Immunochromatographic device for extracting and measuring carbohydrate antigens

Abstract

To provide a method which can perform a measurement with sufficient sensitivity by applying nitrite extraction treatment while taking much of time, in immuno-chromatography for extracting and measuring a carbohydrate antigen by extracting a nitrous acid on an immunochromatographic test piece, and an immunochromatographic device.SOLUTION: In an immunochromatographic device having an immunochromatographic test piece including: a sample pad added with a specimen mixed with a nitrous acid or an acid solution; a sign body region including a sign antibody signing an antibody with respect to a carbohydrate antigen; and a detection region in which the antibody with respect to the carbohydrate antigen is phase-solidified, and measuring the carbohydrate antigen by forming a complex of an antibody-carbohydrate antigen-sign antibody in the detection region, being the immunochromatographic test piece having: a region which is immersed with a neutralization reagent at an upstream side of the sign body region; a region immersed with a solid acid reagent for use in the specimen mixed with the nitrous acid at an upstream side of the region which is immersed with the neutralization reagent; a region immersed with the nitrous acid when using the specimen which is mixed with the acid solution, and extracting and measuring the carbohydrate antigen in the specimen; and a specimen dripping port at an upper part of the sample pad of the test piece composed of a vessel for accommodating the test piece, (i) the immunochromatographic device has a wide specimen dripping port for holding an added specimen sample solution, and promoting the extraction of the carbohydrate antigen by the nitrous acid and the solid acid reagent by supplying the specimen sample solution to the region which is immersed with the solid acid reagent or the nitrous acid in a short period of time, and (ii) a clearance is not formed between the dripping port and the sample pad so as to prevent the specimen from running away from the dripping port.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an immunochromatographic device for extracting and measuring sugar chain antigens, which is capable of performing a nitrite extraction treatment of sugar chain antigens on an immunochromatographic test piece. [Background technology]

[0002] Many of the rapid diagnostic agents based on the immunochromatography method are widely used as a means for quickly and easily measuring viral or bacterial infections and determining treatment plans.

[0003] A rapid diagnostic agent based on a general immunochromatography method can perform a rapid and simple measurement by suspending a specimen in a specimen suspension and then supplying the suspension to an immunochromatography test piece.

[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 carbohydrate antigens.

[0005] For example, a method has been reported in which the immunochromatographic test strip is pre-soaked with sodium nitrite and a neutralizing reagent, and the sample is suspended in an acidic solution such as acetic acid and then supplied to the immunochromatographic test strip, thereby performing the nitrite extraction process on the immunochromatographic test strip (Patent Document 1).

[0006] In another method, an acidic reagent and a neutralizing reagent are impregnated in the immunochromatographic test strip beforehand, and the specimen is suspended in nitrite and then supplied to the immunochromatographic test strip.

[0007] In immunochromatography, it is sometimes necessary to control the chromatographic development speed in the immunochromatographic test piece, and various immunochromatographic methods have been reported, such as those that control the speed by device shape, by the bonding position, by the material used (materials such as nonwoven fabric), and by reagents (Patent Documents 2 and 3). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. WO2005 / 121794 [Patent Document 2] JP 2005-331471 A [Patent Document 3] JP 2005-331463 A Summary of the Invention [Problem to be solved by the invention]

[0009] To detect microorganisms belonging to the genus Streptococcus, such as group A beta-hemolytic streptococci (group A beta-hemolytic streptococci) and oral streptococci, carbohydrate antigens are extracted and measured.

[0010] Nitrite is necessary to extract carbohydrate antigens. Nitrite is unstable on its own, so nitrite extraction is performed by mixing a nitrite solution with an acid solution (acetic acid, citric acid, tartaric acid, etc.) just before use to generate nitrous acid, which is then used to extract the antigens.

[0011] When detecting group A β-hemolytic streptococci (group A β-hemolytic streptococci) by immunochromatography, either a nitrite solution or an acid solution can be applied, dried, and incorporated into an immunochromatographic test piece. In this type of reagent form, the operation of mixing the reagent is unnecessary and the operation is simpler than with liquid reagents, but there is a problem that if the sample develops in a short time after mixing the reagent as in the case of normal immunochromatography, the nitrite extraction process is not performed sufficiently, resulting in insufficient sensitivity.

[0012] In the case of a reagent form in which a liquid reagent is mixed and nitrite is extracted, the sample is usually extracted for 1 to 2 minutes. Therefore, in order to achieve the same sensitivity as the liquid form, it was necessary to hold the sample on the immunochromatographic test strip for 1 to 2 minutes upstream of the neutralization.

[0013] To address this issue, one method involves creating and using a pad made of hydrophobic material that contains nitrous acid or acid to ensure sufficient extraction; however, this poses the problem that if the sample development is too slow, the sample will not be able to completely develop the test piece within the five-minute reaction time.

[0014] One way to solve these problems is to use a thicker material for the pad compared to normal immunochromatographic test strips; however, depending on the thickness and softness of the material, gaps can form at the addition port where the sample is added, causing the sample that should be extracted for 1-2 minutes to spill from the addition port into the container and spread along the sides of the test strip, preventing the reaction from proceeding normally (the acidic sample spreads onto the membrane without being neutralized, causing a non-specific reaction).

[0015] The present invention aims to provide a method and an immunochromatographic device that enable measurement with sufficient sensitivity by performing the nitrite extraction treatment for a sufficient period of time in an immunochromatographic method in which glycan antigens are extracted and measured by nitrite extraction on an immunochromatographic test piece. [Means for solving the problem]

[0016] The present inventors have conducted extensive research into a technique for performing nitrite extraction treatment for a sufficient period of time in an immunochromatographic method for extracting and measuring glycan antigens, which enables nitrite extraction treatment of glycan antigens on an immunochromatographic test piece.

[0017] The inventors have found that the above object can be achieved by adjusting the development time by inclining the support portion of the immunochromatographic device, which is made up of protrusions that support the immunochromatographic test piece, and have thus completed the present invention.

[0018] [1] An immunochromatographic test strip that includes a sample pad for adding a specimen mixed with nitrite or an acidic solution, a label region containing a labeled antibody in which an antibody against a glycan antigen is labeled, and a detection region in which the antibody against the glycan antigen is immobilized, and that forms an antibody-glycan antigen-labeled antibody complex in the detection region to measure the glycan antigen, has a region impregnated with a neutralizing reagent upstream of the label region, and further has a region impregnated with a solid acidic reagent upstream of the region impregnated with the neutralizing reagent when a specimen mixed with nitrite is used, and an immunochromatographic device comprising an immunochromatographic test strip for extracting and measuring a sugar chain antigen in a sample, the test strip having a region impregnated with nitrite when a sample mixed with a reactive solution is used, and a container for storing the test strip, the container comprising an upper lid portion and a lower container portion, the upper lid portion having an upper support made of protrusions supporting the upper side of the immunochromatographic test strip, the lower container portion having a lower support made of protrusions supporting the lower side of the immunochromatographic test strip, and a sample drop port above the sample pad of the immunochromatographic test strip, An immunochromatographic device, characterized in that the protruding portions forming the upper support and / or the lower support that support the immunochromatographic test piece are inclined. [2] The immunochromatographic device of [1], characterized in that the protruding portions that form the upper support and lower support that support the immunochromatographic test piece are inclined. [3] An immunochromatographic device according to [1] or [2], characterized in that the protruding portion of the lower support of the immunochromatographic test piece is inclined downward in the downstream direction of the immunochromatographic test piece. [4] An immunochromatographic device according to any one of [1] to [3], characterized in that the inclination of the protruding portion of the upper support of the immunochromatographic test piece rises in the downstream direction of the immunochromatographic test piece. [5] An immunochromatographic device according to any one of [1] to [4], wherein the glycan antigen is a glycan antigen of a protozoan, a fungus, a bacterium, a mycoplasma, a rickettsia, a chlamydia or a virus. [6] A method for measuring a sugar chain antigen in a sample by immunochromatography using the immunochromatographic device according to any one of [1] to [5], mixing the specimen with a nitrite solution when the immunochromatographic device has an area impregnated with a solid acidic reagent, and mixing the specimen with an acidic solution when the immunochromatographic device has an area impregnated with a nitrite salt, and adding the mixture to a sample pad of the immunochromatographic device; In the region impregnated with the solid acidic reagent or the region impregnated with the nitrite, the glycan 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 region impregnated with the neutralizing reagent, the acidic solution containing the glycan antigen is neutralized. A method for measuring glycan antigens in a specimen by immunochromatography, in which an antibody-glycan antigen-labeled antibody complex is formed in the detection area, promoting extraction of the glycan antigen. Effect of the Invention

[0019] When the immunochromatographic device of the present invention is used, the development time of the specimen sample solution on the immunochromatographic test piece after the addition of the sample can be appropriately adjusted, so that the specimen treatment on the immunochromatographic test piece can be sufficiently performed by the specimen treatment reagent contained on the immunochromatographic test piece. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a diagram showing a schematic structure of an immunochromatographic test strip (one-pad test strip) having a solid acidic reagent region and a neutralizing reagent region. [Diagram 2] FIG. 1 is a diagram showing a schematic structure of an immunochromatographic test strip (two-pad test strip) having a solid acidic reagent region and a neutralizing reagent region. [Diagram 3] FIG. 2 is a perspective view showing the appearance of a storage container of the immunochromatographic device. [Figure 4] FIG. 2 is a cross-sectional view of a storage container of an immunochromatographic device. [Diagram 5] FIG. 2 is a perspective view showing a lid portion of a storage container of an immunochromatographic device, an immunochromatographic test piece, and a container portion of the storage container. [Figure 6]1 is a cross-sectional view showing a lid portion of a storage container of an immunochromatographic device, an immunochromatographic test piece, and a container portion of the storage container. [Figure 7] FIG. 2 is a perspective view of a lid portion of a storage container of an immunochromatographic device. [Figure 8] FIG. 2 is a plan view of a lid portion and a container portion of a storage container of an immunochromatographic device. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention will be described in detail below. The present invention relates to an immunochromatographic device that simplifies the nitrite extraction treatment of a sugar chain antigen so that it can be performed on an immunochromatographic test strip, thereby enabling rapid and accurate measurement of the sugar chain antigen, which is a substance to be detected. The immunochromatographic device comprises an immunochromatographic test strip and a container for storing the same, and can be produced by incorporating the immunochromatographic test strip into the immunochromatographic device. The storage container can prevent deterioration due to, for example, ultraviolet light or moisture in the air. In addition, when a contaminating or infectious specimen sample is used, the storage container can prevent the tester performing the assay from becoming contaminated or infected. For example, a resin case of an appropriate size can be used as the storage container, and the immunochromatographic test strip can be stored in the case. In addition, the surface of the test strip on which the antigen or antibody is immobilized may be covered with a resin film or the like (top laminate).

[0022] The immunochromatographic test piece comprises a support having a detection region where an antibody (antibody 1) that captures the substance to be detected (such as an antigen) is immobilized, a label region having a mobile labeled antibody (antibody 2), a sample pad for adding a specimen, an absorption band that absorbs the developed specimen liquid, and a backing sheet for bonding these components together.

[0023] The number of detection regions and the type of labeled antibody contained in the label region are not limited to one, and by using antibodies corresponding to multiple substances to be detected, two or more antigens can be measured using the same test strip.

[0024] The support is a material capable of immobilizing an antibody for capturing a substance to be detected (antigen), and also capable of not hindering the horizontal passage of liquid. Preferably, the support is a porous thin film (membrane) having capillary action, and is a material capable of transporting liquid and components dispersed therein by absorption. The material constituting the support is not particularly limited, and examples thereof include cellulose, nitrocellulose, cellulose acetate, polyvinylidene difluoride (PVDF), glass fiber, nylon, polyketone, and the like. Of these, a thin film or membrane made of nitrocellulose is more preferable. A membrane with an antibody immobilized thereon is called an antibody-immobilized membrane.

[0025] The labeled region is made of a porous substrate containing a labeled antibody, and the substrate may be made of commonly used materials such as glass fiber or nonwoven fabric. The substrate is preferably in the form of a pad having a thickness of about 0.3 mm to 0.6 mm so that a large amount of labeled antibody can be impregnated therein. A porous substrate impregnated with a labeled antibody and dried is also called a dry pad.

[0026] 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 are often used to label the labeled antibodies. When colored particles such as metal colloid particles, colored polystyrene particles, and colored latex particles are used, the labeling reagents aggregate to cause coloration, and this coloration is measured. Particles with immobilized antibodies are called antibody-immobilized particles.

[0027] The detection region refers to a part of the support where an antibody that captures the substance to be detected (antigen) is immobilized. The detection region is provided with at least one region where an antibody for capturing the antigen is immobilized. The detection region only needs to be included in the support, and the antibody only needs to be immobilized on the support.

[0028] The sample pad is a portion for adding a specimen, and is made of a porous material. The sample pad is the most upstream portion of the immunochromatographic test piece. Commonly used materials such as filter paper, glass fiber, and nonwoven fabric can be used for the sample pad. In order to use a large amount of specimen for immunoassay, the sample pad is preferably in the form of a pad having a thickness of about 0.3 mm to 1 mm. The specimen includes a sample prepared using the specimen, such as a sample obtained by suspending the specimen in another solution.

[0029] The absorption band is a member for absorbing components that are supplied to the support and do not participate in the reaction in the detection area. The material for the absorption band can be a filter paper or sponge with high water retention made of a general natural polymer compound or a synthetic polymer compound, but a material with high water absorption is preferable in order to promote the development of the sample.

[0030] The backing sheet is a member for attaching and fixing all of the above-mentioned materials, i.e., the support, sample pad, label region, absorption band, etc., with partial overlap. The backing sheet is not necessarily required if these materials are arranged and fixed at optimal intervals, but it is generally preferable to use it from the viewpoint of convenience in manufacturing and use.

[0031] The immunochromatographic test strip 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 performed correctly. For example, the control display area is downstream of the detection area, and emits a signal by coloring or the like when a specimen sample passes through the detection area and reaches the control display area. In the control display area, a substance that binds to the antibody bound to the labeled carrier may be immobilized, or a reagent such as a pH indicator that changes color when the specimen reaches the control display area may be immobilized. When the antibody bound to the labeled carrier is a mouse monoclonal antibody, an anti-mouse IgG antibody may be used.

[0032] The size of the immunochromatographic test piece is not limited, but is, for example, about several centimeters to a dozen centimeters in length and several mm to several centimeters in width.

[0033] In the test strip of the above form, the specimen passes through a porous flow path formed by a series of connections of the sample pad, the label region, the support, the detection region, the absorption band, etc. Thus, in this form, all of these constitute the specimen migration region. Depending on the material and form of each constituent material, there may be a form in which the specimen does not penetrate inside the material but passes through the interface, but since the specimen migration region defined in this specification does not matter whether it is inside the material or the interface, test strips of this form are also included in the scope of this specification.

[0034] When measuring glycan antigens in a sample using the immunochromatographic test strip of the present invention, it is necessary to first extract the glycan antigens in the sample. The extraction of glycan antigens is performed by treating a sample containing glycan antigens with nitrous acid. Nitrous acid can be generated by mixing a nitrite such as sodium nitrite with an acid, and the sample containing glycan antigens can be treated with the nitrous acid thus generated. The extracted antigens bind to the antibodies immobilized on the immunochromatographic test strip through an antigen-antibody reaction, but if acid remains in the reaction system, the reaction system becomes acidic and the antigen-antibody reaction is inhibited. Therefore, it is necessary to neutralize the acid in the reaction system.

[0035] In the present invention, in a method for measuring a glycan antigen by mixing a nitrite with an acid to generate nitrous acid, extracting a glycan antigen in a sample with the nitrous acid, neutralizing the nitrous acid, and binding the glycan antigen to an antibody immobilized on an immunochromatographic test strip, the following methods can be used to extract and measure the glycan antigen. In any of the methods, the extraction and neutralization of the glycan antigen with nitrous acid is performed on the immunochromatographic test strip. To extract the glycan antigen with nitrous acid on the immunochromatographic test strip, the immunochromatographic test strip may be impregnated with an acidic reagent or nitrite. To neutralize the glycan antigen on the immunochromatographic test strip, the immunochromatographic test strip may be impregnated with a neutralizing reagent.

[0036] (A) A sample is mixed with an acidic solution in advance and added to the sample pad of an immunochromatographic test strip impregnated with nitrite and a neutralizing reagent. When the mixture reaches the area containing nitrite, the nitrite reacts with the acid to generate nitrous acid, and the glycan antigen in the sample is extracted. The glycan antigen extract is neutralized in the area on the immunochromatographic test strip impregnated with the neutralizing reagent, and the glycan antigen binds to the antibody immobilized on the immunochromatographic test strip and can be detected. In this method, examples of the acidic solution used include acetic acid, hydrochloric acid, malonic acid, malic acid, maleic acid, citric acid, and tartaric acid.

[0037] (B) The specimen is mixed with a nitrite solution and added to the sample pad of an immunochromatographic test strip that has been impregnated with an acidic reagent and a neutralizing reagent. When the mixture reaches the area impregnated with the acidic reagent, the nitrite reacts with the acid, generating nitrous acid, which extracts the glycan antigens in the specimen. The glycan antigen extract is neutralized in the area of ​​the immunochromatographic test strip that has been impregnated with the neutralizing reagent, and the glycan antigens bind to the antibodies immobilized on the immunochromatographic test strip, allowing them to be detected.

[0038] In the immunochromatographic test strip of the present invention for carrying out the above method (A) or (B), an acidic reagent or a nitrate and a neutralizing reagent are impregnated upstream of the label region (upstream of the sample flow, on the side where the sample pad is present), i.e., in the sample pad or between the sample pad and the label region. An immunochromatographic test strip in which a nitrite and a neutralizing reagent are impregnated in the sample pad or between the sample pad and the label region can be used in the above method (A). An immunochromatographic test strip in which an acidic reagent and a neutralizing reagent are impregnated in the sample pad or between the sample pad and the label region can be used in the above method (B). A solid acidic reagent is used as the acidic reagent to be impregnated in the immunochromatographic test strip. These methods make it possible to accurately and specifically measure the substance to be detected in the test sample, regardless of the amount of the test sample to be tested.

[0039] The solid acidic reagent or nitrite may be impregnated into a sample pad, or into a pad made of a porous material such as a nonwoven fabric other than the sample pad, and the resulting solid acidic reagent-impregnated porous material or nitrite-impregnated porous material may be placed between the sample pad and the label region, i.e., upstream of the label region. Here, the region impregnated with the solid acidic reagent or nitrite may or may not be in contact with the sample pad or the label region. In the present invention, the region impregnated with the reagent is also referred to as a pad impregnated with the reagent.

[0040] The neutralizing reagent is disposed downstream of the region impregnated with the solid acidic reagent or nitrite. The neutralizing reagent may be impregnated into a sample pad, a support, or a pad made of a porous material such as a nonwoven fabric other than the support, and the resulting neutralizing reagent-impregnated porous material may be disposed between the region impregnated with the solid acidic reagent or nitrite and the label region. That is, the neutralizing reagent is impregnated into a region upstream of the label region, and the solid acidic reagent or nitrite is impregnated into a region upstream of the neutralizing reagent. Here, the region impregnated with the neutralizing reagent may or may not be in contact with the region impregnated with the solid acidic reagent or nitrite or the label region.

[0041] The area impregnated with a solid acidic reagent is called a solid acidic reagent area, the area impregnated with a nitrite is called a nitrite area, and the area impregnated with a neutralizing reagent is called a neutralizing reagent area or a basic reagent area.

[0042] An immunochromatographic test strip having a solid acidic reagent region or nitrite region and a neutralizing reagent region has, from the upstream, a sample pad, a solid acidic reagent region or nitrite region, a neutralizing reagent region, a label region, a detection region, and an absorption band on a support, and the solid acidic reagent region or nitrite region may be on the sample pad. In addition, the sample pad, the solid acidic reagent region or nitrite region, the neutralizing reagent region, the label region, the detection region, and the absorption band may or may not be in contact with each other between adjacent regions. Furthermore, the solid acidic reagent region or nitrite region, the neutralizing reagent region, and the label region do not necessarily need to be impregnated in separate porous materials, and multiple or all of the regions may be impregnated in the same porous material.

[0043] The solid acidic reagent used in the present invention is solid at room temperature and does not volatilize at high temperatures.

[0044] Preferred solid acidic reagents for use in the present invention include malonic acid, malic acid, maleic acid, citric acid, and tartaric acid.

[0045] In addition, as the preferred solid acidic reagent for use in the present invention, an acid with a high valence, such as citric acid, can be used in a smaller amount for extraction. Also, acids with a smaller acid dissociation constant, such as maleic acid and tartaric acid, are more efficient when used with the same valence.

[0046] Moreover, the preferred solid acidic reagent for use in the present invention is a reagent that does not become discolored on the immunochromatographic test piece, specifically a reagent that is white in the dry state or that is unlikely to become discolored by drying heat or oxidation.

[0047] The nitrites used in the present invention include sodium nitrite, potassium nitrite, and the like.

[0048] The amount of the solid acidic reagent or nitrite used in the present invention, i.e., the amount to be impregnated into the immunochromatographic test strip, is not particularly limited, but is usually about 0.01 μg to 1 mg, and preferably about 0.1 μg to 0.1 mg per immunochromatographic test strip. However, it is preferable to select an optimal amount that provides an effect depending on the type of solid acidic reagent or nitrite used, the composition of the specimen suspension, the amount added, etc.

[0049] To impregnate the sample pad or porous material with the solid acidic reagent or nitrite, the solid acidic reagent or nitrite is first dissolved, applied, and then dried.

[0050] The neutralizing reagent used in the present invention is solid at room temperature and does not volatilize at high temperatures.

[0051] Preferred neutralizing reagents for use in the present invention include Tris base (trishydroxylmethylaminomethane), sodium hydroxide, dipotassium hydrogen phosphate, trisodium citrate, and Good's buffer having a buffering capacity in the alkaline range.

[0052] The amount of neutralizing reagent used in the present invention, i.e., the amount to be impregnated into the immunochromatographic test piece, is not particularly limited, but is usually about 0.01 μg to 1 mg per immunochromatographic test piece, and preferably about 0.1 μg to 0.1 mg. However, it is preferable to select an optimal amount that provides an effect depending on the type of neutralizing reagent used, the composition of the specimen suspension, the amount added, etc.

[0053] To impregnate the sample pad or porous material with the neutralizing reagent, the neutralizing reagent may be dissolved once, the solution applied to the sample pad or porous material, and then dried.

[0054] 1 and 2 are diagrams showing a preferred embodiment of a typical immunochromatographic test strip. The immunochromatographic test strip shown in FIG. 1 and FIG. 2 is an immunochromatographic test strip impregnated with a solid acidic reagent and a neutralizing reagent, but a nitrate may be impregnated instead of the solid acidic reagent, in which case the immunochromatographic test strip is impregnated with a nitrate and a neutralizing reagent. A person skilled in the art can appropriately design and manufacture an immunochromatographic test strip impregnated with a nitrate and a neutralizing reagent. The immunochromatographic test strip is not limited to those shown in FIG. 1 and FIG. 2. In FIG. 1 and FIG. 2, 1 indicates a support, 2 indicates a label region, 3 indicates a detection region, 4 indicates a sample pad, 7 indicates an absorption band, and 8 indicates a backing sheet. A top laminate may be attached to the entire test strip.

[0055] 1A and 2A are top views, and 1B and 2B are cross-sectional views. In the example of FIG. 1, a support 1 on which one detection region 3 is formed, an absorption band 7, a label region 2, a sample pad 4, etc. are laminated on a backing sheet 8 made of resin or the like. As shown in FIG. 1, one end of the absorption band 7 and one end of the support 1, the other end of the support 1 and one end of the label region 2, and the other end of the label region 2 and one end of the sample pad 4 are overlapped, respectively, and a solid acidic reagent is impregnated in the upstream part of the sample pad 4, and a neutralizing reagent is impregnated in the downstream part of the sample pad with a short gap. 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 4 serves both as 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 strip, the solid acidic reagent region and the neutralizing reagent region are provided in the form of one porous material (pad), and therefore it is sometimes called a one-pad test strip. In the example of FIG. 2, the solid acidic reagent region 5 and / or the neutralizing reagent region 6 are present upstream of the label region 2, and are superimposed, thereby forming a continuous lateral flow channel. In the test strip shown in FIG. 2, the solid acidic reagent region 5 also serves as a sample pad. That is, the solid acidic reagent region is present on the sample pad. In this test strip, the solid acidic reagent region and the neutralizing reagent region are provided in the form of two separate porous materials (pads), and therefore it is sometimes called a two-pad test strip. There may be an additional sample pad upstream of the solid acidic reagent region. In addition, in the test strip shown in FIG. 2, the solid acidic reagent region 5 and the neutralizing reagent region 6 are impregnated in different porous materials, but the solid acidic reagent region 5 may be provided in the upstream part of the same porous material (pad), and the neutralizing reagent region 6 may be provided in the downstream part, as in the sample pad of the test strip of FIG. 1.

[0056] The immunochromatographic test piece is stored in a storage container and used as an immunochromatographic device. That is, the immunochromatographic test piece assembled in the storage container is called an immunochromatographic device. Generally, the storage container is composed of a lower container part and an upper lid part. Here, the immunochromatographic test piece is stored in the lower container part, and when performing an immunochromatographic test, the upper lid part is placed on the lower container part. The lower container part and the upper lid part are sometimes simply called the lower part and the upper part of the storage container, respectively. When the immunochromatographic test piece is stored in the storage container, a specimen drop port that can supply a specimen sample solution to the sample pad is located at the top of the sample pad, and when a specimen is added to the specimen drop port of the container, the specimen soaks into the sample pad. Here, the drop port is sometimes called an addition port. Also, when the immunochromatographic test piece is stored in the storage container, a determination part is located above the detection area, and the detection area can be observed through a hole in the determination part of the container.

[0057] When the upper lid is placed over the lower container, the immunochromatographic test strip contained in the lower container is supported immovably by being sandwiched between the protrusions on the upper lid and lower container of the containment container. Therefore, the protrusions on the upper lid and lower container of the containment container are called the upper support and the lower support, respectively, which are made of protrusions.

[0058] The upper support of the upper lid part is provided so as to form the edge of the opening on the back side of the side where the sample is dropped of the dropping port, and furthermore, the upper support of the upper lid part is formed with a protrusion having an incline. The upper support of the upper lid part supports the upper side of the sample pad of the immunochromatographic test piece. When the upper support of the upper lid part presses the upper side of the sample pad of the immunochromatographic test piece from above, the protrusion has an incline such that the height of the protrusion becomes smaller toward the downstream part of the immunochromatographic test piece, that is, the protrusion has an incline that rises in the downstream direction of the immunochromatographic test piece. When the upper lid part is placed on the lower container part, the upper side of the sample pad of the immunochromatographic test piece is pressed from above to fix it and prevent it from moving, and furthermore, it is in close contact with the sample pad to prevent the sample solution from leaking out of the sample pad. Furthermore, when the upper support of the upper lid part presses the sample pad, the pressure becomes weaker from upstream to downstream. The support is sometimes called a support made of inclined protrusions.

[0059] On the other hand, the upper support of the lid portion on the top of the storage container of a conventional immunochromatographic device does not have an inclination and has a flat shape.

[0060] The lower support of the lower container part is formed of a protrusion having an incline provided at a portion corresponding to the specimen drop port of the lower container part. Here, the portion corresponding to the specimen drop port of the lower container part refers to the portion that overlaps with the opening of the specimen drop port when the upper lid part is placed 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 when the upper lid part is placed on the lower container part. 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 provided as a protrusion having an incline at a portion corresponding to the specimen drop port. The lower support has an incline so that the height of the protrusion becomes smaller toward the downstream part of the immunochromatographic test piece when the sample pad of the immunochromatographic test piece is pressed, that is, the lower support has an incline that descends in the downstream direction of the immunochromatographic test piece, and the pressure pressing the sample pad becomes weaker from upstream to downstream. The support is sometimes called a support made of inclined protrusions.

[0061] On the other hand, the lower support of the container below the storage container of the conventional immunochromatographic device does not have an inclination and has a flat shape.

[0062] The lid portion having a support made of protrusions that press and support the upper side of the immunochromatographic test piece with an inclination that rises in the downstream direction of the immunochromatographic test piece is called the lid portion with an inclined upper support, the container portion having a support made of protrusions that press and support the lower side of the immunochromatographic test piece with an inclination that falls in the downstream direction of the immunochromatographic test piece is called the container portion with an inclined lower support, the lid portion that does not have a support made of inclined protrusions is called the conventional lid portion, and the container portion that does not have a support made of inclined protrusions is called the conventional container portion. In this case, the combinations of the upper lid portion and the lower container portion of the storage container of the immunochromatographic device of the present invention include the following combinations A to C. A: Lid portion with an inclined upper support and a conventional container portion B: A lid portion with an upper support having a slope, and a container portion with a lower support having a slope C: A conventional lid portion and a container portion having a sloped lower support In the immunochromatographic device of the present invention, the support having inclined projections may be present in both the upper cover part and the lower container part of the storage container of the immunochromatographic device as in B above, or may be present on only one side as in A and C above. Preferably, it is present on both sides as in B above.

[0063] In this way, the support portion of the upper lid portion and / or the lower container portion that contacts the sample pad of the immunochromatographic test piece has an inclined portion consisting of a protrusion whose height decreases toward the downstream side of the immunochromatographic test piece, so that when the sample pad of the immunochromatographic test piece is sandwiched and pressed between the upper support and the lower support, the upper support and the lower support are in close contact with the immunochromatographic test piece, and the pressing force of the downstream portion is lower than that of the upstream portion. Therefore, the liquid sample added by dripping does not leak out of the immunochromatographic test piece, and the liquid flow path on the downstream side of the sample pad of the immunochromatographic test piece is not strongly pressed by the support, so that the liquid sample flows easily. In addition, the upstream portion of the sample pad is located at a higher position than the downstream portion, so that the liquid sample flows easily due to gravity. As a result of the sample flowing easily, the time when the sample starts to develop can be adjusted to an appropriate speed, so that the most sensitive performance can be achieved within the set judgment time.

[0064] The immunochromatographic device of the present invention has a support consisting of the above-mentioned inclined protrusions in a storage container for the immunochromatographic test piece, and therefore can adjust the development of the sample to an appropriate speed in an immunochromatographic method for extracting and measuring a glycan antigen, which allows nitrous acid extraction treatment of the glycan antigen on the immunochromatographic test piece. The immunochromatographic device of the present invention may also have other structural features related to the mechanism for adjusting the development speed of the specimen sample as follows.

[0065] (1) The immunochromatographic device of the present invention may have a wide drop addition port. For example, the drop addition port of a conventional immunochromatographic device (QuickNavi (trademark)-Strep A, Denka Seiken Co., Ltd.) that stores an immunochromatographic test strip with a vertical length of 5 to 9 cm, preferably 7 cm, and a horizontal length of 0.3 to 0.7 cm, preferably 0.5 cm, is approximately 3.5 mm by approximately 5.5 mm (19.25 mm). 2 ) is provided as a drop addition port of a substantially rectangular shape having a size of about 3.5 mm × about 11 mm (38.5 mm 2The size of the immunochromatographic device and the immunochromatographic test piece stored therein are generally similar, so in an immunochromatographic device that is 3 cm wide and 9 cm long, the drop port is approximately rectangular and has a size of 3 to 5 mm x 8 to 15 mm (24 to 75 mm). 2 ) is an immunochromatographic device with a wide addition port, and can achieve the effects of the immunochromatographic device of the present invention. The longer side of the addition port is the side parallel to the longer side of the chromatography test strip, i.e., the side along the flow direction of the specimen sample solution. The length of the longer side of the addition port is sometimes referred to as the addition port length, and the length of the shorter side is sometimes referred to as the addition port width. The above example is for a roughly rectangular addition port, but the shape of the addition port may be triangular or circular. When the size of the addition port of the immunochromatographic device of the present invention is expressed in terms of area, it is 24 to 75 mm 2 , preferably 35 to 75 mm 2 It is.

[0066] For example, the immunochromatographic device of the present invention stores an immunochromatographic test piece having a vertical length of 5 to 9 cm and a horizontal length of 0.3 to 0.7 cm, and the size of the drop application port is approximately rectangular, 3 to 5 mm (drop application port width) x 8 to 15 mm (drop application port length) (24 to 75 mm). 2 The length of the nozzle is preferably 10 mm or more, more preferably 10 mm. A device with a nozzle length of less than 8 mm, for example 5 mm, is a conventional device with a small nozzle.

[0067] In conventional immunochromatography, when specimens are treated with a specimen treatment reagent provided on an immunochromatographic test piece, the area of ​​the specimen solution in contact with the area impregnated with the specimen treatment reagent is small, so the specimen treatment capacity is weak, and since the specimen treatment is performed while gradually developing the immunochromatographic test piece, there is a difference in the concentration of the specimen treatment reagent between the first sample solution developed and the second sample solution developed, so that the specimen treatment cannot be performed reliably.

[0068] Since the drop port of the immunochromatographic device of the present invention is provided widely, a large amount of specimen solution can be supplied at once in a short time to the area on the immunochromatography impregnated with the specimen processing reagent, i.e., the area impregnated with the solid acidic reagent or the area impregnated with nitrous acid. As a result, extraction of sugar chain antigens on the immunochromatographic test piece can be promoted. The specimen solution that can be supplied at once is 10 μL to 200 μL.

[0069] As a result, there is no difference in concentration of the sample processing reagent due to time lag, and sample processing in immunochromatography can be promoted and performed reliably and efficiently.

[0070] (2) The immunochromatographic device of the present invention may be configured so that there is no gap between the addition port and the sample pad located below it, so that the sample does not escape or spill from the addition port.

[0071] The immunochromatographic device of the present invention has a wide drop port, through which a large amount of specimen sample solution can be supplied at one time. The outer edge of the drop port has a certain height (1 to 5 mm), and the supplied specimen sample solution is temporarily stored within the drop port.

[0072] As a result, it takes time for the entire specimen solution to be absorbed by the immunochromatographic test strip, and the sample solution may not be absorbed by the immunochromatographic test strip and may spill out of the immunochromatographic test strip.

[0073] In the immunochromatographic device of the present invention, the sample pad is supported by a backing sheet with an outer size equal to or larger than the outer size of the drop addition port so that the sample does not escape or spill out of the drop addition port.The backing sheet exerts a force pressing the sample pad against the drop addition port, and the device is configured so that there are no gaps between the drop addition port and the sample pad, thereby preventing sample solution from leaking out.

[0074] Furthermore, if there is a difference in thickness of the sample pad, the thinner parts of the sample pad are likely to have gaps between the drop addition port and the pad, which can easily cause sample solution to leak out.

[0075] In the present invention, the base of the device container is designed to be high in the thin parts of the sample bath and low in the thick parts, so that even if there is a difference in the thickness of the pad, there is no gap between the drop port and the pad, preventing sample solution from leaking out.

[0076] As a result, extraction of sugar chain antigens on the immunochromatographic test piece can be made more efficient.

[0077] (3) In addition, a highly hydrophobic material may be selected as the material (nonwoven fabric) used to manufacture the pad impregnated with the solid acidic reagent or specimen processing reagents such as nitrites and neutralizing reagents of the immunochromatographic test strip, thereby making it possible to adjust the speed of the liquid flow, i.e., the time it takes for the specimen sample solution to spread over the immunochromatographic test strip, to be slow.

[0078] In this case, examples of highly hydrophobic materials include polyethylene, polyester, polystyrene, polypropylene, rayon, and nylon.

[0079] The porous material used in the area to be impregnated with the neutralizing reagent should have a basis weight of 10 to 400 g / m. 2 The thickness of the fabric is 0.1 to 2.0 mm, and the density is 1 cm / m 2 Water absorption per unit is 10-100μl / cm 2 The water absorption speed is 1.0 to 5.0 μl / sec, and the water absorption speed is 1 cm / m 2 The wet fragment was placed in contact with the membrane and left for 5 minutes, after which the water retention was 10-100μl / cm 2 and preferably 1 cm / m 2 The piece is placed in contact with the membrane in a wet state and left to stand for 5 minutes. The liquid spreads over an area of ​​20 mm 2The following are examples of porous materials that have three characteristics: high water absorption, high water retention (liquid retention), and low or sustained release of liquid. Specific examples include filter paper made of cellulose cotton fiber and glass filter paper made of glass fiber. Examples of such filter paper include No. 26-3 made by Toyo Roshi Kaisha, Ltd. In addition, the volume of the filter paper used is large, and it can sufficiently hold the acidic solution that arrives from upstream later than the sample. By using such a porous material, it is possible to impregnate the neutralizing reagent in an amount that can sufficiently neutralize the sample in which the glycan antigen is extracted with nitrous acid generated by the reaction between nitrite and an acidic reagent. In addition, since the pad can absorb and hold a large amount of liquid and has sustained release, it has sufficient neutralizing ability even when the liquid containing nitrous acid remaining upstream of the immunochromatographic test piece develops after the judgment time, and it is possible to prevent the acidic solution from reaching the detection area where the antibody is solidified, and as a result, non-specific reactions are suppressed and glycan antigens can be detected without causing non-specific reactions. On the other hand, in the case of glass filter paper with high water absorption, high water retention, and low water release, specifically GS-25 from Toyo Roshi Kaisha, Ltd., the high water absorption allows it to be impregnated with a larger amount of neutralizing reagent, and the high water retention and low water release prevent the acidic solution from reaching the solid phase detection area. As a result, in the case of a negative result, non-specific reactions are suppressed, and in the case of a positive result, the development of a line color after the judgment time can be prevented.

[0080] The weight of the porous material used in the area to be impregnated with the neutralizing reagent is 10 to 400 g / m 2 Here, "weight" refers to the unit area of ​​fabric (1 m 2 The weight per unit area can be changed appropriately by adjusting the amount and composition of the neutralizing reagent impregnated into the pad. 2 If the density is less than this, the porosity will be high, and the material will be easily torn when impregnated with the neutralizing reagent, making it difficult to handle during immunochromatography production. Therefore, the density is preferably 50 g / m 2 or more, and the basis weight is 300g / m 2In the above cases, the porosity is low, and although it depends on the composition of the neutralizing reagent, the specimen does not smoothly penetrate the material and cannot be mixed with the neutralizing reagent, so the specimen may not be able to penetrate the material more than 300 g / m. 2 The most preferable weight is 250 to 270 g / m 2 It is.

[0081] The "thickness" of the porous material used in the region to be impregnated with the neutralizing reagent is preferably 0.1 to 2.0 mm, but the thickness can be changed as appropriate depending on the amount and composition of the neutralizing reagent to be impregnated into the pad. If the thickness is 0.4 mm or less, the pad will be easily torn when impregnated with the neutralizing reagent, making handling during immunochromatography difficult. Therefore, a preferred thickness is 0.4 to 0.8 mm, and a thickness of about 0.6 mm is more preferred, considering the ease of adjusting the amount of neutralizing reagent to be impregnated and ease of handling during immunochromatography.

[0082] "Water absorption" is 1cm / m 2 Water absorption per unit is 10-100μl / cm 2 and a water absorption speed of 1.0 to 5.0 μl / sec are preferred. The water absorption amount and water absorption speed are measured by preparing 200 μl of a colored solution (1% Tween 20 + Red No. 102) in one cell of a 96-well EIA plate, placing a test piece with a material measuring 5 x 60 mm attached to a backing sheet therein, and measuring the bottom end of the test piece with the end immersed in the solution as the time it takes for the solution to reach the top end of the test piece. After the solution has reached the top end, the test piece is immediately removed and the amount of liquid remaining in the cell is measured. The amount of water absorption is calculated by subtracting the amount of liquid remaining in the cell from 200 μl and calculating the amount of water absorption per 1 cm of the material. 2 The liquid volume is divided by the area, and the water absorption speed is calculated by dividing the amount of water absorbed by the time it takes to reach the top. For the porous material used in the area to be impregnated with the neutralizing reagent, a material with a large water absorption capacity and a slow water absorption speed is preferable. Although it depends on the concentration and water content of the solid acidic reagent, a material with a water absorption capacity of 30 μl / cm 2 In the following cases, it is possible that the neutralizing reagent required for the immunochromatographic test strip may not be fully impregnated. Specifically, the water absorption amount is 30 μl / cm 2It is preferable that the absorption speed of the material of the porous material used in the region impregnated with the neutralizing reagent is 1.0 to 5.0 μl / sec or more. The absorption speed of the material of the porous material used in the region impregnated with the neutralizing reagent affects the time for extracting the glycan antigen in the specimen and the time for neutralizing the test solution after extraction. The time for extracting the glycan antigen can be adjusted to some extent by the material or composition of the solid acidic reagent, but this is not complete, so the absorption speed of the material used in the region impregnated with the neutralizing reagent is an important factor for sufficient extraction and neutralization of the glycan antigen. Specifically, the absorption speed is preferably 1.0 to 5.0 μl / sec, and more preferably 2.0 μl / sec or less.

[0083] "Water retention" is the water retention capacity of the porous material used in the area to be impregnated with the neutralizing reagent. 2 The weight is measured before and after placing the piece on the membrane and adding 70 μl of a solution (1% Tween 20 + Red No. 102) and leaving it for 5 minutes. The amount of water retention varies depending on the composition of the solution added (surfactant and protein amount), but when tested with a 1% Tween 20 solution, the water retention is 10 to 100 μl / cm 2 and more preferably the water retention is 15 μl / cm 2 That's all.

[0084] "Releasability" refers to the release rate of the porous material used in the area to be impregnated with the neutralizing reagent. 2 The fragment is placed on the membrane, 70 μl of a solution (1% Tween 20 + Red No. 102) is added, and the area of ​​the liquid that spreads on the membrane after leaving it for 5 minutes is measured. The area varies depending on the composition of the solution added (surfactant and protein amount), but when tested with a 1% Tween 20 solution, the area is 30 mm 2 The more preferable release is 20 mm. 2 The following is the result.

[0085] For example, the porous material used in the area to be impregnated with the neutralizing reagent has a basis weight of 50 to 300 g / m 2 Or 250~270g / m 2 The thickness is 0.4 to 0.8 mm and the thickness is 1 cm / m 2Water absorption per unit is 30-100μl / cm 2 The water absorption speed is 1.0-2.0μl / sec and 1cm / m 2 The wet fragment is placed in contact with the detection area and left for 5 minutes, after which the water retention is 15-100μl / cm 2 and 1 cm / m 2 The piece is placed in contact with the membrane in a wet state and left to stand for 5 minutes. The liquid spreads over an area of ​​20 mm 2 A material having these characteristics has a high ability to "sufficiently neutralize the acidic solution containing the sugar chain antigen due to the high water absorption and water retention of the area impregnated with the neutralizing reagent, and to prevent the remaining acidic solution from reaching the detection area due to the low or sustained release property of the area impregnated with the neutralizing reagent, or to allow the sufficiently neutralized test solution to continue to spread in the detection area", and therefore has the effect of suppressing non-specific reactions compared to other materials.

[0086] The method of using the device of the present invention will be described below. The following method of use is a method in which a specimen is mixed with a nitrite solution and then measured using an immunochromatographic test method in which the specimen is impregnated with a solid acidic reagent and a neutralizing reagent, but a method in which a specimen is mixed with an acidic solution and then measured using an immunochromatographic test method in which the specimen is impregnated with nitrite and a neutralizing reagent can also be performed by referring to the following explanation of the method of use.

[0087] The measurement is started by contacting and mixing a specimen or a sample prepared using a specimen with a nitrite solution, suspending the specimen in the nitrite solution, and adding it to the specimen inlet of the device. In this case, 5 to 100 μL of the specimen is mixed with 0.01 to 2 mL of 0.1 M to 8 M nitrite, and 5 to 200 μL is added to the inlet. Examples of nitrite include sodium nitrite and potassium nitrite.

[0088] The sample containing the glycan antigen, which is the substance to be detected, is applied to the drop port and moves to the sample pad, and is developed by capillary action into the solid acidic reagent area 5 and the neutralizing reagent area 6 on the sample pad 4, and is then developed horizontally to the label area 2, the support 1, and the absorption band 7 in that order. In the solid acidic reagent area 5, the nitrite mixed with the sample reacts with the solid acidic reagent on the solid acidic reagent area 5, generating free nitrous acid, which extracts the glycan antigen from the sample. The extracted glycan antigen is developed and moved to the neutralizing reagent area 6 together with the acidic developing solution, where the pH of the acidic developing solution containing the glycan antigen is neutralized and adjusted to a neutral range. As a result, the glycan antigen is developed and moved further downstream under neutral conditions. In the label area 2, the labeled antibody is released into the liquid along with the development of the specimen and developed to the support 1. When a glycan antigen is present in a specimen, the glycan antigen is specifically captured by the capture antibody in the detection region 3 of the support 1, and the glycan antigen also reacts specifically with the labeled antibody to form a complex. This results in a sandwich of the antibody via the glycan antigen in the detection region 3, and the labeled antibody-glycan antigen complex can be measured in the detection region 3. The detection region can be observed through the judgment section of the immunochromatographic device.

[0089] According to the method using the immunochromatographic test strip of the present invention, the extraction of glycan antigens in a sample is performed on the immunochromatographic test strip, so there is no need to extract glycan antigens in the sample before measurement using the immunochromatographic test strip, and glycan antigens in a sample can be measured in one step.

[0090] In an immunochromatographic test strip incorporating a pad containing a solid acidic reagent or nitrite dried according to the present invention, extraction can be efficiently performed on the reagent.

[0091] The sample drop port may be made to have approximately the same area as the pad containing the solid acidic reagent (solid acidic reagent area). This allows more sample to come into instantaneous contact with the solid acidic reagent. Furthermore, by using a highly hydrophobic nonwoven fabric for the coating / impregnation member, it is possible to prevent the sample from spreading immediately after addition to the downstream pad impregnated with the neutralizing reagent (neutralizing reagent area). This allows the sample to be held in the sample addition area for 1 to 2 minutes, allowing sufficient antigen extraction.

[0092] In addition, in the present invention, when the solid acidic reagent region or the nitrite region and the neutralizing reagent region overlap and come into contact with each other, a sheet that does not allow liquid to pass therethrough is provided between the solid acidic reagent region or the nitrite region and the neutralizing reagent region so as to be sandwiched therebetween. By providing the sheet, the following three effects can be achieved.

[0093] (A) The movement of the reagent between the two adjacent regions during storage of the test strip can be suppressed. As a result, the reaction caused by contact between the solid acidic reagent or nitrite and the neutralizing reagent can be prevented. As a result, the stability of the reagent can be improved. (B) In addition, when a specimen mixed with nitrite or an acidic reagent is added, the specimen reaches the solid acidic reagent region or the nitrite region and then immediately moves to the neutralizing reagent region, which can prevent insufficient extraction of glycan antigens. In other words, the speed at which the liquid containing the specimen moves from the solid acidic reagent region or the nitrite region to the neutralizing reagent region is reduced, and the time that the liquid containing the specimen remains in the solid acidic reagent region or the nitrite region is extended, resulting in sufficient extraction of glycan antigens and increased measurement sensitivity. (C) Furthermore, when a specimen mixed with nitrite or an acidic reagent is added, the specimen reaches the solid acidic reagent region or the nitrite region and then immediately moves to the neutralizing reagent region, and the liquid containing the specimen flows back into the solid acidic reagent region or the nitrite region, reducing the activity of the solid acidic reagent or nitrite and decreasing the extraction efficiency. In other words, the liquid containing the specimen that has once reached the neutralizing reagent region is prevented from flowing back into the solid acidic reagent region or the nitrite region, preventing a decrease in the activity of the solid acidic reagent or nitrite, allowing the extraction process of sugar chain antigens by nitrite to proceed efficiently and increasing the measurement sensitivity.

[0094] The material of the sheet provided between the solid acidic reagent region or the nitrite region and the neutralizing reagent region is not limited as long as it is a material that does not allow liquid to pass through, and for example, a resin sheet such as a PET (polyethylene terephthalate) sheet or a polyethylene sheet can be used. A resin sheet is also called a resin film.

[0095] The sheet may be provided so that the solid acidic reagent region or the nitrite region and the neutralizing reagent region are partially in contact with each other and overlap each other. In this case, it is preferable that the length of the overlapping portion between the solid acidic reagent region or the nitrite region and the neutralizing reagent region is as short as possible, for example, 5 mm or less, preferably 3 mm or less, and more preferably 2 mm or less.

[0096] Alternatively, the sheet may be provided so that the solid acidic reagent area or nitrite area does not come into contact with the neutralizing reagent area, and a pad impregnated with the acidic reagent or nitrite may be shaped to cover the PET sheet. In this case, the neutralizing area is completely covered by the sheet, and the solid acidic reagent area or nitrite area is provided on the sheet. In this case, the liquid in the solid acidic reagent area or nitrite area does not move directly to the neutralizing area, but flows over the sheet and then reaches the neutralizing reagent area.

[0097] The sheet may be provided, for example, only between the solid acidic reagent region or the nitrite region and the neutralizing reagent region. On the other hand, when a resin sheet is attached as a top laminate sheet so as to cover the upper side of the immunochromatographic test piece, the top laminate sheet may be attached so as to cover the areas other than the solid acidic reagent region or the nitrite region, or the neutralizing reagent area other than the sample pad when the solid acidic reagent region or the nitrite region also serves as a sample pad, the label region, the support, the detection region, and the absorption band, and the solid acidic reagent region or the nitrite region may be attached to the top of the top laminate sheet attached to the top of the neutralizing reagent portion.

[0098] In the method of the present invention, the biological sample to be used as a specimen is not particularly limited, but examples thereof include body fluids such as serum, plasma, blood, urine, stool, saliva, tissue fluid, cerebrospinal fluid, and swabs, and dilutions thereof.

[0099] In the method using the immunochromatographic device of the present invention, the substance to be measured is a glycan antigen that can be measured by immunoassay, that is, an assay using an antigen-antibody reaction. Examples of antigens include polysaccharides, which are glycan antigens present in bacterial cell walls extracted by nitrous acid extraction treatment. Protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, viruses, etc. that contain these substances can also be measured. The method using the immunochromatographic test strip of the present invention can confirm whether or not a glycan antigen derived from a protozoa, fungus, bacteria, mycoplasma, rickettsia, chlamydia, virus, etc. is contained in a biological sample of a subject, and if a glycan antigen is contained, it can be determined that the subject is suffering from an infectious disease caused by a protozoa, fungus, bacteria, mycoplasma, rickettsia, chlamydia, virus, etc. For example, the presence or absence of infection with group A beta-hemolytic streptococcus (Streptococcus pyogenes), Escherichia coli, Legionella, Campylobacter, etc. can be detected. EXAMPLES

[0100] The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples. In the following examples, % indicates w / v % unless otherwise specified.

[0101] Example 1: Examination of the size of the drip nozzle 1. Immobilization of anti-Streptococcus pyogenes (group A beta-hemolytic streptococcus) antibodies onto nitrocellulose membrane (support) A solution of anti-Streptococcus pyogenes antibody diluted with purified water to 1.0 mg / mL and anti-rabbit IgG antibody were prepared, and the anti-Streptococcus pyogenes antibody was applied in a line shape to the sample pad side of a nitrocellulose membrane backed with a PET (polyethylene terephthalate) film, and the anti-rabbit IgG antibody was applied in a line shape to the absorption band side. The nitrocellulose membrane was then dried at 45°C for 30 minutes to obtain an anti-Streptococcus pyogenes antibody-immobilized membrane. This membrane is referred to as the "antibody-immobilized membrane" in this example.

[0102] 2. Immobilization of anti-Streptococcus pyogenes antibodies onto colored polystyrene particles Anti-Streptococcus pyogenes antibody was diluted with purified water to 1.0 mg / mL, and colored polystyrene particles were added to this to make 0.1%. After stirring, carbodiimide was added to make 1% and further stirred. The supernatant was removed by centrifugation, and the mixture was resuspended in 50 mM Tris (pH 9.0) and 3% BSA to obtain a 0.04% suspension of anti-Streptococcus pyogenes antibody-bound colored polystyrene particles. These particles are referred to as "antibody-immobilized particles" in this example.

[0103] 3. Coating and drying of anti-Streptococcus pyogenes antibody-conjugated colored polystyrene particles A predetermined amount of the antibody-immobilized particle suspension prepared in 2 was applied to a nonwoven fabric and dried for 30 minutes at 45° C. The resulting nonwoven fabric is referred to as a “dry pad” in this example.

[0104] 4. Preparation of neutralizing reagent (basic reagent) pad As a neutralizing reagent (basic reagent), 3M Trizma (trademark) Base, 1.5% TritonX100 was applied to 30 μL / cm of filter paper (Toyo Roshi; No. 26-3).

[0105] 5. Preparation of Solid Acid Reagent Pads As a solid acidic reagent, 1.0 M tartaric acid, 0.5% TritonX100 was applied to a nonwoven fabric (Unitika; Elves (registered trademark)) at 13 μL / cm. Immediately after application, the fabric was dried at 45° C. for 1 hour to obtain a nonwoven fabric impregnated with a solid acidic reagent.

[0106] 6. Preparation of immunochromatographic test strips for detecting Streptococcus pyogenes The antibody-immobilized membrane prepared in 1, the dry pad prepared in 3, the neutralizing reagent (basic reagent) pad prepared in 4, and the solid acidic reagent pad prepared in 5 were laminated with other members (backing sheet, absorption band) and cut to a width of 5 mm to prepare a Streptococcus pyogenes test strip. In this embodiment, the test strip using the solid acidic reagent and neutralizing reagent (basic reagent)-impregnated filter paper as the sample pad is called the "immunochromatographic test strip of the present invention." The immunochromatographic test strip is provided with, from upstream along the flow of the specimen, a solid acidic reagent-impregnated nonwoven fabric, a neutralizing reagent (basic reagent)-impregnated nonwoven fabric, a dry pad (labeled body region), an antibody-immobilized membrane (detection region), and an absorption band.

[0107] 7. Immunochromatography Device The immunochromatographic test piece of the present invention was incorporated into a storage container ((1) small drop port) with a drop port length (length of the long side) of 5 mm to produce the immunochromatographic device 1, and was then incorporated into a storage container ((2) large drop port) with a drop port length (length of the long side) of 10 mm to produce the immunochromatographic device (2). The storage containers of the immunochromatographic devices (1) and (2) are conventionally used storage containers, and neither the lower container part nor the upper lid part has a support consisting of protrusions with an inclined shape that the storage container of the lower immunochromatographic device has. The lower container part of the conventionally used storage container is referred to as the conventional container part, and the upper lid part of the conventionally used storage container is referred to as the conventional lid part.

[0108] In addition, the lower container part of the containment vessel, which has a support made of protrusions that press and support the lower side of the immunochromatographic test piece with a slope that slopes down in the downstream direction of the immunochromatographic test piece (referred to as the container part with a sloped lower support) and the upper lid part of the containment vessel, which has a support made of protrusions that press and support the upper side of the immunochromatographic test piece with a slope that slopes up in the downstream direction of the immunochromatographic test piece (lid part with a sloped upper support), were combined with the above-mentioned conventional container part and conventional lid part as shown below to prepare immunochromatographic devices (3) to (5) incorporating the immunochromatographic test piece, and the following tests were performed. The length of the drop addition port of the upper lid part of immunochromatographic devices (3) to (5) was 10 mm. (3) A lid portion having an inclined upper support and a conventional container portion (4) A lid portion having an upper support with a slope, and a container portion having a lower support with a slope (5) Conventional lid portion and container portion with inclined lower support

[0109] 8. Sample Streptococcus pyogenes was cultured and the culture solution was diluted with physiological saline to a bacterial count of 1.0 × 10 7 CFU / mL was adjusted. In addition, physiological saline was used as a negative sample.

[0110] 9. Measurement 20 μL of the specimen was suspended in 180 μL of sodium nitrite solution (2.0 M NaNO3, 1% Tween20), of which 75 μL was added to the drop port of the immunochromatographic device of the present invention. In addition, as in the conventional method, the specimen was suspended in a nitrite extract made by mixing sodium nitrite and hydrochloric acid, and then 50 μL of the specimen suspension neutralized with a Tris solution was added to the drop port of the conventional immunochromatographic device (in which neither the acidic reagent nor the neutralizing reagent was immobilized). After 10 minutes, the presence or absence and the degree of deposition of colored polystyrene particles on the predetermined position where the anti-Streptococcus pyogenes antibody was immobilized were scored with a code of +++, ++, + for strong signals, ± for cases where it was difficult to judge, and - for cases where no signal was observed.

[0111] 10.Results JPEG0007634062000001.jpg107162

[0112] The immunochromatographic devices (2), (3), (4), and (5) with larger drop ports were able to start development sooner and therefore give more rapid positive results compared to the immunochromatographic device (1) with a smaller drop port.

[0113] Furthermore, the immunochromatographic devices (3), (4), and (5) in which the upper lid portion and / or the lower container portion had a support having inclined protrusions gave stronger signals after 10 minutes than the immunochromatographic devices (1) and (2) that did not have a support having inclined protrusions. In particular, the immunochromatographic device (4) in which the upper lid portion and the lower container portion had a support having inclined protrusions gave stronger signals after 10 minutes than the immunochromatographic devices (3) and (5) in which only one of the upper lid portion or the lower container portion had a support having inclined protrusions. [Explanation of symbols]

[0114] 1. Support (including detection area) 2 Label region 3. Detection Area 4 Sample Pad 5. Solid Acid Reagent Area 6 Neutralization Reagent Area 7 Absorption Bands 8 Backing sheet 9 Immunochromatographic test strips 10 Containment Vessel 11. Lid of containment vessel 12. Containment vessel section 13 Droplet port of the device 14 Device Judgment Section 15 Protruding part of the containment vessel lid 16 Slope of containment vessel [Industrial Applicability]

[0115] The immunochromatographic device of the present invention can be used to detect infection with group A β-hemolytic streptococcus with high sensitivity.

Claims

1. The immunochromatographic test strip includes a sample pad for adding a specimen mixed with nitrite or an acidic solution, a label region containing a labeled antibody obtained by labeling an antibody against a glycan antigen, and a detection region in which the antibody against the glycan antigen is immobilized, and in which an antibody-glycan antigen-labeled antibody complex is formed in the detection region to measure the glycan antigen. The test strip has a region impregnated with a neutralizing reagent upstream of the label region, and further has a region impregnated with a solid acidic reagent upstream of the region impregnated with the neutralizing reagent when a specimen mixed with nitrite is used, and the acid an immunochromatographic device comprising an immunochromatographic test strip for extracting and measuring a sugar chain antigen in a sample, the test strip having a region impregnated with nitrite when a sample mixed with a reactive solution is used, and a container for storing the test strip, the container comprising an upper lid portion and a lower container portion, the upper lid portion having an upper support made of protrusions supporting the upper side of the immunochromatographic test strip, the lower container portion having a lower support made of protrusions supporting the lower side of the immunochromatographic test strip, and a sample drop port above the sample pad of the immunochromatographic test strip, When the upper lid is placed over the lower container, the immunochromatographic test strip contained in the lower container is supported by the protrusions on the upper lid and the lower container of the storage container so that it does not move. The protrusion portion that forms the upper support that supports the immunochromatographic test piece is inclined, and the upper support is provided so as to form the edge of an opening on the back side of the side of the drop port where the sample is dropped, and is formed with a protrusion having an incline, and when the upper side of the sample pad of the immunochromatographic test piece is pressed from above, the protrusion has an incline such that the height of the protrusion becomes smaller toward the downstream part of the immunochromatographic test piece, i.e., the incline rises in the downstream direction of the immunochromatographic test piece, and when the sample pad is pressed, the pressure becomes weaker from upstream to downstream, An immunochromatographic device characterized in that the lower support is formed with an inclined protrusion provided at a portion corresponding to the sample addition port of the lower container part, wherein the portion corresponding to the sample addition port of the lower container part refers to the portion which overlaps with the opening of the sample addition port when the upper lid part is placed over the lower container part.

2. The immunochromatographic device according to claim 1, 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.

3. A method for measuring a sugar chain antigen in a sample by immunochromatography using the immunochromatographic device according to claim 1 or 2, comprising: mixing the specimen with a nitrite solution when the immunochromatographic device has an area impregnated with a solid acidic reagent, and mixing the specimen with an acidic solution when the immunochromatographic device has an area impregnated with a nitrite salt, and adding the mixture to a sample pad of the immunochromatographic device; In the region impregnated with the solid acidic reagent or the region impregnated with the nitrite, the glycan 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 region impregnated with the neutralizing reagent, the acidic solution containing the glycan antigen is neutralized. A method for measuring glycan antigens in a specimen by immunochromatography, in which an antibody-glycan antigen-labeled antibody complex is formed in the detection area, promoting extraction of the glycan antigen.

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