Immunochromatographic test kit for extracting and measuring sugar chain antigens and capable of controlling analyte development

Abstract

To provide an immunochromatographic test kit for controlling the development of analytes on an immunochromatographic test piece and for appropriately controlling processing using acidic reagents, nitrite, and neutralizing reagents, and an immunochromatography method using the kit.SOLUTION: An immunochromatographic test kit includes an immunochromatographic test piece that includes a sample pad to which an analyte mixed with (i) a nitrite solution or an acidic solution and (ii) nitrite or an acidic solution is added, a marker region that includes a marker antibody marking an antibody for sugar chain antigens, and a detection region in which the antibody for sugar chain antigens is immobilized, the immunochromatographic test piece forming an antibody-sugar chain antigen-marker antibody complex in the detection region and measuring the sugar chain antigens, the immunochromatographic test piece being for extracting and measuring sugar chain antigens in the analyte and having a region impregnated with a neutralizing reagent, upstream of the marker region, and upstream of the region impregnated with the neutralizing reagent, a region impregnated with a solid acidic reagent when an analyte mixed with nitrite is used, or a region impregnated with nitrite when an analyte mixed with acidic solution is used. The nitrite or acidic solution includes at least one selected from the group consisting of polyoxyethylene octyl phenyl ether, a quaternary ammonium compound, phosphoric acid, sodium hydroxide, N-acetyl-L-cysteine, NaCl, PVA, BSA, and NaBr.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an immunochromatographic test kit for extracting and measuring a sugar chain antigen, which enables nitrite extraction treatment of the sugar chain antigen on an immunochromatographic test strip.

Background Art

[0002] Many rapid diagnostic agents based on the principle of immunochromatography are widely used as a means to quickly and easily measure viral or bacterial infections and determine treatment strategies.

[0003] A general rapid diagnostic agent based on the principle of immunochromatography can be quickly and easily measured by suspending a sample in a sample suspension and then supplying the suspension to an immunochromatographic test strip.

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

[0005] For example, a method has been reported in which nitrite extraction treatment is performed on an immunochromatographic test strip only by an operation of suspending a sample in an acidic solution such as acetic acid and supplying it to the immunochromatographic test strip by previously including sodium nitrite and a neutralizing reagent in the immunochromatographic test strip (Patent Document 1).

[0006] There is also a method in which an acidic reagent and a neutralizing reagent are previously included in an immunochromatographic test strip, and a sample is suspended in a nitrite and supplied to the immunochromatographic test strip.

[0007] Furthermore, a method for efficiently performing nitrite extraction treatment of a sugar chain antigen on an immunochromatographic test strip has been reported (Patent Documents 2 to 4).

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

[0009] Many of the currently commercially available antigen detection reagents for group A beta-hemolytic streptococcus (Strep A) based on the principle of immunochromatography employ a technique in which acidic nitrous acid generated by mixing a nitrite solution (R1) and an acid solution (R2) is neutralized and reacted with a dry neutralizing agent (R3) on a detection reagent (test strip) for immunochromatography. However, a one-step technique is also known in which R1 or R2 is applied to a pad to omit the preparation and extraction steps of R1 and R2. In a one-step Strep A antigen detection reagent, since the extraction step is performed on the test device, it is essential to ensure a certain extraction time after sample dropping. However, when using a highly viscous specimen, a phenomenon was observed in which the sample solution remained on the device for too long. As a result, there were problems such as (1) the sample not developing, (2) the sample developing but the sensitivity at 5 minutes after the start of measurement decreasing due to delayed development, and (3) the sample developing but a non-specific reaction occurring.

[0010] An object of the present invention is to provide an immunochromatography test kit for controlling the development of a specimen on an immunochromatography test strip and appropriately controlling the treatment with an acidic reagent, nitrite, and a neutralizing reagent, and an immunochromatography method using the kit. [Means for Solving the Problems]

[0011] The present inventors have intensively studied a method for controlling the development of a specimen on an immunochromatography test strip and appropriately controlling the treatment with an acidic reagent, nitrite, and a neutralizing reagent.

[0012] As a result, by adding compounds such as surfactants, salts, chemically synthesized substances (PVA), and proteinaceous substances (BSA) to the specimen suspension in advance, the start time of development is shortened, line determination becomes possible from an early time zone, and non-specific reactions are also reduced, leading to the completion of the present invention.

[0013] That is, the present invention is as follows. [1] (i) A nitrite solution or an acidic solution, and (ii) An immunochromatographic test strip for extracting and measuring a sugar chain antigen in a specimen, which includes a sample pad to which a specimen mixed with a nitrite or an acidic solution is added, a labeled body region containing a labeled antibody labeled with an antibody against a sugar chain antigen, and a detection region in which an antibody against the sugar chain antigen is immobilized. In the detection region, a complex of antibody-sugar chain antigen-labeled antibody is formed to measure the sugar chain antigen. The test strip has a region impregnated with a neutralizing reagent upstream of the labeled body region, and further has a region impregnated with a solid acidic reagent when using a specimen mixed with nitrite upstream of the region impregnated with the neutralizing reagent, and has a region impregnated with nitrite when using a specimen mixed with an acidic solution. An immunochromatographic test kit comprising the immunochromatographic test strip, wherein the nitrite or the acidic solution contains one or more selected from the group consisting of polyoxyethylene octyl phenyl ether, quaternary ammonium compounds, phosphoric acid, sodium hydroxide, N-acetyl-L-cysteine, NaCl, PVA, BSA, and NaBr. [2] The immunochromatographic test kit according to [1], wherein a region impregnated with a solid acidic reagent or nitrite exists on the sample pad. [3] The immunochromatographic test kit according to [1] or [2], wherein the solid acidic reagent is selected from the group consisting of malonic acid, malic acid, maleic acid, citric acid, and tartaric acid. [4] The immunochromatographic test kit according to any one of [1] to [3], wherein the neutralizing reagent is tris(hydroxymethyl)aminomethane or sodium hydroxide. [5] The immunochromatographic test kit according to any one of [1] to [4], wherein the sugar chain antigen is a sugar chain antigen of protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia or virus. [6] The immunochromatographic test kit according to any one of [1] to [5], wherein the polyoxyethylene octylphenyl ether is Triton X and the quaternary ammonium compound is benzalkonium chloride or benzethonium chloride.

[0014] [7] A method for measuring a sugar chain antigen in a specimen by an immunochromatographic method using the immunochromatographic test kit according to any one of [1] to [6], when the immunochromatographic test piece has a region impregnated with a solid acidic reagent, mixing the specimen with a nitrous acid solution, and when the immunochromatographic test piece has a region impregnated with nitrite, mixing the specimen with an acidic solution, and adding the mixture to the sample pad of the immunochromatographic test piece, in the region impregnated with the solid acidic reagent or the region impregnated with nitrite, the sugar chain antigen is extracted from the specimen by the action of nitrous acid generated by the reaction between the nitrite and the solid acidic reagent, and in the region impregnated with the neutralizing reagent, the acidic solution containing the sugar chain antigen is neutralized, In the detection region, a method for measuring a sugar chain antigen in a specimen by controlling the speed and direction of development of the specimen on an immunochromatographic test piece by an immunochromatographic method, in which a complex of an antibody-sugar chain antigen-labeled antibody is formed, and controlling the treatment with an acidic reagent, nitrite and a neutralizing reagent.

Effect of the Invention

[0015] By adding any one of a surfactant, a salt, a proteinaceous substance such as N-acetyl-L-cysteine and BSA, and other chemical synthetic agents to a nitrite solution (R1) or an acidic solution, even when using a highly viscous throat swab specimen, it is possible to provide a device that prevents insufficient sensitivity and non-specific reactions due to poor development or delayed development.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Embodiments for Carrying Out the Invention

[0017] Hereinafter, the present invention will be described in detail. The present invention relates to an immunochromatographic test kit that simplifies the nitrite extraction treatment of a carbohydrate antigen on an immunochromatographic test strip and enables rapid and accurate measurement of the carbohydrate antigen as the substance to be detected.

[0018] The immunochromatographic test kit includes an immunochromatographic test strip impregnated with nitrite or an acidic reagent and a nitrous acid solution or an acidic solution.

[0019] The immunochromatographic test strip 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 labeled body region having a movable labeled antibody (antibody 2), a sample pad for adding a specimen, an absorption band for absorbing the developed test liquid, a backing sheet for bonding these members together, and the like.

[0020] The immunochromatographic test strip of the present invention may be stored in a storage container, and the storage container can prevent deterioration caused by, for example, ultraviolet rays or moisture in the air. Also, when using a specimen sample with contamination or infectivity, the storage container can prevent the tester performing the assay from being contaminated or infected. For example, a resin case of an appropriate size can be used as the storage container, and the device of the present invention can be stored in the case. Also, the surface of the test strip on which the antigen or antibody is immobilized may be covered with a resin film or the like (PET sheet). The storage container and the test strip stored therein may be collectively referred to as an immunochromatographic device.

[0021] Note that the number of detection regions and the types of labeled antibodies contained in the labeled body region are not limited to one, and by using antibodies corresponding to a plurality of substances to be detected, two or more antigens can be measured using the same test strip.

[0022] The support is a material having the performance of immobilizing an antibody for capturing the substance to be detected (antigen), and also having the performance of not preventing the liquid from passing horizontally. Preferably, it is a porous thin film (membrane) having capillary action, and is a material capable of transporting the liquid and the components dispersed therein by absorption. The material forming the support is not particularly limited, and examples include cellulose, nitrocellulose, cellulose acetate, polyvinylidene difluoride (PVDF), glass fiber, nylon, polyketone, and the like. Among these, those using nitrocellulose as a thin film or membrane are more preferable. The membrane on which the antibody is immobilized is called an antibody-immobilized membrane.

[0023] The labeling body region is composed of a porous substrate containing a labeled antibody, and materials such as commonly used glass fibers or non-woven fabrics can be used as the substrate material. The substrate is preferably in the form of a pad with a thickness of about 0.3 mm to 0.6 mm in order to impregnate a large amount of labeled antibody. The porous substrate impregnated with the labeled antibody and dried is also called a dry pad.

[0024] For the labeling of labeled antibodies, enzymes such as alkaline phosphatase or horseradish peroxidase, metal colloids such as gold colloids, silica particles, cellulose particles, magnetic particles, fluorescent particles, colored polystyrene particles, and colored latex particles are often used. When using colored particles such as metal colloid particles, colored polystyrene particles, or colored latex particles, since these labeling reagents aggregate to cause coloring, this coloring is measured. The particles immobilized with antibodies are called antibody-immobilized particles.

[0025] The detection region refers to a partial region of a support on which 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 can be immobilized on the support.

[0026] The sample pad is a site for adding a specimen and is a porous material. The sample pad is the site at the most upstream of the immunochromatographic test strip. Materials such as commonly used filter paper, glass fibers, or non-woven fabrics can be used. In order to use a large amount of specimen for immunoassay, it is preferably in the form of a pad with a thickness of about 0.3 mm to 1 mm. The specimen includes samples obtained by suspending the specimen in other solutions, as well as samples prepared using the specimen.

[0027] The absorption zone is a member for absorbing components that are supplied to the support and did not participate in the reaction in the detection region. As the material, filter paper, sponge, etc. with high water retention composed of general natural polymer compounds, synthetic polymer compounds, etc. can be used, but those with high water absorption are preferred for promoting the development of the specimen.

[0028] The backing sheet is a member for attaching and fixing all of the aforementioned materials, namely the support, sample pad, labeling region, absorption band, etc., with partial overlap. The backing sheet is not necessarily required as long as these materials are arranged and fixed at an optimal interval, but it is generally preferred for manufacturing or convenience in use.

[0029] The immunochromatographic test strip of the present invention may further have a control display region (member). The control display region is a site indicating that the test has been accurately performed. For example, the control display region is present downstream of the detection region, and when the specimen sample passes through the detection region and reaches the control display region, it emits a signal by coloring or the like. Substances that bind to the antibody to which the labeling carrier is bound may be immobilized in the control display region, or reagents such as pH indicators that change color when the specimen arrives may be immobilized. When the antibody to which the labeling carrier is bound is a mouse monoclonal antibody, an anti-mouse IgG antibody may be used.

[0030] The size of the immunochromatographic test strip is not limited. For example, the longitudinal length is several cm to a dozen or so cm, and the lateral length is about several mm to several cm.

[0031] In the test strip of the above form, the specimen passes through a porous flow path formed by a series of connections such as the sample pad, labeling region, support, detection region, absorption band, etc. Therefore, in this form, all of these become the specimen movement 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. However, since the specimen movement region defined in this specification does not matter whether it is inside or at the interface of the material, the test strip of this form is also included in the scope of this specification.

[0032] When measuring a sugar chain antigen in a specimen using the immunochromatographic test strip of the present invention, it is necessary to first 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 generated by mixing a nitrite such as sodium nitrite with an acid, and the specimen containing the sugar chain antigen may be treated with the nitrous acid thus generated. The extracted antigen is bound to the antibody immobilized on the immunochromatographic test strip by an antigen-antibody reaction. At this time, 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.

[0033] In the present invention, in a method of mixing a nitrite and an acid to generate nitrous acid, extracting a sugar chain antigen in a specimen with the nitrous acid, neutralizing the acid, and then binding the sugar chain antigen to an antibody immobilized on an immunochromatographic test strip to measure the sugar chain antigen, the following methods can be mentioned as methods for extracting and measuring the sugar chain antigen. In any method, the extraction and neutralization of the sugar chain antigen with nitrous acid are performed on the immunochromatographic test strip. In order to perform the extraction of the sugar chain antigen with nitrous acid on the immunochromatographic test strip, an acidic reagent or a nitrite may be included on the immunochromatographic test strip. In order to perform the neutralization on the immunochromatographic test strip, the immunochromatographic test strip may be impregnated with a neutralizing reagent.

[0034] (A) Mix the specimen with an acidic solution in advance and add it to the sample pad of an immunochromatographic test strip impregnated with a nitrite and a neutralizing reagent. When the mixed solution reaches the region impregnated with the nitrite, the nitrite reacts with the acid to generate nitrous acid, and the sugar chain antigen in the specimen is extracted. The extract of the sugar chain antigen is neutralized in the region impregnated with the neutralizing reagent on the immunochromatographic test strip, and the sugar chain antigen binds to the antibody immobilized on the immunochromatographic test strip and can be detected. In this method, examples of the acidic solution to be used include acetic acid, hydrochloric acid, malonic acid, malic acid, maleic acid, citric acid, tartaric acid, and the like.

[0035] (B) The sample is mixed with a nitrite solution in advance and added to the sample pad of an immunochromatographic test strip impregnated with an acidic reagent and a neutralizing reagent. When the mixed solution reaches the region impregnated with the acidic reagent, nitrite reacts with the acid to generate nitrous acid, and the sugar chain antigen in the sample is extracted. The extract of the sugar chain antigen is neutralized in the region impregnated with the neutralizing reagent on the immunochromatographic test strip, and the sugar chain antigen binds to the antibody immobilized on the immunochromatographic test strip and can be detected.

[0036] In the immunochromatographic test strip of the present invention for performing the above method (A) or (B), upstream of the labeled region (upstream of the flow of the sample and on the side where the sample pad is present), that is, an immunochromatographic test strip impregnated with nitrite in the sample pad or between the sample pad and the labeled region can be used in the method (A) above. Also, an immunochromatographic test strip impregnated with an acidic reagent in the sample pad or between the sample pad and the labeled region can be used in the method (B) above. Note that a solid acidic reagent is used as the acidic reagent. By these methods, it becomes possible to accurately and specifically measure the analyte in the test sample regardless of the amount of the test sample used for the test.

[0037] The region impregnated with the solid acidic reagent is called the solid acidic reagent region, the region impregnated with the acidic reagent is called the acidic reagent region, and the region impregnated with the neutralizing reagent is called the neutralizing reagent region.

[0038] The solid acidic reagent or nitrite may be impregnated into the sample pad, or impregnated into a pad made of a porous material such as a non-woven fabric separate from the sample pad, and the obtained solid acidic reagent-impregnated porous material or nitrite-impregnated porous material may be disposed between the sample pad and the labeled region, that is, on the upstream side of the labeled region. Here, the region impregnated with the solid acidic reagent or nitrite and the sample pad or the labeled region may or may not be in contact. In the present invention, the region impregnated with the reagent is also referred to as the reagent-impregnated pad.

[0039] The neutralizing reagent is disposed downstream of the region impregnated with the solid acidic reagent or nitrite. The neutralizing reagent may be impregnated in a support, or impregnated in a pad made of a porous material such as a non-woven fabric separate from the support, and the obtained neutralizing reagent-impregnated porous material may be disposed between the region impregnated with the solid acidic reagent or nitrite and the labeling region. That is, it has a region impregnated with the neutralizing reagent upstream of the labeling region, and further has a region impregnated with the solid acidic reagent or nitrite upstream of the region impregnated with the neutralizing reagent.

[0040] Here, the region impregnated with the neutralizing reagent and the region impregnated with the solid acidic reagent or nitrite or the labeling region may or may not be in contact.

[0041] Specific examples of the material of the porous material used in the region impregnated with the nitrite reagent, acidic reagent or neutralizing reagent include filter paper made of cotton fibers of cellulose and glass filter paper made of glass fibers.

[0042] The immunochromatographic test strip having a solid acidic reagent region or nitrite region and a neutralizing reagent region has, on a support, a sample pad, a solid acidic reagent region or nitrite region, a neutralizing reagent region, a labeling region, a detection region and an absorption zone from upstream, and the solid acidic reagent region or nitrite region may be on the sample pad. Also, the sample pad, the solid acidic reagent region or nitrite region, the neutralizing reagent region, the labeling region, the detection region and the absorption zone may or may not be in contact with adjacent regions.

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

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

[0045] In addition, as a preferable solid acidic reagent used in the present invention, for example, an acid with a high valence such as citric acid can be extracted in a smaller amount. Also, if they have the same valence, those with a smaller acid dissociation constant, such as maleic acid and tartaric acid, are more efficient.

[0046] In addition, as a preferable solid acidic reagent used in the present invention, a reagent that is not colored on the immunochromatographic test strip, specifically, a reagent that is white in the dry state or is not easily colored by dry heat or oxidation is preferable.

[0047] Examples of the nitrite used in the present invention include sodium nitrite and potassium nitrite.

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

[0049] To impregnate the solid acidic reagent or nitrite into the sample pad or porous material, the solid acidic reagent or nitrite is dissolved once, applied, and dried. For example, a 0.1 to several M solution of the solid acidic reagent or nitrite may be applied and dried.

[0050] The neutralizing reagent used in the present invention is solid at room temperature and does not volatilize at high temperatures. The neutralizing reagent is also referred to as a basic reagent.

[0051] Examples of the preferable neutralizing reagent used in the present invention include tris base (tris(hydroxymethyl)aminomethane), sodium hydroxide, dipotassium hydrogen phosphate, trisodium citrate, and Good buffers having a buffering capacity in the alkaline region.

[0052] The amount of the neutralizing reagent used in the present invention, that is, the amount impregnated into the immunochromatographic test strip, is not particularly limited, but is usually about 0.01 μg to 1 mg per test strip of the immunochromatographic test strip, preferably about 0.1 μg to 0.1 mg. However, it is preferable to select the optimal amount that can achieve the effect depending on the type of the neutralizing reagent used, the composition and addition amount of the specimen suspension, and the like.

[0053] To impregnate the neutralizing reagent into the porous material, the neutralizing reagent may be dissolved once, the solution may be applied to the porous material, and then dried. For example, a neutralizing reagent solution of 0.1 to several M, preferably 0.5 to several M may be applied and dried.

[0054] Figs. 1 to 3 are diagrams showing a preferable form of a typical immunochromatographic test strip. The immunochromatographic test strip shown in Fig. 1 is an immunochromatographic test strip impregnated with a solid acidic reagent and a neutralizing reagent, but it may be impregnated with nitrite instead of the solid acidic reagent. In this case, it becomes an immunochromatographic test strip impregnated with nitrite and a neutralizing reagent. That is, in the following description of the test strip, a nitrite reagent region may be provided instead of the solid acidic reagent region. A person skilled in the art can appropriately design and manufacture an immunochromatographic test strip impregnated with nitrite and a neutralizing reagent. Note that the immunochromatographic test strip is not limited to those shown in Figs. 1 to 3. In Fig. 1, 1 indicates a support, 2 indicates a labeled body region, 3 indicates a detection region, 7 indicates an absorption band, and 8 indicates a backing sheet.

[0055] Figures 1 and 2 show test pieces in which the solid acidic reagent region 5 also serves as the sample pad 10. Figure 1A is a top view, and Figure 1B is a cut-away cross-sectional view. In the example of Figure 1, the solid acidic reagent region 5 and / or the neutralizing reagent region 6 are present upstream of the labeling body region 2, and they are superimposed, thereby forming a continuous lateral flow channel. In the test piece shown in Figure 1, the solid acidic reagent region 5 also serves as the sample pad 10. That is, the solid acidic reagent region 5 is present on the sample pad 10. In this test piece, since the solid acidic reagent region 5 and the neutralizing reagent region 6 are provided on two separate porous materials (pads), it may be called a two-pad test piece. A sample pad may further be present upstream of the solid acidic reagent region. The solid acidic reagent region and the sample pad may be present separately.

[0056] In the test piece of Figure 2, the sample pad 10 also serves as the solid acidic reagent region 5 and the neutralizing reagent region 6, and the solid acidic reagent region 5 and the neutralizing reagent region 6 are present on the sample pad 10. Figure 2A is a top view, Figure 2B is a cut-away cross-sectional view, and Figure 2C shows the positional relationship between the neutralizing reagent region 6 and the solid acidic reagent region 5 or the nitrous acid reagent region. In this test piece, since the solid acidic reagent region 5 and the neutralizing reagent region 6 are provided on one porous material (pad), it may be called a one-pad test piece.

[0057] In the test piece of Figure 3, the sample pad 10 exists separately from the solid acidic reagent region 5 and the neutralizing reagent region 6, the solid acidic reagent region 5 also serves as the neutralizing reagent region 6, and the sample pad exists separately. The sample pad 10 is not impregnated with reagents and functions only as a sample pad. Figure 3A is a top view, Figure 3B is a cut-away cross-sectional view, and Figure 3C shows the positional relationship between the neutralizing reagent region 6 and the solid acidic reagent region 5 or the nitrous acid reagent region. The test piece of Figure 3 can be called a two-pad test piece because the porous material (pad) that also serves as the solid acidic reagent region and the neutralizing reagent region and the sample pad are provided as two separate porous materials (pads). Although not shown in the figure, the three of the sample pad 10, the solid acidic reagent region 5, and the neutralizing reagent region 6 may exist separately.

[0058] The measurement is started by bringing a specimen or a sample prepared using the specimen into contact and mixing with a nitrite solution, suspending the specimen in the nitrite solution, and adding it to the sample pad 10, the sample pad 10 also serving as the solid acidic reagent region 5, or the sample pad 10 also serving as the neutralizing reagent region 6 and the solid acidic reagent region 5. At this time, 5 to 100 μL of the specimen and 0.01 to 2 mL of 0.1 M to 8 M nitrite are mixed, and 5 to 200 μL may be supplied to the sample pad 10, the sample pad 10 also serving as the solid acidic reagent region 5, or the sample pad 10 also serving as the neutralizing reagent region 6 and the solid acidic reagent region 5. Examples of the nitrite include sodium nitrite and potassium nitrite.

[0059] A sample containing a sugar chain antigen, which is a substance to be detected, provided to the sample pad 10, the sample pad 10 also serving as the solid acidic reagent region 5, or the sample pad 10 also serving as both the neutralizing reagent region 6 and the solid acidic reagent region 5, is developed by capillary action to the sample pad 10, the sample pad 10 also serving as the solid acidic reagent region 5, or the sample pad 10 also serving as both the neutralizing reagent region 6 and the solid acidic reagent region 5 and the neutralizing reagent region 6. At this time, a top laminate sheet, which is a PET sheet, exists between the solid acidic reagent region 5 and the neutralizing reagent region 6. For this reason, the flow of the sample from the solid acidic reagent region 5 to the neutralizing reagent region 6 is suppressed, and the sample once entering the neutralizing reagent region 6 can be prevented from flowing back to the solid acidic reagent region 5. Next, the sample is sequentially developed horizontally to the labeled body region 2, the support 1, and the absorption zone 7. In the solid acidic reagent region 5, nitrite mixed in the sample reacts with the solid acidic reagent on the solid acidic reagent region 5, generating free nitrous acid, and the sugar chain antigen is extracted from the sample by the action of the nitrous acid. The extracted sugar chain antigen is developed and transferred to the neutralizing reagent region 6 together with the acidic developing solution, and the pH of the acidic developing solution containing the sugar chain antigen is neutralized and adjusted to the neutral range in the neutralizing reagent region 6. As a result, the sugar chain antigen is further developed and transferred downstream under neutral conditions. In the labeled body region 2, the labeled antibody is released into the liquid and developed to the support 1 as the sample is developed. When the sugar chain antigen is present in the sample, in the detection region 3 of the support 1, the sugar chain antigen is specifically captured by the capture antibody, and the sugar chain antigen also forms a complex with the labeled antibody by a specific reaction. Thereby, a sandwich of antibodies via the sugar chain antigen is established in the detection region 3, and the labeled antibody-sugar chain antigen complex can be measured in the detection region 3.

[0060] The immunochromatographic test kit of the present invention contains a nitrite solution (R1) or an acidic solution (R2) for suspending the specimen. Even when a highly viscous specimen such as a throat swab specimen is used in the nitrite solution (R1) or the acidic solution (R2) for suspending the specimen, a substance that prevents insufficient sensitivity and non-specific reactions due to poor development or delayed development is added. Such substances include surfactants, salts, proteinaceous substances such as N-acetyl-L-cysteine (NALC) and BSA (bovine serum albumin), phosphoric acid, sodium hydroxide, polyvinyl alcohol (PVA), and other chemically synthesized agents. The surfactant is not limited, and any of nonionic surfactants, cationic surfactants, and anionic surfactants can be used. Specifically, polyoxyethylene octylphenyl ethers such as Triton X-100 and Triton X-114; quaternary ammonium compounds such as benzalkonium chloride and benzethonium chloride; polyoxyethylene sorbitan fatty acid esters such as Tween 20 and Tween 80; polyoxyethylene alkyl ethers such as Brij-35 and Brij-58; cholic acid-based having a steroid skeleton such as sodium cholate and deoxycholic acid; and anionic surfactants such as sodium lauryl sulfate and sodium dodecylbenzenesulfonate. Among these, polyoxyethylene octylphenyl ethers such as Triton X-100 and Triton X-114; and quaternary ammonium compounds such as benzalkonium chloride and benzethonium chloride are preferred. Examples of the salt include NaCl and NaBr. An example of the addition concentration of the above-exemplified compounds is shown below.

[0061] Triton X-100 (Tx-100): 0.5 to 5%, preferably 1 to 3%, more preferably 2% Benzalkonium chloride: 5 to 20%, preferably 5 to 15%, more preferably 10% Benzethonium chloride: 10 mM to 200 mM, preferably 50 to 150 mM, more preferably 100 mM Phosphate buffer: 100 to 500 mM, preferably 150 to 350 mM, more preferably 250 mM NaOH: 0.05 N to 3 N, preferably 0.05 N to 2 N, more preferably 0.1 N to 1 N NALC: 10 mM to 200 mM, preferably 50 to 150 mM, more preferably 100 mM NaCl: 1 to 5 N, preferably 2 to 4 N, more preferably 3 N BSA: 0.5 to 3%, preferably 0.5 to 2%, more preferably 1% NaBr: 10 mM to 200 mM, preferably 50 to 150 mM, more preferably 100 mM

[0062] Among these, the effect of PVA is particularly high, and the saponification degree of PVA is preferably 70 to 90 mol%. Also, the concentration of PVA showed an effect at 0.01% to 5%, but 0.01% to 0.05% is more preferable.

[0063] According to the method using the immunochromatographic test kit of the present invention, since the extraction of the sugar chain antigen in the sample is performed on the immunochromatographic test strip, it is not necessary to extract the sugar chain antigen in the sample in advance before the measurement using the immunochromatographic test strip, and the sugar chain antigen in the sample can be measured in one step.

[0064] In the method using the immunochromatographic test kit of the present invention, the biological sample to be the sample is not particularly limited, and examples include body fluids such as serum, plasma, blood, urine, feces, saliva, tissue fluid, cerebrospinal fluid, swab solution, etc. or dilutions thereof.

[0065] In the method using the immunochromatographic test kit of the present invention, the analyte to be measured is a sugar chain antigen that can be measured by an immunoassay, that is, an assay using an antigen-antibody reaction. Examples of the antigen include polysaccharides and the like, which are sugar chain antigens present on the cell wall of bacteria extracted by nitrite extraction treatment. Protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, viruses, etc. containing these substances can also be measured. By the method using the immunochromatographic test strip of the present invention, it is possible to confirm whether or not a sugar chain antigen derived from protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, viruses, etc. is contained in a biological sample of a subject. If a sugar chain antigen is contained, it can be determined that the subject is suffering from an infectious disease caused by protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia, viruses, etc. For example, it is possible to detect the presence or absence of infection with group A β-hemolytic streptococcus (Streptococcus pyogenes), Escherichia coli, Legionella, Campylobacter, etc.

[0066] The kit of the present invention is not limited to the case where the immunochromatographic test strip is impregnated with nitrite or an acidic reagent and a neutralizing reagent, and can also be used when impregnating the immunochromatographic test strip with a plurality of, that is, two to n reagents having different effects and reagents that should avoid reactions during storage.

Example

[0067] The present invention will be specifically described by the following examples, but the present invention is not limited by these examples. In the following examples, % indicates w / v% unless otherwise specified.

[0068] Example 1 Example of an immunochromatographic test strip for measuring group A streptococcus The following were used as nitrite, solid acidic reagent, and neutralizing reagent. Nitrite (liquid reagent) 3.0 M sodium nitrite Solid acidic reagent (impregnated in the immunochromatographic test strip) 1.0 M citric acid Neutralizing reagent (impregnated in immunochromatographic test strips), 3.0 M Tris base (Trizma BASE)

[0069] 1. Immobilization of anti-Streptococcus pyogenes (Group A β-hemolytic streptococcus) antibody on nitrocellulose membrane (support) A solution of diluted anti-Streptococcus pyogenes antibody and anti-rabbit IgG antibody were prepared. The anti-Streptococcus pyogenes antibody was linearly applied to the sample pad side of the nitrocellulose membrane lined with a PET film, and the anti-rabbit IgG antibody was linearly applied to the absorption band side. Thereafter, the nitrocellulose membrane was dried to obtain an anti-Streptococcus pyogenes antibody-immobilized membrane. This membrane is referred to as the "antibody-immobilized membrane" in this example.

[0070] 2. Immobilization of anti-Streptococcus pyogenes antibody on colored polystyrene particles Colored polystyrene particles were added to the diluted anti-Streptococcus pyogenes antibody solution. After stirring, carbodiimide was added and stirring was continued. The supernatant was removed by centrifugation and resuspended in a buffer to obtain a suspension of anti-Streptococcus pyogenes antibody-bound colored polystyrene particles. These particles are referred to as "antibody-immobilized particles (colored polystyrene)" in this example.

[0071] 3. Application and drying of anti-Streptococcus pyogenes antibody-immobilized colored polystyrene particles A predetermined amount of the suspension of antibody-immobilized colored polystyrene particles prepared in 2 was applied to a non-woven fabric and dried. The obtained non-woven fabric is referred to as "C.Pad" in this example.

[0072] 4. Application of neutralizing reagent (basic reagent) The above neutralizing reagent (basic reagent) was applied to filter paper.

[0073] 5. Preparation of non-woven fabric impregnated with solid acidic reagent The above solid acidic reagent was applied to a nonwoven fabric. Immediately after the application, it was dried to obtain a solid acidic reagent-impregnated nonwoven fabric as a solid acidic reagent-impregnated region.

[0074] 6. Preparation of an immunochromatographic test strip for detecting Streptococcus pyogenes First, an antibody-immobilized membrane (support) was attached to a portion 20 mm from the downstream on the backing sheet of the strip.

[0075] An absorption band for absorbing liquid was attached downstream of the membrane. A 10-mm-wide glass fiber (C.Pad) coated with antibody-binding colored polystyrene particles was attached upstream of the membrane with a 2-mm overlap with the membrane.

[0076] A pad impregnated with a neutralizing reagent (neutralizing reagent-impregnated region) was attached so that the overlap with the C.Pad was 4 mm.

[0077] A top laminate sheet (60 mm), which is a PET sheet, was attached in accordance with the upper part of the immunochromatographic test strip.

[0078] Finally, a pad impregnated with a solid acidic reagent (solid acidic reagent-impregnated region) was attached upstream (lower end) of the immunochromatographic test strip such that the top laminate sheet was interposed between the pad impregnated with the solid acidic reagent and the pad impregnated with the neutralizing reagent, and the pad impregnated with the solid acidic reagent and the pad impregnated with the neutralizing reagent were in contact.

[0079] The structure of the immunochromatographic test strip thus prepared is shown in FIG. 4. Also, a schematic diagram is shown in FIG. 5 together with the distances (mm) between the respective parts. In FIGS. 4 and 5, the pad impregnated with the solid acidic reagent and the pad impregnated with the neutralizing reagent are shown as being separated without contact even at a site where there is no top laminate between the two pads, but actually, the two pads are in contact at the portion without the top laminate.

[0080] Example 2 The immunochromatographic test strip of Example 1 was placed in a test device case (a resin case for storing the immunochromatographic test strip). A solution containing only 3.0 M sodium nitrite solution was used as a control, and solutions of each condition were prepared by adding Triton X-100, benzalkonium chloride, benzetonium chloride, phosphoric acid, sodium hydroxide, NALC, NaCl, PVA, BSA, and NaBr to the 3 M sodium nitrite solution at the concentrations shown in Table 1. Using a negative human nasal mucus specimen known to cause non-specific reactions, specimens were prepared with the sodium nitrite solution and the sodium nitrite solutions added with the above substances (Solution 4: Specimen 1) and used as samples. 75 μL of the prepared sample was dropped and judged after 5 minutes.

[0081] As a result, compared with the control condition without addition, the start time of development was shortened in any condition, and line judgment became possible from an earlier time zone. Also, the degree of non-specific reaction (the intensity of color development of non-specific lines) was reduced compared with the control. In particular, in the condition with PVA added, the development was also improved, and the non-specific suppression effect was the highest (Table 1).

[0082]

Table 1

[0083] Example 3 The immunochromatographic test strip of Example 1 was placed in a test device case. As the sodium nitrite solution, (1) 2 M sodium nitrite solution (without PVA) and (2) 2 M sodium nitrite + 0.5% PVA were prepared. Specimens were prepared with each sodium nitrite solution in which viable Streptococcus pyogenes was added to a human throat swab specimen and used as samples. 75 μL of the prepared sample was dropped and judged after 5 minutes.

[0084] As a result, in the condition with PVA, the start time of development was shortened. In the case of a positive specimen, positive judgment became possible from an earlier time zone, and the line color development at 5 minutes of judgment also resulted in a strong color. Also, when a negative specimen was tested, the frequency of non-specific reaction occurrence decreased by about 4% (Table 2).

[0085]

Table 2

Explanation of Symbols

[0086] 1 Support (including detection area) 2 Label area 3 Detection area 5 Solid acidic reagent area 6 Neutralizing reagent area 7 Absorption band 8 Backing sheet 9 Top laminate sheet 10 Sample pad

Industrial Applicability

[0087] The infection of group A β-hemolytic streptococcus can be accurately detected using the immunochromatographic device of the present invention.

Claims

1. (i) A nitrite solution or an acidic solution, and (ii) A sample pad to which a specimen mixed with a nitrite or an acidic solution is added, a labeled body region containing a labeled antibody labeled with an antibody against a sugar chain antigen, and a detection region in which an antibody against the sugar chain antigen is immobilized, and forming an antibody-sugar chain antigen-labeled antibody complex in the detection region to measure the sugar chain antigen. An immunochromatographic test strip, which has a region impregnated with a neutralizing reagent upstream of the labeled body region, and further has a region impregnated with a solid acidic reagent when using a specimen mixed with nitrite upstream of the region impregnated with the neutralizing reagent, and has a region impregnated with nitrite when using a specimen mixed with an acidic solution. An immunochromatographic test strip for extracting and measuring a sugar chain antigen in a specimen An immunochromatographic test kit comprising A nitrite or an acidic solution contains benzalkonium chloride which is a quaternary ammonium compound, enabling line determination from an early time zone and reducing non-specific reactions. An immunochromatographic test kit.

2. The immunochromatographic test kit according to claim 1, wherein a region impregnated with a solid acidic reagent or nitrite exists on the sample pad.

3. The immunochromatographic test kit according to claim 1 or 2, wherein the solid acidic reagent is selected from the group consisting of malonic acid, malic acid, maleic acid, citric acid, and tartaric acid.

4. The immunochromatographic test kit according to any one of claims 1 to 3, wherein the neutralizing reagent is tris(hydroxymethyl)aminomethane or sodium hydroxide.

5. The immunochromatographic test kit according to any one of claims 1 to 4, 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.

6. (i) A nitrite solution or an acidic solution, and (ii) a sample pad to which a sample mixed with a nitrite or an acidic solution is added, a labeled body region containing a labeled antibody labeled with an antibody against a sugar chain antigen, and a detection region in which the antibody against the sugar chain antigen is immobilized, and an immunochromatographic test piece for measuring a sugar chain antigen by forming a complex of antibody-sugar chain antigen-labeled antibody in the detection region, having a region impregnated with a neutralizing reagent upstream of the labeled body region, and further having a region impregnated with a solid acidic reagent when using a sample mixed with nitrite upstream of the region impregnated with the neutralizing reagent, and having a region impregnated with nitrite when using a sample mixed with an acidic solution, an immunochromatographic test kit for extracting and measuring a sugar chain antigen in a sample, wherein the nitrite or the acidic solution comprises one or more selected from the group consisting of polyoxyethylene octylphenyl ether and benzalkonium chloride, a method for measuring a sugar chain antigen in a sample by an immunochromatographic method using the immunochromatographic test kit, when the immunochromatographic test piece has a region impregnated with a solid acidic reagent, mixing the sample with a nitrous acid solution, and when the immunochromatographic test piece has a region impregnated with nitrite, mixing the sample with an acidic solution, and adding to the sample pad of the immunochromatographic test piece, in the region impregnated with the solid acidic reagent or the region impregnated with nitrite, the sugar chain antigen is extracted from the sample by the action of nitrous acid generated by the reaction of nitrite with the solid acidic reagent, in the region impregnated with the neutralizing reagent, the acidic solution containing the sugar chain antigen is neutralized, in the detection region, a complex of antibody-sugar chain antigen-labeled antibody is formed. By the immunochromatographic method, controlling the speed and direction of the development of the sample on the immunochromatographic test piece, and controlling the treatment with the acidic reagent, nitrite and neutralizing reagent, line determination can be performed from an early time zone, and non-specific reactions are reduced. A method for measuring a sugar chain antigen in a sample.

7. The method according to claim 6, wherein there is a region impregnated with a solid acidic reagent or nitrite on the sample pad of the immunochromatographic test piece.

8. The method according to claim 6 or 7, wherein the solid acidic reagent impregnated in the immunochromatographic test strip is selected from the group consisting of malonic acid, malic acid, maleic acid, citric acid and tartaric acid.

9. The method according to any one of claims 6 to 8, wherein the neutralizing reagent impregnated in the immunochromatographic test strip is tris (hydroxymethyl) aminomethane or sodium hydroxide.

10. The method according to any one of claims 6 to 9, wherein the carbohydrate antigen is a carbohydrate antigen of protozoa, fungi, bacteria, mycoplasma, rickettsia, chlamydia or virus.

11. The method according to any one of claims 6 to 10, wherein the polyoxyethylene octylphenyl ether is Triton X (registered trademark).

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