An immunochromatographic test strip for extracting and measuring sugar chain antigens, which can control the development of a sample by impregnating a hydrophobic material with a nitrite or a solid acidic reagent.

Abstract

To provide an immunochromatographic test piece for controlling development of a specimen on the immunochromatographic test piece and appropriately controlling treatment with acidic reagent, nitrite and neutralizing reagent, and immunochromatographic method using the test piece.SOLUTION: An immunochromatographic test piece including a sample pad for adding a specimen mixed with nitrite or acid solution, a labeled body area containing a labeled antibody formed by labeling antibody against a carbohydrate antigen, and a detection area immobilizing an antibody against the carbohydrate antigen, forming a complex of antibody-carbohydrate antigen-labeled antibody in the detection area and measuring the carbohydrate antigen; the immunochromatographic test piece for extracting and measuring the carbohydrate antigen in the specimen, includes an area impregnated with a neutralizing reagent on an upstream side of the labeled body area, and includes on a further upstream side of the area impregnated with the neutralizing reagent, an area impregnated with the solid acidic reagent when using a specimen mixed with the nitrite, and an area impregnated with the nitrite when using the specimen mixed with the acid solution. The immunochromatographic test piece is characterized in using a hydrophobic material in the area impregnated with the nitrite or solid acid reagent.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for extracting sugar chain antigens from an immunochromatographic test piece using nitrous acid. The present invention relates to an immunochromatographic test strip for extracting and measuring carbohydrate antigens. [Background technology]

[0002] Many rapid diagnostic drugs based on the immunochromatography method are capable of quickly detecting viral or bacterial infections. It is easily measured and is widely used as a means of determining treatment plans.

[0003] A typical rapid diagnostic drug based on immunochromatography is a method in which a sample is suspended in a sample suspension. The suspension is then supplied to an immunochromatographic test piece for rapid and easy measurement.

[0004] Group A beta-hemolytic streptococci (group A beta-hemolytic streptococci), oral streptococci, and other Streptococcus species In order to detect microorganisms belonging to the group, it is necessary to extract and measure the carbohydrate antigens.

[0005] For example, the immunochromatographic test strip is pre-soaked with sodium nitrite and a neutralizing agent. The sample is suspended in an acidic solution such as acetic acid and then fed to the immunochromatographic test strip to obtain nitrite. A method has been reported in which acid extraction treatment is performed on an immunochromatographic test piece (Patent Document 1).

[0006] In addition, the immunochromatographic test strip is pre-soaked with an acidic reagent and a neutralizing reagent, and the specimen is then soaked in nitrite. Another method is to supply the antibody to the immunochromatographic test strip by suspending it in salt.

[0007] Furthermore, a method for efficiently extracting glycan antigens from immunochromatographic test strips using nitrite has been reported. It has been reported (Patent Documents 2 to 4).

[0008] However, when using an on-device extraction mechanism, in order to achieve the target performance, , it was necessary to ensure a consistent extraction time on the device. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. WO2005 / 121794 [Patent Document 2] JP 2018-151329 A [Patent Document 3] JP 2018-151330 A [Patent Document 4] JP 2018-151331 A Summary of the Invention [Problem to be solved by the invention]

[0010] Currently available immunochromatographic detection assays for group A β-hemolytic streptococci (Strep A) antigens Many drugs are made by mixing a nitrite solution (R1) with an acid solution (R2) to generate acidic nitrite, which is then injected into the bloodstream. Neutralize and react with the dry neutralizer (R3) on the immunochromatographic detection reagent (test strip). The technology uses a pad that can be applied with R1 or R2, allowing for the preparation of R1 and R2 on demand. A one-step technique that omits the steps of extraction and ELISA is also known. For drugs, the extraction process is carried out on the test device, so after the sample is dropped, a certain extraction time is required. However, when using a mechanism for extracting data on the device, To achieve this performance, it was necessary to guarantee a constant extraction time on the device. To ensure a consistent extraction time, the pad to be soaked must be made of a hydrophilic, thick material. However, depending on the viscosity of the sample, the retention time varies. In addition, the extraction time was reduced by holding the sample on a highly hydrophobic pad. It is possible to ensure this, but depending on the viscosity of the sample, the extraction time may vary. The sample never developed, and the liquid did not fully develop within the 5-minute test time. There was a problem.

[0011] The present invention controls the development of a sample on an immunochromatographic test piece, and separates an acidic reagent, nitrite, and neutral Immunochromatographic test strip for appropriately controlling treatment with a neutralizing reagent and immunoassay using said test strip The object of the present invention is to provide a immunochromatographic method. [Means for solving the problem]

[0012] The present inventors controlled the development of the sample on the immunochromatographic test strip and determined the presence of an acidic reagent and nitrite. We conducted extensive research into methods to appropriately control the treatment with neutralizing reagents.

[0013] As a result, the use of a hydrophobic material for the material impregnated with the solid acidic reagent or nitrite They found that this improved the reaction time.

[0014] In addition, by adjusting the concentration of the surfactant applied to the material, the optimal nitrite extraction time can be determined. It has been found that the signal intensity can be adjusted by controlling the signal intensity.

[0015] That is, the present invention is as follows. [1] A sample pad for adding a sample mixed with nitrite or an acidic solution, and a sample pad for detecting carbohydrate antigens. The labeled antibody is labeled with an antibody against the sugar chain antigen. The detection region is included, and a complex of an antibody, a sugar chain antigen, and a labeled antibody is formed in the detection region, and the sugar chain antigen is detected. An immunochromatographic test strip for measuring an antigen, the neutralizing reagent being impregnated upstream of the label region. and a sample mixed with nitrite upstream of the neutralizing reagent-impregnated region. When using a solid acidic reagent, a region is impregnated with the solid acidic reagent, and a specimen mixed with an acidic solution is used. When the sample is used, the area is impregnated with nitrite, and the area is used to extract and measure carbohydrate antigens in the sample. An immunochromatographic test strip for The method is characterized in that a hydrophobic material is used in the area impregnated with nitrite or a solid acidic reagent. An immunochromatographic test strip. [2] A hydrophobic material that has been impregnated with nitrite or a solid acidic reagent is impregnated with a surfactant. [1] Immunochromatographic test strip. [3] The immunoassay of [2], in which the surfactant is polyoxyethylene octylphenyl ether. Chromatographic test strips. [4] Polyoxyethylene octylphenyl ether is Triton X-100. Nochromatographic test piece. [5] The hydrophobic material impregnated with nitrite or solid acidic reagent is polyester, The group consisting of polyethylene, rayon, nylon, and mixtures of any two or more of them An immunochromatographic test strip selected from [1] to [4]. [6] There is an area on the sample pad that is impregnated with a solid acidic reagent or nitrite. , any of the immunochromatographic test strips [1] to [5]. [7] The solid acidic reagent consists of malonic acid, malic acid, maleic acid, citric acid and tartaric acid. An immunochromatographic test strip according to any one of [1] to [6] selected from the group consisting of: [8] The neutralizing agent is tris(hydroxymethylaminomethane) or sodium hydroxide. , any of the immunochromatographic test strips [1] to [7]. [9] The carbohydrate antigen is a protozoan, fungal, bacterial, mycoplasmal, rickettsial, chlamydial or is an immunochromatographic test strip of any of [1] to [8], which is a viral glycan antigen.

[10] Any of the immunochromatographic test strips [1] to [9] and a nitrite solution or an acidic solution An immunochromatographic test kit comprising:

[11] Using any one of the immunochromatographic test strips [1] to

[10] , A method for measuring a carbohydrate antigen in a sample, comprising the steps of: When the immunochromatographic test strip has an area impregnated with a solid acidic reagent, the specimen is nitrite-containing. When the immunochromatographic test strip has an area impregnated with nitrite, it is possible to detect it by mixing it with an acid solution. Mixing the sample with an acidic solution and adding the mixture to the sample pad of the immunochromatographic test strip. fruit, In the area impregnated with the solid acidic reagent or the area impregnated with the nitrite, The carbohydrate antigens are extracted from the specimen by the action of nitrous acid generated by the reaction of the solid acidic reagent. In the region impregnated with the neutralizing reagent, the acidic solution containing the carbohydrate antigen is neutralized; Immunochromatography, in which an antibody-carbohydrate antigen-labeled antibody complex is formed in the detection area This allows the speed and direction of the sample development on the immunochromatographic test strip to be controlled, and the acidic reagent and A method for measuring carbohydrate antigens in a sample by controlling treatment with nitrite and neutralizing reagents. Effect of the Invention

[0016] In the one-step StrepA antigen detection reagent, nitrite or solid acidic reagent is impregnated. When using a hydrophobic material as a substrate, the concentration of the surfactant can be adjusted to reduce the solid It allows the time required for the acidic reagent and nitrite treatment to be adjusted to the optimum, resulting in As a result, we developed an immunochromator that can achieve the desired performance (fast line coloring time and high sensitivity). Test pieces are provided. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a diagram showing a schematic structure of an immunochromatographic test strip having a solid acidic reagent region and a neutralizing reagent region, in which the solid acidic reagent region also serves as a sample pad (two-pad test strip). [Diagram 2] FIG. 1 is a schematic diagram showing the structure of an immunochromatographic test strip having a solid acidic reagent region and a neutralizing reagent region, the solid acidic reagent region and the neutralizing reagent region also serving as a sample pad (one-pad test strip). [Diagram 3] FIG. 1 is a schematic diagram showing the structure of an immunochromatographic test strip having a solid acidic reagent region and a neutralizing reagent region, in which the solid acidic reagent region also serves as the neutralizing reagent region and a sample pad is provided separately (two-pad test strip). [Figure 4] FIG. 1 is a diagram showing a schematic structure of an immunochromatographic test piece having a solid acidic reagent region and a neutralizing reagent region, in which a PET sheet is attached between the solid acidic reagent region and the neutralizing reagent region. [Diagram 5] FIG. 1 is a schematic diagram showing the structure of an immunochromatographic test piece having a solid acidic reagent region and a neutralizing reagent region, in which a PET sheet is attached between the solid acidic reagent region and the neutralizing reagent region, and shows the distance between each part. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention will be described in detail below.

[0019] The present invention simplifies the nitrite extraction process for sugar chain antigens so that it can be performed on an immunochromatographic test strip. This immunochromatography enables rapid and accurate measurement of the target substance, the carbohydrate antigen. The present invention relates to a kit comprising said immunochromatographic test strip and a nitrite solution or an acidic solution.

[0020] The immunochromatographic test strip has an antibody (antibody 1) immobilized on it that captures the substance to be detected (such as an antigen). a support having a detection region formed by displacing a mobile labeled antibody (antibody 2); The sample pad to which the sample is added, the absorption band to which the developed sample liquid is absorbed, and these components are affixed together. It is equipped with a backing sheet etc. for assembly.

[0021] The immunochromatographic test piece of the present invention may be contained in a storage container. This makes it possible to prevent deterioration caused by, for example, ultraviolet rays and moisture in the air. When using a sample with a potential for contamination, the containment vessel may cause contamination or infection to the tester performing the assay. For example, a resin case of an appropriate size can be used as a containment vessel. The device of the present invention may be stored in the case. The surface of the containment vessel may be covered with a resin film (PET sheet). The test piece and the resulting assembly are sometimes referred to as an immunochromatographic device.

[0022] The number of detection regions and the type of labeled antibody contained in the labeled region are limited to one. By using antibodies corresponding to multiple target substances, two or more antigens can be detected in the same test. It can be measured on a piece.

[0023] The support is a material that has the ability to immobilize an antibody to capture the target substance (antigen). The material has the ability to pass liquid horizontally without being hindered by capillary action. It is a porous thin film (membrane) that absorbs and transports liquids and the components dispersed in them. The material of the support is not particularly limited, and may be, for example, cellulose. cellulose, nitrocellulose, cellulose acetate, polyvinylidene difluoride (PVD F), glass fiber, nylon, polyketone, etc. Among these, nitrocellulose It is more preferable to use a thin film or membrane made of the antibody. This is called an antibody-immobilized membrane.

[0024] The labeled region is made of a porous substrate containing a labeled antibody. The substrate may be a glass fiber or a nonwoven fabric. In order to impregnate the labeled antibody, it is preferable that the thickness of the pad is about 0.3 mm to 0.6 mm. The porous substrate that has been impregnated with labeled antibodies and dried is also called a dry pad.

[0025] For labeling antibodies, enzymes such as alkaline phosphatase and horseradish peroxidase are used. enzymes, metal colloids such as gold colloids, silica particles, cellulose particles, magnetic particles, fluorescent particles, etc. Light particles, colored polystyrene particles, colored latex particles, etc. are often used. Metal When using colloidal particles, colored polystyrene particles, colored latex particles, etc. In the method, coloration occurs due to aggregation of these labeling reagents, and this coloration is measured. The particles on which the antibodies are immobilized are called antibody-immobilized particles.

[0026] The detection area is a part of the support where the antibody that captures the substance to be detected (antigen) is immobilized. The detection area is provided with at least one area where an antibody for capturing an antigen is immobilized. The detection region may be contained in the support, and the antibody may be immobilized on the support.

[0027] The sample pad is a porous material that is used to apply a sample. The head is the most upstream part of the immunochromatographic test strip. Filter paper, glass fiber, nonwoven fabric, etc. can be used. For this purpose, it is preferable that the sample is in the form of a pad having a thickness of about 0.3 mm to 1 mm. This also includes samples prepared using specimens, such as samples obtained by floating in water.

[0028] The absorption band is a portion for absorbing components that are supplied to the support and do not participate in the reaction in the detection area. The material is a water-retentive material made of general natural polymer compounds and synthetic polymer compounds. Although high-absorbency filter paper, sponge, etc. can be used, in order to promote the development of the sample, The one is preferred.

[0029] The backing sheet is made of all the materials mentioned above, i.e., the support, the sample pad, the label area, etc. It is a component for attaching and fixing the absorbent band, etc. with partial overlap. If these materials are placed and fixed at optimal intervals, the use of tape sheets is not necessarily required. However, it is generally preferred to use it because of the convenience in manufacturing and use.

[0030] The immunochromatographic test strip of the present invention may further include a control display area (member). The control area is a part that shows that the test was performed correctly. The detection zone is downstream of the detection zone, and the sample passes through the detection zone and reaches the control zone. In some cases, a signal is generated by coloring, etc. In the control display area, the antibody bound to the labeled carrier is The binding substance may be immobilized, or a pH indicator that changes color when the sample arrives may be used. The antibody bound to the labeling carrier is a mouse monoclonal antibody. In the case of a local antibody, an anti-mouse IgG antibody may be used.

[0031] The size of the immunochromatographic test piece is not limited, but for example, the length is several centimeters to several tens of centimeters, the width is The length is about a few mm to a few cm.

[0032] In the test strip of the above form, the specimen is disposed in the sample pad, the label region, the support, the detection region, The absorption bands, etc. are connected in series through the porous flow path. Depending on the material and shape of each component, the sample may move through the material. Although there may be cases where the analyte does not penetrate the inside but passes through the interface, the analyte migration region defined in this specification is the region of the material. Since it does not matter whether the test piece is inside or at the interface of the material, test pieces of this form are also included in the scope of this specification.

[0033] When measuring sugar chain antigens in a sample using the immunochromatographic test strip of the present invention, the sample is first It is necessary to extract the glycan antigens from the samples. Nitrous acid is produced by mixing nitrites such as sodium nitrite with acid. Nitrite can be generated by the above method, and a sample containing a carbohydrate antigen is treated with the generated nitrite. The extracted antigen undergoes an antigen-antibody reaction with the antibody immobilized on the immunochromatographic test piece. However, if acid remains in the reaction system, the reaction system becomes acidic, and the antigen-antibody bond is formed. The reaction is inhibited. Therefore, it is necessary to neutralize the acid in the reaction system.

[0034] In the present invention, nitrite is mixed with an acid to generate nitrous acid, and the nitrous acid is used to detect the nitrite in the specimen. The carbohydrate antigens are extracted, the acid is neutralized, and the carbohydrate antigens are then attached to the antibodies immobilized on the immunochromatographic test strip. A method for measuring glycan antigens by binding glycan antigens and extracting and measuring the glycan antigens The following methods are listed as examples of such methods. In each method, the extraction of carbohydrate antigens using nitrous acid is performed. The extraction of carbohydrate antigens with nitrous acid is performed on the immunochromatographic test strip. To perform the test on a test strip, an acidic reagent or nitrite is applied to the immunochromatographic test strip. In order to perform neutralization on the immunochromatographic test strip, the neutralization It is sufficient to impregnate the substrate with a cleaning agent.

[0035] (A) An immunochromatographic test sample that has been mixed with an acidic solution and impregnated with nitrite and a neutralizing reagent. Add the mixture to the sample pad of the test strip. When the mixture reaches the area impregnated with nitrite, The salt reacts with the acid to generate nitrous acid, which extracts the carbohydrate antigens in the sample. The immunochromatographic test strip is neutralized in the area impregnated with the neutralizing reagent, and the carbohydrate antigen is The acidic solution used in this method binds to the antibody immobilized on the mat test strip and can be detected. Examples of the acid include acetic acid, hydrochloric acid, malonic acid, malic acid, maleic acid, citric acid, and tartaric acid. can be. (B) Immunochromic gel that has been mixed with the specimen and nitrite solution and impregnated with acidic and neutralizing reagents When the mixture reaches the area impregnated with the acidic reagent, Nitrate reacts with acid to generate nitrous acid, which extracts carbohydrate antigens from the sample. The liquid is neutralized in the area of ​​the immunochromatographic test strip that is impregnated with a neutralizing reagent, and the carbohydrate antigen is It binds to the antibody immobilized on the chromatographic test strip and can be detected.

[0036] In the immunochromatographic test strip of the present invention for carrying out the above-mentioned method (A) or (B), Upstream of the body region (upstream of the sample flow, on the side where the sample pad is located), i.e. In the case where nitrite is impregnated in the sample pad or between the sample pad and the label region, The immunochromatographic test strip can be used in the above method (A). Or an immunochromatographic test strip in which an acidic reagent is impregnated between the sample pad and the label area. can be used in the above method (B). As the acidic reagent, a solid acidic reagent can be used. By these methods, the substance to be detected in the test sample is subjected to the test of the test sample. It becomes possible to perform accurate and specific measurements regardless of the amount.

[0037] The area impregnated with the solid acidic reagent is called the solid acidic reagent area. The area is referred to as the acidic reagent area, and the area impregnated with the neutralizing reagent is referred to as the neutralizing reagent area.

[0038] The solid acidic reagent or nitrite may be impregnated in a sample pad or may be added to a sample pad. The resulting solid acid is impregnated into a pad made of a porous material such as a nonwoven fabric, which is different from the pad. A reagent-impregnated porous material or a nitrite-impregnated porous material is disposed between the sample pad and the label region. , i.e., upstream of the label region. Here, a solid acidic reagent or a nitrite salt The area impregnated with the label may or may not be in contact with the sample pad or label area. In the present invention, the area impregnated with the reagent is also referred to as a reagent-impregnated pad.

[0039] The neutralizing reagent is disposed downstream of the region where the solid acidic reagent or nitrite is impregnated. The neutralizing reagent may be impregnated into the support, or may be attached to a porous material such as a nonwoven fabric separate from the support. The resulting neutralizing agent-impregnated porous material is then impregnated with a pad of a solid acidic reagent or nitrous acid. Alternatively, the salt may be placed between the region impregnated with the salt and the label region. a region impregnated with a neutralizing reagent in the flow, and a solid The substrate has an area impregnated with an acidic reagent or a nitrite.

[0040] Here, the area impregnated with the neutralizing reagent and the area impregnated with the solid acidic reagent or nitrite are The label regions may or may not be contiguous.

[0041] The material of the porous material in the area to be impregnated with the solid acidic reagent or nitrite is hydrophobic. Specifically, nonwoven fabric made of polyester and nonwoven fabric made of polyethylene are used. Woven fabric, nonwoven fabric made of rayon, nonwoven fabric made of nylon, or any two of these Nonwoven fabrics, etc. that are a mixture of one or more hydrophobic materials, or any two or more of them Examples of the nonwoven fabric include a nonwoven fabric that is a mixture of the above hydrophobic material and hydrophilic material.

[0042] It is also preferable to impregnate a hydrophobic material with a surfactant. There are no limitations on the surfactant, but any of nonionic surfactants, cationic surfactants, and anionic surfactants can be used. Specifically, Triton X-100 (Registered Trademark) , Triton X-114 (Registered Trademark) Polyoxyethylene octylphenyl ethers such as benzalkonium chloride and benzethonium chloride; quaternary ammonium compounds such as Tween 20 (Registered Trademark) , Tween 80 (Registered Trademark)Polyoxyethylene sorbitan fatty acid esters such as Brij-35 (Registered Trademark) , Brij-58 (Registered Trademark) Polyoxyethylene alkyl ethers such as sodium cholate and deoxycholic acid; cholate-based surfactants having a steroid skeleton such as sodium lauryl sulfate and sodium dodecylbenzenesulfonate; and other anionic surfactants. Among these, Triton X-100 (Registered Trademark) , Triton X-114 (Registered Trademark) Polyoxyethylene octylphenyl ethers such as Triton X-100 are preferred. The optimum concentration of the surfactant varies depending on the type, but if it is too high, the sensitivity decreases (the extraction time is too short, so the sensitivity decreases), and if it is too low, no effect is seen. (Registered Trademark) In the case of , 0.1 to 2% is effective, and 0.2 to 0.5% is even better. However, this value varies depending on the amount applied per test, the type of surfactant, and the hydrophobicity of the material to which it is applied.

[0043] The material of the porous material used in the region to be impregnated with the neutralizing reagent is specifically cellulose. Examples include filter paper made from cotton fibers and glass filter paper made from glass fibers.

[0044] Immunochromatographic test with solid acidic or nitrite reagent area and neutralizing reagent area The strip is provided on a support with a sample pad, a solid acidic reagent area or a nitrite area, It has a neutralization reagent region, a label region, a detection region and an absorption band, and is a solid acidic reagent region or a nitrous acid reagent region. The salt region may be on the sample pad. Also, the sample pad, the solid acidic reagent region Alternatively, the nitrite region, the neutralizing reagent region, the label region, the detection region and the absorption band are adjacent regions. It doesn't matter if they are in contact with each other or not.

[0045] The solid acidic reagent used in the present invention is solid at room temperature and is It is non-volatile.

[0046] The preferred solid acidic reagents for use in the present invention include malonic acid, malic acid, maleic acid, Examples of acids include citric acid and tartaric acid.

[0047] In addition, examples of solid acidic reagents that can be used in the present invention include citric acid. If an acid with a higher valence is used, a smaller amount can be extracted. Also, if the acid has the same valence, the acid dissociation constant is smaller. Small acids, such as maleic acid and tartaric acid, are efficient.

[0048] In addition, the preferred solid acidic reagent for use in the present invention is Specifically, reagents that are white in the dry state or that are resistant to coloring due to drying heat or oxidation. A reagent that is easy to use is preferred.

[0049] The nitrites used in the present invention include sodium nitrite, potassium nitrite, etc. can be.

[0050] The amount of the solid acidic reagent or nitrite used in the present invention, i.e., the amount of the immunochromatographic reagent The amount of the immunochromatographic test piece to be impregnated is not particularly limited, but is usually about 100 g / ml per one test piece. The amount is about 0.01 μg to 1 mg, and preferably about 0.1 μg to 0.1 mg. The effect varies depending on the type of solid acidic reagent or nitrite used, the composition of the sample suspension, and the amount added. It is preferable to select an optimum amount to be used.

[0051] To impregnate the sample pad or porous material with a solid acidic reagent or nitrite, A solid acidic reagent or nitrite is dissolved, applied, and dried. For example, a solid solution of 0.1 to several M is used. An acidic reagent or a nitrite solution may be applied and allowed to dry.

[0052] The neutralizing reagent used in the present invention is solid at room temperature and volatilizes at high temperatures. Neutralizing reagents are also called basic reagents.

[0053] The preferred neutralizing reagent for use in the present invention is a tris base (trishydroxymethyl Aminomethane), sodium hydroxide, dipotassium hydrogen phosphate, trisodium citrate, An example of such a buffer is the Good's buffer, which has buffering capacity in the alkali range.

[0054] The amount of neutralizing reagent 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 per immunochromatographic test piece. The concentration is preferably about 0.1 μg to 0.1 mg. However, depending on the type of neutralizing reagent used, It is preferable to select the optimal amount that provides the desired effect depending on the composition of the sample suspension and the amount added. .

[0055] To impregnate the porous material with the neutralizing reagent, first dissolve the neutralizing reagent and then apply the solution to the porous material. For example, the neutralizing agent may be applied to the sample at a concentration of 0.1 to several M, preferably 0.5 to several M. The drug solution is applied and allowed to dry.

[0056] 1 to 3 are diagrams showing a preferred embodiment of a typical immunochromatographic test piece. The immunochromatographic test strip shown in Fig. 1 is an immunochromatographic test strip impregnated with a solid acidic reagent and a neutralizing reagent. The test strip is a nitrite test strip, but it may be impregnated with nitrite instead of the solid acidic reagent. In this case, The immunochromatographic test strip is impregnated with nitrite and neutralizing reagent. In the description of the strip, a nitrite reagent area may be provided in place of the solid acid reagent area. Those skilled in the art can appropriately design an immunochromatographic test strip impregnated with nitrite and a neutralizing reagent. The immunochromatographic test strip is not limited to those shown in Figures 1 to 3. In FIG. 1, 1 is the support, 2 is the label region, 3 is the detection region, 7 is the absorption band, and 8 is the indicates the backing sheet.

[0057] 1 and 2 show a test strip in which a solid acidic reagent area 5 also serves as a sample pad 10. FIG. 1A is a top view, and FIG. 1B is a cutaway cross-sectional view. In the example of FIG. 1, the fixed Shape: Acidic reagent area 5 and / or neutralizing reagent area 6 are present and overlapped; This creates a continuous lateral flow channel. In this embodiment, the solid acidic reagent area 5 also serves as the sample pad 10. A drug region 5 is present on the sample pad 10. In this test strip, a solid acidic reagent region Since the area 5 and the neutralizing reagent area 6 are provided on two separate porous materials (pads), Sometimes called a two-pad test strip. An additional sample pad is placed upstream of the solid acidic reagent area. There may be a separate solid acidic reagent area and a sample pad.

[0058] In the test strip of FIG. 2, the sample pad 10 serves both as the solid acidic reagent region 5 and the neutralizing reagent region 6. The solid acidic reagent area 5 and the neutralizing reagent area 6 are present on the sample pad 10. FIG. 2A is a top view, FIG. 2B is a cutaway view, and FIG. 2C is a diagram showing the neutralizing reagent region 6 and the solid acidic reagent. The positional relationship between the drug region 5 and the nitrite reagent region is shown. Since the reagent area 5 and the neutralization reagent area 6 are provided on a single porous material (pad), It is sometimes called a single pad test specimen.

[0059] In the test strip of FIG. 3, the sample pad 10 is separated from the solid acidic reagent region 5 and the neutralizing reagent region 6. The solid acidic reagent area 5 also serves as the neutralizing reagent area 6, and the sample pad is separate. The sample pad 10 is not impregnated with a reagent and is used as a sample pad only. FIG. 3A is a top view, FIG. 3B is a cutaway cross-sectional view, and FIG. 3C is a view of the neutralizing reagent region 6 and the solidified The shape of the test strip is shown in FIG. 3. The shape of the test strip is shown in FIG. The porous material (pad) that serves as the acidic reagent area and the neutralizing reagent area and the sample pad are separate. Since it is provided as two porous materials (pads), it is called a two-pad test specimen. Although not shown in the figure, the sample pad 10, the solid acidic reagent area 5 and the neutralization reagent There may be three separate drug regions 6 .

[0060] The measurement is carried out by contacting and mixing the specimen or a sample prepared using the specimen with a nitrite solution, and The sample is suspended in a nitrite solution and serves as a sample pad 10 and a solid acidic reagent area 5. A pad 10 or a sample pad 10 having both a neutralizing reagent area 6 and a solid acidic reagent area 5. The test is started by adding 5 to 100 μL of sample and 0.0 M to 8 M nitrite. Mix 1 to 2 mL of the solution, and pour 5 to 200 μL into the sample pad 10, which also serves as the solid acidic reagent area 5. Pull pad 10 or sample pad 1 serving as both neutralizing reagent area 6 and solid acidic reagent area 5 0. Examples of nitrite include sodium nitrite and potassium nitrite. .

[0061] A sample pad 10, a sample pad 10 also serving as a solid acidic reagent region 5, or a neutralization reagent The substance to be detected is provided on the sample pad 10 which serves as both the drug region 6 and the solid acidic reagent region 5. The sample containing the carbohydrate antigen is drawn by capillary action into the sample pad 10 and the solid acidic reagent area 5. or a sample pad 10 serving as both the neutralizing reagent area 6 and the solid acidic reagent area 5. The solid-state acidic reagent area 5 and the neutralizing reagent area 6 are then applied to the sample pad 10 and the neutralizing reagent area 6. Between the neutralizing reagent regions 6, there is a top laminate sheet which is a PET sheet. Therefore, the flow of the sample from the solid acidic reagent area 5 to the neutralizing reagent area 6 is suppressed, and once the sample is neutralized, This can prevent the sample that has entered the solid acidic reagent area 6 from flowing back into the solid acidic reagent area 5. The sample is then spread horizontally through the label region 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 and the solid acidic reagent on the solid acidic reagent area 5 are The reaction of the acidic reagent with glycan ions produces free nitrous acid, which then removes the glycan antibodies from the sample. The extracted carbohydrate antigen is developed and migrates to the neutralizing reagent area 6 together with the acidic developing solution. In the neutralizing reagent area 6, the pH of the acidic developing solution containing the sugar chain antigen is neutralized and adjusted to a neutral range. As a result, the carbohydrate antigen migrates further downstream under neutral conditions. As the sample develops, the labeled antibody is released into the liquid and spreads onto the support 1. When a sugar chain antigen is present in the detection region 3 of the support 1, the sugar chain antigen is captured by a capture antibody in the detection region 3 of the support 1. The sugar chain antigen reacts specifically with the labeled antibody to form a complex. As a result, a sandwich of the antibody via the sugar chain antigen is formed in the detection area 3, and the labeled antibody - the carbohydrate antigen complex can be measured in detection area 3.

[0062] According to the method using the immunochromatographic test strip of the present invention, the extraction of sugar chain antigens in a sample is performed by immunochromatography. Since the measurement is performed on an immunochromatographic test strip, it is necessary to perform the measurement in advance before using the immunochromatographic test strip. Therefore, there is no need to extract the glycan antigens in the sample, and the glycan antigens in the sample can be measured in one step. This can be done.

[0063] In the method using the immunochromatographic test piece of the present invention, the biological sample to be used as the specimen is not particularly limited. Not specified, but bodily fluids such as serum, plasma, blood, urine, feces, saliva, tissue fluid, cerebrospinal fluid, swabs, etc. Dilutions thereof are included.

[0064] In the method using the immunochromatographic test piece of the present invention, the substance to be detected is an immunochromatographic test piece. It is a carbohydrate antigen that can be measured by immunoassay, that is, an assay that utilizes an antigen-antibody reaction. The antigen is a sugar chain antigen present in the bacterial cell wall, which is extracted by nitrite extraction treatment. These substances include protozoa, fungi, bacteria, mycoplasma, and lycopersicon gracilis. Kettsia, chlamydia, viruses, etc. can also be measured. The method detects protozoa, fungi, bacteria, mycoplasma, and rickettsia in the subject's biological sample. It is necessary to check whether the carbohydrate antigens derived from bacteria, chlamydia, viruses, etc. are included. If carbohydrate antigens are present, the subject may be a protozoan, fungal, bacterial, mycoplasmal, It can be determined that you are suffering from an infectious disease caused by rickettsia, chlamydia, viruses, etc. For example, group A beta-hemolytic streptococcus (Streptococcus pyogenes), Escherichia coli, Legionella It is possible to detect the presence or absence of infection with Campylobacter, etc.

[0065] The method using the immunochromatographic test piece of the present invention is to add nitrite or This is not limited to the case where an acidic reagent and a neutralizing reagent are impregnated, but also applies to multiple immunochromatographic test pieces. That is, impregnated with 2 to n reagents with different effects that should not react during storage. It can also be used to EXAMPLES

[0066] The present invention will be described in detail with reference to the following examples. Not limited to this. In the following examples, % indicates w / v % unless otherwise specified.

[0067] Example 1 Example of immunochromatographic test strip for measuring group A streptococci The following were used as nitrite, solid acidic reagent, and neutralizing reagent: Nitrite (liquid reagent) 2.0M sodium nitrite Solid acidic reagent (impregnated into immunochromatographic test strip) 500mM citric acid, 0.0~2.5% TritonX-100 (Registered Trademark) Neutralizing reagent (impregnated into immunochromatographic test strip) 3.0M Tris hydrochloride (Trizma BASE)

[0068] 1. Nitrocellulose membrane of anti-Streptococcus pyogenes (group A beta-hemolytic streptococcus) antibody Immobilization on a support A diluted solution of anti-Streptococcus pyogenes antibody and anti-rabbit IgG antibody were prepared and then used for the PET filter. The sample pad side of the nitrocellulose membrane backed with Streptococcus pyogenes was coated with anti-Streptococcus pyogenes. The nitrosogenes antibody was applied in a line on the absorption band side, and anti-rabbit IgG antibody was applied in a line on the absorption band side. The cellulose membrane was then dried to obtain an anti-Streptococcus pyogenes antibody-immobilized membrane. In this example, this membrane is referred to as an "antibody-immobilized membrane."

[0069] 2. Immobilization of anti-Streptococcus pyogenes antibodies onto colored polystyrene particles Colored polystyrene particles were added to the anti-Streptococcus pyogenes antibody dilution solution, stirred, and then carbo Add diimide and mix further. Remove the supernatant by centrifugation, resuspend in buffer, and use anti-St A suspension of colored polystyrene particles bound to the Reptococcus pyogenes antibody was obtained. In the examples, they are referred to as "antibody-immobilized particles (colored polystyrene)".

[0070] 3. Coating and drying of anti-Streptococcus pyogenes antibody-conjugated colored polystyrene particles A predetermined amount of the antibody-immobilized colored polystyrene particle suspension prepared in step 2 is applied to a nonwoven fabric and dried. The resulting nonwoven fabric is referred to as "C. Pad" in this example.

[0071] 4. Application of neutralizing reagent (basic reagent) The above neutralizing reagent (basic reagent) was applied to a filter paper.

[0072] 5. Preparation of Solid Acid Reagent-impregnated Pads The above solid acidic reagent is applied to a non-woven fabric (polyester / polyethylene blend) made of hydrophobic material. After application, the solid acidic reagent was immediately dried to form a solid acidic reagent impregnated area. A drug-impregnated pad was obtained.

[0073] 6. Preparation of immunochromatographic test strips for detecting Streptococcus pyogenes First, place the antibody-immobilized membrane (support) on the backing sheet of the strip from the downstream for 20 It was attached to the mm part.

[0074] An absorbent strip was attached downstream of the membrane to absorb the liquid. Upstream of the membrane, antibody-conjugated colored polystyrene was applied with a 2 mm overlap between the membrane and the membrane. A 10 mm wide glass fiber (C.Pad) coated with polyethylene particles was attached.

[0075] C.Pad and the pad soaked in neutralizing reagent are overlapped by 4 mm. A neutralizing reagent-impregnated area was attached.

[0076] A top laminate sheet (60 mm) made of PET is placed on top of the immunochromatographic test piece. mm) was attached.

[0077] Finally, the pad impregnated with the solid acidic reagent (solid acidic reagent impregnated area) is A top laminate sheet is placed on the pad impregnated with the acid, and a solid acid test sample is placed on the pad. The pad impregnated with the drug and the pad impregnated with the neutralizing reagent are placed in contact with each other. It was attached to the upstream (bottom) end of the test piece.

[0078] The structure of the immunochromatographic test piece thus prepared is shown in Figure 4. Also, a schematic diagram is shown in Figure 5. The figure is shown together with the distance (mm) between each part. In addition, in Fig. 4 and Fig. 5, the solid acidic reagent The pad impregnated with the neutralizing agent and the pad impregnated with the neutralizing agent are placed between the two pads with a top laminate. Even in areas where there is no top lamination, it is shown as being separated and not in contact. The two pads are in contact where there is no seat.

[0079] Triton X-100, a solid acidic reagent (Registered Trademark) Immunochromatographic test strips with concentrations of (1) 0.0%, (2) 0.1%, (3) 0.2%, (4) 0.5%, (5) 1.0%, (6) 1.5%, (7) 2.0%, and (8) 2.5% were obtained.

[0080] Example 2 Streptococcus pyogenes was added to the immunochromatographic test strip prepared in Example 1 at 10 5 Floating in cfc / mL The sample was diluted two-fold with the 2M sodium nitrite solution and 75 μL of the diluted sample was dropped onto the sample, and the progress was observed.

[0081] The higher the concentration of surfactant, the longer it takes to start development (the time until the sample becomes visible in the test window). The time was shortened.

[0082] In addition, the extraction time of carbohydrate antigens by nitrous acid varies depending on the concentration of the surfactant, as in (3) and (4). By controlling the time, it was possible to adjust the signal strength for each deployment time (Table 1 ).

[0083] [Table 1] [Explanation of symbols]

[0084] 1. Support (including detection area) 2 Label region 3. Detection Area 5. Solid Acid Reagent Area 6 Neutralization Reagent Area 7 Absorption Bands 8 Backing sheet 9 Top Laminate Sheet 10 Sample Pad [Industrial Applicability]

[0085] Accurate detection of group A β-hemolytic streptococcus infection using the immunochromatographic device of the present invention It is possible.

Claims

1. An immunochromatographic test strip for extracting and measuring a sugar chain antigen in a sample, the immunochromatographic test strip comprising a sample pad for adding a sample mixed with nitrite or an acidic solution, a label region containing a labeled antibody obtained by labeling an antibody against a sugar chain antigen, and a detection region in which an antibody against the sugar chain antigen is immobilized, and in which an antibody-sugar chain antigen-labeled antibody complex is formed in the detection region to measure the sugar chain antigen, the immunochromatographic test strip having a region impregnated with a neutralizing reagent upstream of the label region, and further having a region impregnated with a solid acidic reagent upstream of the region impregnated with the neutralizing reagent when a sample mixed with nitrite is used, and a region impregnated with nitrite when a sample mixed with an acidic solution is used, An immunochromatographic test strip characterized by using a hydrophobic nonwoven fabric (a mixture of polyester and polyethylene) impregnated with a surfactant in an area impregnated with nitrite or a solid acidic reagent.

2. 2. The immunochromatographic test strip according to claim 1, wherein the surfactant is Triton X-100 (registered trademark).

3. 3. The immunochromatographic test strip according to claim 1, wherein a region impregnated with a solid acidic reagent or a nitrite is present on the sample pad.

4. The immunochromatographic test strip according to any one of claims 1 to 3, wherein the solid acidic reagent is selected from the group consisting of malonic acid, malic acid, maleic acid, citric acid and tartaric acid.

5. The immunochromatographic test strip according to any one of claims 1 to 4, wherein the neutralizing reagent is trishydroxylmethylaminomethane or sodium hydroxide.

6. 6. The immunochromatographic test piece 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.

7. An immunochromatographic test kit comprising the immunochromatographic test strip according to any one of claims 1 to 6 and a nitrite solution or an acidic solution.

8. A method for measuring a sugar chain antigen in a sample by immunochromatography using the immunochromatographic test strip according to any one of claims 1 to 6, comprising: mixing the specimen with a nitrite solution when the immunochromatographic test piece has an area impregnated with a solid acidic reagent, and mixing the specimen with an acidic solution when the immunochromatographic test piece has an area impregnated with a nitrite, and adding the mixture to a sample pad of the immunochromatographic test piece; 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 of measuring carbohydrate antigens in a sample by controlling the speed and direction of the development of the sample on the immunochromatographic test piece, in which an antibody-carbohydrate antigen-labeled antibody complex is formed in the detection area, and by controlling the treatment with an acidic reagent, nitrite, and neutralizing reagent.

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