[0125] In addition, in the present invention, when the solid acidic reagent region or the nitrite region and the neutralizing reagent region overlap and contact each other, a sheet material through which liquid cannot pass is sandwiched between the solid acidic reagent region or the nitrite region and the neutralizing reagent region. By providing the sheet material, the following three effects can be achieved.

[0126] (A) When the test piece is stored, the reagent can be suppressed from moving between two adjacent regions. As a result, the solid acidic reagent or nitrite can be prevented from contacting with the neutralizing reagent and causing a reaction. As a result, the stability of the reagent can be improved.

[0127] (B) In addition, when a sample mixed with a nitrite or an acidic reagent is added, the sample reaches the solid acidic reagent area or the nitrite area and then immediately moves to the neutralizing reagent area, thereby preventing insufficient extraction of sugar chain antigens. That is, the speed at which the liquid containing the sample moves from the solid acidic reagent area or the nitrite area to the neutralizing reagent area is reduced, and the time for the liquid containing the sample to stay in the solid acidic reagent area or the nitrite area is extended, resulting in sufficient extraction of sugar chain antigens and improved measurement sensitivity.

[0128] (C) It is also possible to prevent, when a specimen mixed with nitrite or an acidic reagent is added, the specimen reaches the solid acidic reagent area or the nitrite area and then immediately moves to the neutralizing reagent area, and the liquid containing the specimen after the movement flows back to the solid acidic reagent area or the nitrite area, thereby reducing the activity of the solid acidic reagent or nitrite and reducing the extraction efficiency. That is, the liquid containing the specimen that reaches the neutralizing reagent area is prevented from flowing back to the solid acidic reagent area or the nitrite area, thereby preventing the activity of the solid acidic reagent or nitrite from being reduced, so that the extraction process of the sugar chain antigen using nitrous acid is carried out efficiently, and the measurement sensitivity is improved.

[0129] The material of the sheet provided between the solid acid reagent area or the nitrite area and the neutralizing reagent area is not limited as long as it is a material through which liquid cannot pass, and resin sheets such as PET (polyethylene terephthalate) sheets and polyethylene sheets can be used. Resin sheets are also called resin films.

[0130] The sheet material can be arranged in a manner that the solid acidic reagent region or the nitrite region and the neutralizing reagent region are partially in contact and overlapped. In this case, the length of the overlapping portion of the solid acidic reagent region or the nitrite region and the neutralizing reagent region is preferably as short as possible, for example, less than 5 mm, preferably less than 3 mm, and more preferably less than 2 mm.

[0131] In addition, sheet material can also be arranged in a way that solid acidic reagent region or nitrite region and neutralizing reagent region do not contact, and is made into a shape that the pad impregnated with acidic reagent or nitrite covers the PET sheet material. In this case, neutralization region is fully covered by sheet material, and solid acidic reagent region or nitrous acid region are arranged on this sheet material. In this case, liquid in solid acidic reagent region or nitrous acid region can not directly move to neutralization region, but arrives at neutralization reagent region after flowing through on sheet material.

[0132] The sheet material may be provided only between, for example, the solid acidic reagent region or the nitrite region and the neutralizing reagent region. On the other hand, when the resin sheet material is pasted as the top laminate sheet material in a manner covering the upper side of the immunochromatographic test piece, the top laminate sheet material may be pasted in a manner covering the neutralizing reagent region, the labeling body region, the support body, the detection region, and the absorption band except the solid acidic reagent region or the nitrite region (except the sample pad when the solid acidic reagent region or the nitrite region also serves as the sample pad), and the solid acidic reagent region or the nitrite region is pasted on the upper part of the top laminate sheet material to which the upper part of the neutralizing reagent portion is pasted.

[0133] In the method of the present invention, the biological sample as a specimen is not particularly limited, and examples thereof include body fluids such as serum, plasma, blood, urine, feces, saliva, tissue fluid, cerebrospinal fluid, and swab fluid, or dilutions thereof.

[0134] In the method using the immunochromatographic device of the present invention, the substance to be measured is a sugar chain antigen that can be measured by an immunoassay, i.e., an assay utilizing an antigen-antibody reaction. As antigens, polysaccharides of sugar chain antigens present on the cell wall of bacteria extracted by nitrous acid extraction, etc. can be cited. Protozoa, fungi, bacteria, mycoplasmas, rickettsia, chlamydia, viruses, etc. containing these substances can also be measured. By using the method of the immunochromatographic test piece of the present invention, it is possible to confirm whether the biological sample of the subject contains sugar chain antigens from protozoa, fungi, bacteria, mycoplasmas, rickettsia, chlamydia, viruses, etc., and in the case of containing sugar chain antigens, it is possible to judge whether the subject suffers from an infection caused by protozoa, fungi, bacteria, mycoplasmas, rickettsia, chlamydia, viruses, etc. For example, it is possible to detect the presence or absence of infection with group A beta-hemolytic streptococcus (Streptococcus pyogenes), Escherichia coli, Legionella, Campylobacter, etc.

[0135] Example

[0136] The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

[0137] In the following examples, % means w / v % unless otherwise specified.

[0138] Example 1: Study on the size of the dripping port

[0139] 1. Immobilization of anti-Streptococcus pyogenes (group A β-hemolytic streptococcus) antibodies on nitrocellulose membrane (support)

[0140] A liquid obtained by diluting anti-Streptococcus pyogenes antibody to 1.0 mg / mL with pure water and anti-rabbit IgG antibody were prepared, and the anti-Streptococcus pyogenes antibody was linearly coated on the sample pad side of a nitrocellulose membrane supported by a PET (polyethylene terephthalate) film, and the anti-rabbit IgG antibody was linearly coated on the absorption band side. Then, 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 is referred to as an "antibody-immobilized membrane".

[0141] 2. Immobilization of Anti-Streptococcus pyogenes Antibodies onto Colored Polystyrene Particles

[0142] Anti-Streptococcus pyogenes antibody was diluted to 1.0 mg / mL with pure water, and colored polystyrene particles were added to 0.1% and stirred. Then, carbodiimide was added to 1% and stirred again. The supernatant was removed by centrifugation and resuspended in 50 mM Tris (pH 9.0) and 3% BSA to obtain a colored polystyrene particle suspension bound to 0.04% anti-Streptococcus pyogenes antibody. In this example, the particles are referred to as "antibody-immobilized particles".

[0143] 3. Coating and Drying of Anti-Streptococcus pyogenes Antibody-Conjugated Colored Polystyrene Particles

[0144] A predetermined amount of the antibody-immobilized particle suspension prepared in 2 was applied to a nonwoven fabric and dried at 45° C. for 30 minutes. In this example, the obtained nonwoven fabric is referred to as a “drying mat”.

[0145] 4. Preparation of neutralizing reagent (alkaline reagent) gasket

[0146] 3M Trizma (trade name) Base (Tris alkali) and 1.5% TritonX100 as a neutralizing agent (alkaline agent) were applied to 30 μL / cm filter paper (Toyo Filter Paper; No. 26-3).

[0147] 5. Preparation of solid acid reagent gasket

[0148] 1.0 M tartaric acid and 0.5% TritonX100 as solid acid reagents were applied to nonwoven fabric (Unitika; ELEVES (registered trademark)) at 13 μL / cm and dried at 45°C for 1 hour immediately after application to obtain nonwoven fabric impregnated with a solid acid reagent.

[0149] 6. Preparation of immunochromatographic test strips for the detection of Streptococcus pyogenes

[0150] The antibody-immobilized membrane prepared in 1, the drying pad prepared in 3, the neutralizing agent (alkaline agent) pad prepared in 4, and the solid acidic agent pad prepared in 5 are bonded to other components (backing plate, absorption band) and cut into 5 mm width to prepare a Streptococcus pyogenes test piece. In this embodiment, the test piece using the filter paper impregnated with the solid acidic agent and the neutralizing agent (alkaline agent) as the sample pad is referred to as the "immunochromatographic test piece of the present invention". It should be pointed out that the immunochromatographic test piece has a solid acidic agent impregnated non-woven fabric, a neutralizing agent (alkaline agent) impregnated non-woven fabric, a drying pad (labeling body area), an antibody-immobilized membrane (detection area), and an absorption band from the upstream along the flow trajectory of the specimen.

[0151] 7. Immunochromatographic Devices

[0152] The immunochromatographic test piece of the present invention is assembled in a storage container having a dropping port length (length of the long side) of 5 mm ((1) the dropping port is small) to produce the immunochromatographic device of 1, and is assembled in a storage container having a dropping port length (length of the long side) of 10 mm ((2) the dropping port is large) to produce the immunochromatographic device of (2). The storage container of the immunochromatographic devices of (1) and (2) is a storage container currently used, and neither the lower container portion nor the upper cover portion has a support body including a protrusion having an inclined shape, which is possessed by the storage container of the immunochromatographic device below. The lower container portion of the storage container currently used is referred to as the existing container portion, and the upper cover portion of the storage container currently used is referred to as the existing cover portion.

[0153] In addition, the lower container portion of the storage container and the upper cover portion of the storage container are combined with the above-mentioned existing container portion and the existing cover portion in the following manner, wherein the lower container portion of the storage container is a container portion having a support body including a protrusion for pressing and supporting the lower side of the immunochromatographic test piece (also referred to as a container portion having a lower support body with an inclination), and the protrusion has a downward inclination in the downstream direction of the immunochromatographic test piece; and the upper cover portion of the storage container is a cover portion of a support body including a protrusion for pressing and supporting the upper side of the immunochromatographic test piece (an upper support body with an inclination cover portion), and the protrusion has an upward inclination in the downstream direction of the immunochromatographic test piece, thereby manufacturing immunochromatographic devices (3) to (5) assembled with the immunochromatographic test piece, and implementing the following test. The dripping port length of the upper cover portion of the immunochromatographic device (3) to (5) is 10 mm.

[0154] (3) The upper support has an inclined cover portion and an existing container portion

[0155] (4) The upper support has an inclined cover portion, and the lower support has an inclined container portion

[0156] (5) The existing lid and the lower support have an inclined container portion

[0157] 8. Specimens

[0158] Cultivate Streptococcus pyogenes and adjust the culture medium to a bacterial count of 1.0 × 10 7 CFU / mL.

[0159] In addition, normal saline was used as a negative specimen.

[0160] 9. Determination

[0161] 20 μL of specimens are suspended in 180 μL of sodium nitrite solution (2.0M NaNO3, 1% Tween20), and 75 μL of them are added to the dropping port of the immunochromatographic device of the present invention. In addition, as an existing method, after suspending the specimen in the nitrous acid extract mixed with sodium nitrite and hydrochloric acid, the 50 μL specimen suspension after neutralization by Tri solution is added to the dropping port of the immunochromatographic device (without immobilized acidic reagent and neutralizing reagent) of the existing method. After 10 minutes, the scoring code is used to evaluate whether there is stacking of colored polystyrene particles and the degree of stacking on the prescribed position of the immobilized anti-Streptococcus pyogenes antibody, and the strong signal degree is successively as +++, ++, +, the situation that is difficult to judge is as ±, and the situation that no signal is found is as -.

[0162] 10. Results

[0163] Device Condition

[0164] (1) Small dripping port (2) The dripping mouth is big, (3) The dripping port is large and the upper support is tilted (4) The dripping port is large, the upper support is tilted, and the lower support is tilted (5) The dripping port is large and the lower support is tilted

[0165] Positive specimens

[0166] Expand time (1) (2) (3) (4) (5) 1min - - - - - 2min - ± + ± + 3min - + + + + 4min - + + + + 5min ± + + + + 6min ± + + + + 7min + + + ++ + 8min + + + ++ + 9min + + ++ ++ + 10min + + ++ +++ ++ Expand start time (seconds) 155 47 46 50 48

[0167] Compared with the immunochromatographic device (1) having a smaller dropping port, the immunochromatographic devices (2), (3), (4) and (5) having larger dropping ports have an earlier development start time, and thus can determine positivity more quickly.

[0168] In addition, compared with the immunochromatographic device not having (1) and (2) including a support having inclined protrusions, the immunochromatographic device having (3), (4) and (5) including a support having inclined protrusions in the upper cover part and / or the lower container part significantly obtained a signal after 10 minutes. In particular, the immunochromatographic device having (4) including a support having inclined protrusions in the upper cover part and the lower container part significantly obtained a signal after 10 minutes compared with the immunochromatographic device having (3) and (5) including a support having inclined protrusions in only one of the upper cover part or the lower container part.

[0169] Explanation of symbols

[0170] 1 Support body (including detection area)

[0171] 2 Marker body region

[0172] 3 Detection area

[0173] 4 Sample pad

[0174] 5 Solid acid reagent area

[0175] 6 Neutralization reagent area

[0176] 7 Absorption band

[0177] 8 Backing board

[0178] 9 Immunochromatographic test strips

[0179] 10 Storage Container

[0180] 11 Cover portion of storage container

[0181] 12 Container part of storage container

[0182] 13. Device drop-in port

[0183] 14 Device determination unit

[0184] 15. Protrusion of the cover portion of the storage container

[0185] 16. Inclined portion of the container portion of the storage container

[0186] Industrial Applicability

[0187] The immunochromatographic device of the present invention can detect group A beta-hemolytic streptococcal infection with high sensitivity.

[0188] All publications, patents and patent applications cited in this specification are directly incorporated into this specification by reference.

Claims

1. An immunochromatographic device, characterized in that: The invention comprises an immunochromatographic test piece for extracting and measuring sugar chain antigens in a specimen and a container for storing the test piece. The immunochromatographic test piece comprises: Sample pad, used to add specimen mixed with nitrite or acid solution; a marker region, comprising a marker antibody, wherein the marker antibody is labeled with an antibody against a sugar chain antigen; and The detection area has an antibody against the sugar chain antigen immobilized on the solid phase. The immunochromatographic test piece forms a complex of antibody-sugar chain antigen-labeled antibody in the detection area to measure sugar chain antigen, has an area impregnated with a neutralizing agent upstream of the labeling area, and has an area impregnated with a solid acidic reagent when a sample mixed with nitrite is used, and has an area impregnated with nitrite when a sample mixed with an acidic solution is used, upstream of the area impregnated with the neutralizing agent. The container includes an upper cover portion and a lower container portion, the upper cover portion having an upper support body including a protrusion supporting the upper side of the immunochromatographic test piece, and the lower container portion having a lower support body including a protrusion supporting the lower side of the immunochromatographic test piece. The upper part of the sample pad of the test piece has a sample dropping port. In the immunochromatographic device, The protrusion portion of the lower support body that supports the immunochromatographic test piece is inclined. Furthermore, the lower support body has an inclination such that the height of the protrusion decreases toward the downstream portion of the immunochromatographic test piece when the sample pad of the immunochromatographic test piece is pressed, that is, an inclination downward in the downstream direction of the immunochromatographic test piece, and the pressure of pressing the sample pad becomes weaker from the upstream to the downstream. The protrusion portion of the upper support body that supports the immunochromatographic test piece is inclined. Furthermore, the upper support body is arranged in a manner that forms an edge of an opening portion on the back side of the side where the sample is dropped at the dropping port, and is formed by an inclined protrusion. The upper support body has an inclination that makes the height of the protrusion decrease toward the downstream part of the immunochromatographic test piece when the upper side of the sample pad of the immunochromatographic test piece is pressed from above, that is, an upward inclination along the downstream direction of the immunochromatographic test piece, and when the sample pad is pressed, the pressure becomes weaker from the upstream to the downstream.

2. The immunochromatographic device according to claim 1, wherein The sugar chain antigen is a sugar chain antigen of a protozoa, fungus, bacteria, mycoplasma, rickettsia, chlamydia or virus.

3. A method for determining sugar chain antigens in a sample, using the immunochromatographic device according to claim 1 or 2 to determine the sugar chain antigens in the sample by immunochromatography, the method comprising: When the immunochromatographic device has a region impregnated with a solid acidic reagent, the sample is mixed with a nitrous acid solution; when the immunochromatographic device has a region impregnated with a nitrite, the sample is mixed with an acidic solution and added to a sample pad of the immunochromatographic device. In the area impregnated with the solid acidic reagent or the area impregnated with the nitrite, the sugar chain antigen is extracted from the specimen by the action of nitrous acid generated by the reaction of the nitrite with the solid acidic reagent, and in the area impregnated with the neutralizing reagent, the acidic solution containing the sugar chain antigen is neutralized. In the detection area, a complex of antibody-sugar chain antigen-labeled antibody is formed, which promotes the extraction of sugar chain antigen and determines the sugar chain antigen in the specimen by immunochromatography.

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