Immunochromatographic test strip for extracting and measuring sugar chain antigens capable of controlling sample development by impregnating a hydrophilic material with nitrite or a solid acidic reagent

Abstract

The present invention provides an immunochromatographic test piece for extracting and measuring sugar chain antigens, which can control the development of samples by impregnating nitrite or solid acidic reagent into hydrophilic raw materials. The immunochromatographic test piece includes: a sample pad for adding a sample mixed with nitrite or an acidic solution, a label region containing a labeled antibody that labels an antibody for sugar chain antigens, and a detection region in which the antibody for sugar chain antigens is solid-phased, a complex of antibody-sugar chain antigen-labeled antibody is formed in the detection region, and sugar chain antigens are measured, an area impregnated with a neutralizing reagent is provided upstream of the label region, and further upstream of the area impregnated with the neutralizing reagent, when a sample mixed with nitrite is used, an area impregnated with a solid acidic reagent is provided, when a sample mixed with an acidic solution is used, an area impregnated with nitrite is provided, and a hydrophilic raw material is used in the area impregnated with nitrite or a solid acidic reagent.

Description

Technical Field

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

[0002] Most rapid diagnostics based on immunochromatography are widely used as a means to quickly and easily detect viral or bacterial infections and determine treatment strategies.

[0003] In general, a rapid diagnostic drug based on the principle of immunochromatography is a method of suspending a sample in a sample suspension and then supplying the suspension to an immunochromatographic test strip, thereby enabling rapid and simple measurement.

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

[0005] For example, a method has been reported in which sodium nitrite and a neutralizing agent are contained in advance in an immunochromatographic test strip, and a sample is simply suspended in an acidic solution such as acetic acid and then supplied to the immunochromatographic test strip, thereby performing nitrous acid extraction on the immunochromatographic test strip (Patent Document 1).

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

[0007] Furthermore, methods for efficiently performing nitrous acid extraction treatment of sugar chain antigens on immunochromatographic test pieces have been reported (Patent Documents 2 to 4).

[0008] However, when using a mechanism for extraction on a device, in order to achieve the target performance, a certain extraction time must be guaranteed on the device.

[0009] Prior art literature

[0010] Patent Literature

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

[0012] Patent Document 2: Japanese Patent Application Publication No. 2018-151329

[0013] Patent Document 3: Japanese Patent Application Publication No. 2018-151330

[0014] Patent Document 4: Japanese Patent Application Publication No. 2018-151331 Summary of the invention

[0015] Technical problem to be solved by the invention

[0016] Most of the immunochromatographically based group A beta-lysing Strep A antigen detection reagents currently on the market use a technique in which the acidic nitrous acid generated by mixing a nitrite solution (R1) and an acid solution (R2) is reacted by neutralizing the acidic nitrous acid with a neutralizing agent (R3) in a dry state on the immunochromatographic detection reagent (test piece). However, there is also a known technique in which the necessary preparation and extraction steps of R1 and R2 are omitted by coating R1 or R2 on a pad. In a one-step group A beta-lysing Strep A antigen detection reagent, the extraction step is performed on the test equipment, so a certain extraction time must be guaranteed after the sample is dripped. However, in order to achieve a one-step method, there is no means to solve both the reaction time and performance (sensitivity / specificity).

[0017] An object of the present invention is to provide an immunochromatographic test strip for controlling the development of a sample on an immunochromatographic test strip and appropriately controlling the treatment with an acidic reagent, nitrite, and a neutralizing reagent, and an immunochromatographic method using the test strip.

[0018] Technical solutions for solving technical problems

[0019] The inventors of the present invention have conducted intensive studies on a method for controlling the spread of a sample on an immunochromatographic test piece and appropriately controlling the treatment with an acidic reagent, nitrite, and a neutralizing reagent.

[0020] As a result, it was found that the reaction time and sensitivity can be improved by using a hydrophilic material for impregnation of a solid acidic reagent or nitrite.

[0021] It was also found that by using a raw material with a thickness of 0.2 mm to 1.0 mm and a grammage of 30 g / m 2 ~131g / m 2 raw materials, thereby improving reaction time and sensitivity.

[0022] That is, the present invention is as follows.

[0023] [1] An immunochromatographic test strip, characterized in that it is used to extract and measure sugar chain antigens in a sample, comprising: a sample pad for adding a sample mixed with nitrite or an acidic solution; a marker region comprising a marker antibody that labels an antibody against the sugar chain antigen; and a detection region in which the antibody against the sugar chain antigen is solid-phased, a complex of antibody-sugar chain antigen-marker antibody is formed in the detection region, and the sugar chain antigen is measured, wherein upstream of the marker region there is a region impregnated with a neutralizing reagent, and upstream of the region impregnated with the neutralizing reagent there is a region impregnated with a solid acidic reagent when a sample mixed with nitrite is used, and when a sample mixed with an acidic solution is used, there is a region impregnated with nitrite,

[0024] A hydrophilic raw material is used in the area impregnated with nitrite or solid acid reagent.

[0025] [2] The immunochromatographic test piece according to [1], wherein the hydrophilic raw material impregnated with nitrite or solid acid reagent has a gram weight of 30 g / m 2 ~131g / m 2 , and the thickness is 0.2mm~1.0mm.

[0026] [3] The immunochromatographic test piece according to [1] or [2], wherein the hydrophilic raw material impregnated with the nitrite or the solid acidic reagent is selected from the group consisting of glass fiber, filter paper, nonwoven fabric, and a mixture of any two or more thereof.

[0027] [4] The immunochromatographic test piece according to any one of [1] to [3], wherein the sample pad includes a region impregnated with a solid acidic reagent or nitrite.

[0028] [5] The immunochromatographic test piece according to any one of [1] to [4], wherein the solid acid reagent is selected from the group consisting of malonic acid, malic acid, maleic acid, citric acid and tartaric acid.

[0029] [6] The immunochromatographic test piece according to any one of [1] to [5], wherein the neutralizing agent is tris(hydroxymethyl)aminomethane or sodium hydroxide.

[0030] [7] The immunochromatographic test piece according to any one of [1] to [6], wherein the sugar chain antigen is a sugar chain antigen of a protozoa, fungus, bacteria, mycoplasma, rickettsia, chlamydia or virus.

[0031] [8] An immunochromatographic test kit comprising the immunochromatographic test piece described in any one of [1] to [7] and a nitrite solution or an acidic solution.

[0032] [9] A method for measuring sugar chain antigens in a sample, comprising measuring sugar chain antigens in a sample by immunochromatography using the immunochromatographic test piece according to any one of [1] to [7],

[0033] The method for measuring sugar chain antigens in a sample comprises: mixing the sample with a nitrous acid solution when the immunochromatographic test piece has an area impregnated with a solid acidic reagent, mixing the sample with an acidic solution when the immunochromatographic test piece has an area impregnated with nitrite, and adding the acidic solution to a sample pad of the immunochromatographic test piece,

[0034] In the area impregnated with the solid acidic reagent or the area impregnated with nitrite, the sugar chain antigen is extracted from the sample by the action of nitrous acid generated by the reaction of nitrite and the solid acidic reagent.

[0035] In the area impregnated with the neutralizing agent, the acidic solution containing the sugar chain antigen is neutralized.

[0036] In the detection area, a complex of antibody-sugar chain antigen-marker antibody is formed. The method for measuring sugar chain antigens in a sample controls the development speed and / or development direction of the sample on an immunochromatography test piece by immunochromatography, controls the treatment based on an acidic reagent, nitrite and a neutralizing reagent, and measures the sugar chain antigens in the sample.

[0037] This specification includes the disclosure of Japanese Patent Application No. 2019-012835 which is the basis of priority of this application.

[0038] Effects of the invention

[0039] In a one-step group A beta-lysin antigen detection reagent, the raw material for impregnation with nitrite or solid acidic reagent is selected in consideration of the thickness, gram weight, and water absorption of the raw material, so that the time required for treatment with solid acidic reagent and nitrite can be adjusted to the optimal time. As a result, an immunochromatographic test strip capable of exerting the target performance (early line color development time and high sensitivity) is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a diagram schematically showing the structure of an immunochromatographic test piece 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 piece).

[0041] Figure 2 The diagram schematically shows the structure of an immunochromatographic test piece (a single pad test piece) in which the solid acidic reagent region and the neutralizing reagent region also serve as a sample pad among the immunochromatographic test pieces having the solid acidic reagent region and the neutralizing reagent region.

[0042] Figure 3The diagram schematically shows the structure of an immunochromatographic test piece 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 the sample pad exists alone (two-pad test piece).

[0043] Figure 4 This is a diagram schematically 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.

[0044] Figure 5 The diagram schematically shows 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 distances between the respective parts. DETAILED DESCRIPTION

[0045] Hereinafter, the present invention will be described in detail.

[0046] The present invention relates to an immunochromatographic test strip or a kit comprising the immunochromatographic test strip and a nitrous acid solution or an acidic solution, which simplifies the nitrous acid extraction treatment of sugar chain antigens so that it can be performed on an immunochromatographic test strip and can rapidly and accurately measure sugar chain antigens as test substances.

[0047] The immunochromatographic test piece comprises: a support having a detection area where an antibody (antibody 1) that captures a substance to be detected (antigen, etc.) is immobilized, a labeling area having a movable labeling antibody (antibody 2), a sample pad for adding a sample, an absorption band for absorbing the sample liquid after development, a backing sheet for bonding these components together, etc.

[0048] The immunochromatographic test strip of the present invention can be stored in a storage container, which can prevent degradation caused by, for example, ultraviolet rays or moisture in the air. In addition, when using a contaminating or infectious sample specimen, the storage container can prevent the tester performing the analysis from being contaminated or infected. For example, a resin box of an appropriate size can be used as a storage container, and the device of the present invention can be stored in the box. In addition, the surface of the test strip on which the antigen or antibody is fixed can also be covered with a resin film (PET sheet). Sometimes the storage container and the test strip stored therein are collectively referred to as an immunochromatographic device.

[0049] It should be noted that the number of detection regions and the type of labeling antibody contained in the label region are not limited to one, and two or more antigens can be measured on the same test strip by using a plurality of antibodies corresponding to the detection substance.

[0050] The support is a material having the performance of fixing antibodies for capturing the detected substance (antigen), and has the performance of not hindering the passage of liquid in the horizontal direction. It is preferably a porous film (diaphragm) with capillary action, a material that can be transported by absorbing liquid and the components dispersed therein. The material forming the support is not a particularly limited material, for example, cellulose, nitrocellulose, cellulose acetate, polyvinylidene fluoride (PVDF), glass fiber, nylon, polyketone, etc. can be cited. Among them, nitrocellulose is preferably used as a material of the film or diaphragm. The diaphragm on which the antibody is fixed is called an antibody immobilized diaphragm.

[0051] The marker region is formed by a porous substrate containing a marker antibody, and the material of the substrate can be glass fiber, nonwoven fabric, etc., which are generally used. The substrate is impregnated with a large amount of marker antibodies, so it is preferably in the form of a pad with a thickness of about 0.3 mm to 0.6 mm. The porous substrate impregnated with the marker antibody and dried is also called a dry pad.

[0052] The labeling of labeled antibodies often uses enzymes such as alkaline phosphatase or horseradish peroxidase, metal colloids such as colloidal gold, silica particles, cellulose particles, magnetic particles, fluorescent particles, dyed polystyrene particles, and dyed latex particles. When dyed particles such as metal colloid particles, dyed polystyrene particles, and dyed latex particles are used, these labeling agents aggregate to cause staining, and the staining is measured. The particles to which the antibodies are fixed are called antibody-immobilized particles.

[0053] The detection region refers to a region of a portion of the support body where an antibody for capturing the substance to be detected (antigen) is fixed. The detection region is provided with at least one region where an antibody for capturing the antigen is fixed. The detection region may be included in the support body, and the antibody may be fixed on the support body.

[0054] The sample pad is a porous material used to add a sample. The sample pad is located at the uppermost part of the immunochromatographic test piece. The material can use filter paper, glass fiber, nonwoven fabric, etc. that are generally used. In order to be used for immunoassays of a large number of samples, it is preferably a pad with a thickness of about 0.3 mm to 1 mm. The sample also includes a sample prepared using a sample such as a sample obtained by suspending the sample in another solution.

[0055] The absorption band is used to absorb the components supplied to the support and not involved in the reaction in the detection area. The material can be filter paper, sponge, etc. with high water retention formed from general natural polymer compounds, synthetic polymer compounds, etc., but in order to promote the development of the sample, a highly water-absorbent material is preferred.

[0056] The backing sheet is a component for attaching and fixing all the aforementioned materials, i.e., the support, sample pad, marker area, absorption band, etc., in an overlapping manner. As long as these materials are arranged and fixed at the most suitable intervals, the backing sheet is not necessary, but it is generally preferred to use it from the convenience of manufacturing or use.

[0057] In the immunochromatographic test piece of the present invention, there may further be a control display area (component). The control display area is a part that shows that the test is correctly implemented. For example, the control display area is present downstream of the detection area, and the sample specimen passes through the detection area and sends a signal by staining when it reaches the control display area. In the control display area, a substance that binds to an antibody bound to a labeled carrier may be solid-phased, and a reagent such as a pH indicator that changes color when the sample arrives may also be solid-phased. In the case where the antibody bound to the labeled carrier is a mouse monoclonal antibody, an anti-mouse IgG antibody can be used.

[0058] The size of the immunochromatographic test piece is not limited, and may be, for example, several centimeters to tens of centimeters in length and several millimeters to several centimeters in length.

[0059] In the test piece of the above-mentioned form, the sample passes through a porous flow path formed by a series of connections such as a sample pad, a marker area, a support body, a detection area, and an absorption band. Therefore, in this form, these all become sample movement areas. Through the material and form of each constituent material, it is possible to have a form in which the sample passes on the surface without penetrating the inside of the material, but the sample movement area defined in this specification is not related to the inside or surface of the material, so the test piece of this form is also included in the scope of this specification.

[0060] When the sugar chain antigen in the sample is measured using the immunochromatographic test piece of the present invention, it is first necessary to extract the sugar chain antigen in the sample. The extraction of the sugar chain antigen is performed by treating the sample containing the sugar chain antigen with nitrous acid. Nitrous acid can be produced by mixing nitrites such as sodium nitrite with an acid, and the sample containing the sugar chain antigen is treated with nitrous acid produced by such an operation. The extracted antigen is combined with the antibody immobilized on the immunochromatographic test piece through an antigen-antibody reaction. At this time, when the acid remains in the reaction system, the reaction system is acidic, which hinders the antigen-antibody reaction. Therefore, it is necessary to neutralize the acid in the reaction system.

[0061] In the present invention, after nitrite and acid are mixed to generate nitrous acid, sugar chain antigens in the sample are extracted by the nitrous acid and the acid is neutralized, the sugar chain antigens are bound to the antibody solid-phased on the immunochromatographic test piece and the sugar chain antigens are measured. In such a method, the following methods can be cited as methods for extracting sugar chain antigens and measuring. In any method, the extraction and neutralization of sugar chain antigens by nitrous acid are both performed on the immunochromatographic test piece. In order to perform the extraction of sugar chain antigens by nitrous acid on the immunochromatographic test piece, it is sufficient to include an acidic reagent or nitrite on the immunochromatographic test piece in advance. In order to perform neutralization on the immunochromatographic test piece, it is sufficient to impregnate the immunochromatographic test piece with a neutralizing reagent in advance.

[0062] (A) The sample and the acidic solution are mixed in advance and added to the sample pad of the immunochromatographic test piece impregnated with nitrite and a neutralizing agent. When the mixed solution reaches the area impregnated with nitrite, the nitrite reacts with the acid to produce nitrous acid, thereby extracting the sugar chain antigen in the sample. The extract of the sugar chain antigen is neutralized in the area impregnated with the neutralizing agent on the immunochromatographic test piece, and the sugar chain antigen is combined with the antibody solid-phased on the immunochromatographic test piece and can be detected. In this method, the acidic solution used may include acetic acid, hydrochloric acid, malonic acid, malic acid, maleic acid, citric acid, tartaric acid, and the like.

[0063] (B) The sample and nitrite solution are mixed in advance, and the sample pad of the immunochromatographic test piece impregnated with an acidic reagent and a neutralizing reagent is added. When the mixed solution reaches the area impregnated with the acidic reagent, the nitrite reacts with the acid to produce nitrous acid, thereby extracting the sugar chain antigen in the sample. The sugar chain antigen extract is neutralized in the area impregnated with the neutralizing reagent on the immunochromatographic test piece, and the sugar chain antigen binds to the antibody immobilized on the immunochromatographic test piece and can be detected.

[0064] In order to carry out the immunochromatographic test piece of the present invention of the method of (A) or (B) mentioned above, the immunochromatographic test piece that is impregnated with nitrite upstream of the marker area (upstream of the sample flow, there is a sample pad side), i.e., in the sample pad or between the sample pad and the marker area, can be used for the method of (A) mentioned above. In addition, the immunochromatographic test piece that is impregnated with an acidic reagent in the sample pad or between the sample pad and the marker area can be used for the method of (B) mentioned above. It should be noted that, as an acidic reagent, a solid acidic reagent can be used. By these methods, regardless of the amount of the test substance in the test sample supplied to the test sample, specificity can be correctly determined.

[0065] The region impregnated with the solid acidic reagent is referred to as a solid acidic reagent region, the region impregnated with the acidic reagent is referred to as an acidic reagent region, and the region impregnated with the neutralizing reagent is referred to as a neutralizing reagent region.

[0066] As for the solid acidic reagent or nitrite, it can be impregnated into the sample pad, or it can be impregnated into a pad formed of a porous material such as a non-woven fabric other than the sample pad. The porous material impregnated with the solid acidic reagent or the porous material impregnated with the nitrite can be arranged between the sample pad and the marker area, that is, on the upstream side of the marker area. Here, the area impregnated with the solid acidic reagent or nitrite and the sample pad or the marker area can be in contact or not in contact. In the present invention, the area impregnated with the reagent is also referred to as a pad impregnated with the reagent.

[0067] The neutralizing agent is arranged at a place downstream of the area impregnated with the solid acidic agent or nitrite. As for the neutralizing agent, it can be impregnated in the support body, or it can be impregnated in a pad formed of a porous material such as a non-woven fabric other than the support body, and the porous material impregnated with the neutralizing agent can be arranged between the area impregnated with the solid acidic agent or nitrite and the marking body area. That is, there is an area impregnated with the neutralizing agent upstream of the marking body area, and then there is an area impregnated with the solid acidic agent or nitrite upstream of the area impregnated with the neutralizing agent.

[0068] Here, the region impregnated with the neutralizing agent and the region impregnated with the solid acidic agent or nitrite or the marker region may or may not be in contact with each other.

[0069] As the raw material of the porous material of the region impregnated with the solid acid reagent or nitrite, a hydrophilic raw material is used. Specifically, glass filter paper formed by glass fiber, filter paper formed by cotton, non-woven fabric formed by rayon, non-woven fabric formed by nylon, or a mixture of any two or more of these hydrophilic raw materials, filter paper, non-woven fabric, etc., or a mixture of these hydrophilic raw materials and hydrophobic raw materials, filter paper, non-woven fabric, etc. can be cited.

[0070] The raw material of the porous material used in the area impregnated with the solid acid reagent or nitrite preferably has a gram weight of 30 g / m 2 ~131g / m 2 In addition, the raw material preferably has a thickness of 0.2 mm to 1.0 mm.

[0071] Specific examples of the raw material of the porous material of the region to be impregnated with the neutralizing agent include filter paper made of cotton, glass filter paper made of glass fiber, and the like.

[0072] The immunochromatographic test piece having a solid acidic reagent region or a nitrite region and a neutralizing reagent region has a sample pad, a solid acidic reagent region or a nitrite region, a neutralizing reagent region, a marker region, a detection region and an absorption band on a support body from upstream, and may have a solid acidic reagent region or a nitrite region on the sample pad. In addition, as for the sample pad, the solid acidic reagent region or the nitrite region, the neutralizing reagent region, the marker region, the detection region and the absorption band, adjacent regions may or may not be in contact with each other.

[0073] The solid acidic reagent used in the present invention is a solid substance at room temperature and a non-volatile substance at high temperature.

[0074] Preferred solid acidic reagents used in the present invention include malonic acid, malic acid, maleic acid, citric acid, and tartaric acid.

[0075] In addition, as the preferred solid acidic reagent used in the present invention, as long as use the acid with many valences such as citric acid, promptly can extract with less quantity.In addition, if the valence is the same, then the acid with smaller acid dissociation constant, such as maleic acid, tartaric acid efficiency is good.

[0076] Furthermore, as a preferred solid acidic reagent used in the present invention, a reagent that does not stain the immunochromatographic test piece, specifically, a reagent that is white in a dry state or that is difficult to stain under drying heat or oxidation is preferred.

[0077] Examples of the nitrite used in the present invention include sodium nitrite and potassium nitrite.

[0078] The amount of the solid acidic reagent or nitrite used in the present invention, i.e., the amount impregnated into the immunochromatographic test piece, is not particularly limited. Usually, the amount per immunochromatographic test piece is about 0.01 μg to 1 mg, preferably about 0.1 μg to 0.1 mg. It is most preferred to select the most suitable amount that can achieve the effect according to the type of solid acidic reagent or nitrite used, the composition of the sample suspension, and the amount added.

[0079] In order to impregnate the sample pad or porous material with the solid acid reagent or nitrite, the solid acid reagent or nitrite is first dissolved, applied and dried. For example, a 0.1 to several M solid acid reagent or nitrite solution may be applied and dried.

[0080] The neutralizing agent used in the present invention is a substance that is solid at room temperature and does not volatilize at high temperature. The neutralizing agent is also called an alkaline agent.

[0081] Preferred neutralizing agents used in the present invention include Tris base (trishydroxymethylaminomethane), sodium hydroxide, dipotassium hydrogen phosphate, trisodium citrate, and Good's buffer having a buffering capacity in the alkaline region.

[0082] The amount of the neutralizing agent used in the present invention, i.e., the amount impregnated into the immunochromatographic test piece, is not particularly limited. Usually, the amount per immunochromatographic test piece is about 0.01 μg to 1 mg, preferably about 0.1 μg to 0.1 mg. It is most preferred to select the most suitable amount that can achieve the effect according to the type of neutralizing agent used, the composition of the sample suspension, the amount added, etc.

[0083] In order to impregnate the porous material with the neutralizing agent, the neutralizing agent may be dissolved, the solution may be applied to the porous material, and then dried. For example, 0.1 to several M, preferably 0.5 to several M, of the neutralizing agent solution may be applied and dried.

[0084] Figures 1 to 3 This is a diagram showing a preferred embodiment of a typical immunochromatographic test piece. Figure 1 The immunochromatographic test piece shown is an immunochromatographic test piece impregnated with a solid acidic reagent and a neutralizing reagent, but it can also be impregnated with nitrite instead of the solid acidic reagent. In this case, it becomes an immunochromatographic test piece impregnated with nitrite and a neutralizing reagent. That is, in the following description of the test piece, there is a case where a nitrite reagent area is provided instead of a solid acidic reagent area. If a person skilled in the art is present, it is possible to appropriately design and manufacture an immunochromatographic test piece impregnated with nitrite and a neutralizing reagent. It should be noted that the immunochromatographic test piece is not limited to Figures 1 to 3 In the figure, 1 refers to the support, 2 refers to the marking area, 3 refers to the detection area, 7 refers to the absorption band, and 8 refers to the backing sheet.

[0085] Figure 1 and 2 The test piece in which the solid acid reagent area 5 also serves as the sample pad 10 is shown. Figure 1 A is the top view, Figure 1 B is a cutaway sectional view. Figure 1 In the example, there is a solid acidic reagent area 5 and / or a neutralizing reagent area 6 upstream of the marker area 2, which overlap to form a continuous lateral flow chromatography flow path. Figure 1 In the test piece shown, the solid acid reagent area 5 also serves as a sample pad 10. That is, the solid acid reagent area 5 is present on the sample pad 10. On this test piece, the solid acid reagent area 5 and the neutralizing reagent area 6 are respectively arranged on two sheets of porous materials (pads), so it is sometimes called a two-pad test piece. In the upstream of the solid acid reagent area, a sample pad can be present. The solid acid reagent area and the sample pad can exist separately.

[0086] At once Figure 2 In the test piece, the sample pad 10 serves as both the solid acidic reagent area 5 and the neutralizing reagent area 6 , and the solid acidic reagent area 5 and the neutralizing reagent area 6 exist on the sample pad 10 . Figure 2 A is the top view, Figure 2 B is a cutaway sectional view, Figure 2 C shows the positional relationship between the neutralization reagent area 6 and the solid acidic reagent area 5 or the nitrous acid reagent area. In this test piece, the solid acidic reagent area 5 and the neutralization reagent area 6 are provided on a single porous material (pad), so it is sometimes called a single pad test piece.

[0087] At once Figure 3 For the test piece, the sample pad 10 exists separately from the solid acidic reagent area 5 and the neutralizing reagent area 6. The solid acidic reagent area 5 also serves as the neutralizing reagent area 6, and the sample pad exists alone. The sample pad 10 is not impregnated with a reagent and functions only as a sample pad. Figure 3 A is the top view, Figure 3 B is a cutaway sectional view, Figure 3 C shows the positional relationship between the neutralization reagent area 6 and the solid acidic reagent area 5 or the nitrous acid reagent area. Figure 3 For the test piece, the porous material (pad) serving as the solid acid reagent area and the neutralizing reagent area and the sample pad are provided on two separate porous materials (pads), so it can be called a two-pad test piece. Although not shown in the figure, the sample pad 10, the solid acid reagent area 5 and the neutralizing reagent area 6 can exist separately.

[0088] The measurement is started by bringing the sample or the specimen prepared using the sample into contact with the nitrite solution and mixing it, suspending the sample in the nitrite solution, and adding and supplying it to the sample pad 10, the sample pad 10 serving as the solid acidic reagent area 5, or the sample pad 10 serving as both the neutralization reagent area 6 and the solid acidic reagent area 5. At this time, 5 to 100 μL of the sample and 0.01 to 2 mL of 0.1 M to 8 M nitrite are mixed, and 5 to 200 μL are supplied to the sample pad 10, the sample pad 10 serving as the solid acidic reagent area 5, or the sample pad 10 serving as both the neutralization reagent area 6 and the solid acidic reagent area 5. Examples of nitrite include sodium nitrite and potassium nitrite.

[0089] The sample containing the sugar chain antigen as the substance to be detected supplied to the sample pad 10, the sample pad 10 that doubles as the solid acidic reagent area 5, or the sample pad 10 that doubles as the neutralizing reagent area 6 and the solid acidic reagent area 5 is developed to the sample pad 10, the sample pad 10 that doubles as the solid acidic reagent area 5, or the sample pad 10 that doubles as the neutralizing reagent area 6 and the solid acidic reagent area 5 and the neutralizing reagent area 6 by capillary action. At this time, between the solid acidic reagent area 5 and the neutralizing reagent area 6, there is a top laminate sheet as a PET sheet. Thus, the flow of the sample from the solid acidic reagent area 5 to the neutralizing reagent area 6 can be suppressed, and the sample that has temporarily entered the neutralizing reagent area 6 can be prevented from flowing back to the solid acidic reagent area 5. Next, the sample is developed horizontally to the marker area 2, the support body 1, and the absorption band 7 in sequence. In the solid acidic reagent area 5, the nitrite mixed in the sample reacts with the solid acidic reagent on the solid acidic reagent area 5 to produce free nitrous acid, and the sugar chain antigen is extracted from the sample by the action of the nitrous acid. The extracted sugar chain antigen moves to the neutralizing reagent area 6 together with the acidic developing solution, and the pH of the acidic developing solution containing the sugar chain antigen is neutralized in the neutralizing reagent area 6 and adjusted to the neutral region. As a result, the sugar chain antigen moves downstream under neutral conditions. In the marker area 2, while the sample sample is being developed, the labeled antibody is released into the liquid and developed toward the support 1. In the case where the sugar chain antigen exists in the sample sample, the sugar chain antigen is specifically captured by the capture antibody in the detection area 3 of the support 1. In addition, the sugar chain antigen also forms a complex with the labeled antibody through a specific reaction. Thus, a sandwich body of the antibody via the sugar chain antigen is realized in the detection area 3, and the labeled antibody-sugar chain antigen complex can be measured in the detection area 3.

[0090] According to the method using the immunochromatographic test strip of the present invention, sugar chain antigens in a sample can be extracted on the immunochromatographic test strip, so there is no need to extract sugar chain antigens in the sample before measurement using the immunochromatographic test strip, and sugar chain antigens in the sample can be measured in one step.

[0091] In the method using the immunochromatographic test piece of the present invention, the biological sample serving as a sample is not particularly limited, and examples thereof include body fluids such as serum, plasma, blood, urine, feces, saliva, tissue fluid, cerebrospinal fluid, and swab fluid, or dilutions thereof.

[0092] In the method using the immunochromatographic test piece of the present invention, the substance to be detected that is the object of determination is a sugar chain antigen that can be determined by utilizing immunoassay, i.e., analysis of antigen-antibody reaction. As antigens, polysaccharides that can be extracted by nitrous acid extraction treatment and are present in the cell wall of bacteria as sugar chain antigens can be cited. Protozoa, fungi, bacteria, mycoplasmas, rickettsia, chlamydia, viruses, etc. containing these substances can also be determined. By using the method of the immunochromatographic test piece of the present invention, it is possible to confirm whether the biological sample of the subject contains sugar chain antigens from protozoa, fungi, bacteria, mycoplasmas, rickettsia, chlamydia, viruses, etc., and in the case of containing sugar chain antigens, it is possible to judge whether the subject suffers from an infection caused by protozoa, fungi, bacteria, mycoplasmas, rickettsia, chlamydia, viruses, etc. For example, it is possible to detect the presence or absence of infection by group A beta-hemolytic streptococcus (Streptococcus pyogenes), Escherichia coli, Legionella, Campylobacter, etc.

[0093] The method of using the immunochromatographic test piece of the present invention is not limited to the case where nitrite or an acidic reagent and a neutralizing reagent are impregnated into the immunochromatographic test piece, and can also be used when a plurality of reagents, namely 2 to n reagents having different effects and to avoid reactions during storage, are impregnated into the immunochromatographic test piece.

[0094] Example

[0095] The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.

[0096] In the following examples, unless otherwise specified, % represents weight to volume ratio (w / v %).

[0097] Example 1

[0098] Example of immunochromatographic test piece for group A streptococcal lysogeny

[0099] The following substances were used as nitrite, solid acid reagent and neutralizing agent.

[0100] Nitrite (liquid reagent) 2.0M sodium nitrite

[0101] Solid acid reagent (impregnated into immunochromatographic test strip) 500mM citric acid

[0102] Neutralizing agent (impregnated into immunochromatographic test strip) 3.0MTris base (Trizma BASE)

[0103] 1. Immobilization of anti-Streptococcus pyogenes (group A β-hemolytic streptococcus) antibodies on nitrocellulose membrane (support)

[0104] A dilution of anti-group A β-hemolytic streptococcus antibody and anti-rabbit IgG antibody were prepared, and anti-group A β-hemolytic streptococcus antibody was linearly coated on the sample pad side of a nitrocellulose membrane sheet lined with a PET film, and anti-rabbit IgG antibody was linearly coated on the absorption band side. After that, the nitrocellulose membrane sheet was dried to obtain an anti-group A β-hemolytic streptococcus antibody-immobilized membrane sheet. In this example, this membrane sheet is referred to as an "antibody-immobilized membrane sheet".

[0105] 2. Immobilization of Anti-Group A β-Hemolytic Streptococcus Antibodies on Dyed Polystyrene Particles

[0106] Dyed polystyrene particles were added to the anti-group A β-hemolytic streptococcus antibody dilution solution, stirred, and then carbodiimide was added, and further stirred. The supernatant was removed by centrifugation, and the suspension was resuspended in a buffer solution to obtain an anti-group A β-hemolytic streptococcus antibody-bound dyed polystyrene particle suspension. In this example, the particles are referred to as "antibody-immobilized particles (dyed polystyrene)".

[0107] 3. Coating and Drying of Anti-Group A β-Hemolytic Streptococcus Antibody-Conjugated Dyed Polystyrene Particles

[0108] A predetermined amount of the antibody-immobilized dyed polystyrene particle suspension prepared in 2 was applied to a nonwoven fabric and dried. The obtained nonwoven fabric is referred to as "C.Pad" in this example.

[0109] 4. Application of neutralizing agent (alkaline agent)

[0110] The above-mentioned neutralizing agent (alkaline agent) is applied to filter paper.

[0111] 5. Preparation of solid acid reagent impregnation pad

[0112] The solid acidic agent is applied to two materials, a hydrophobic nonwoven fabric (polyester / polyethylene blend) and a hydrophilic glass fiber, and dried immediately after application to obtain a solid acidic agent impregnation pad as a solid acidic agent impregnation area.

[0113] 6. Preparation of immunochromatographic test strips for detection of group A β-hemolytic streptococci

[0114] First, the antibody-immobilized membrane (support) was attached to the strip-shaped backing sheet at a portion 20 mm downstream.

[0115] Downstream of the membrane, an absorbent tape is attached for absorbing the liquid.

[0116] A 10 mm wide glass fiber (C.Pad) coated with antibody-binding dyed polystyrene particles was attached upstream of the membrane so as to overlap the membrane by 2 mm.

[0117] The pad impregnated with the neutralizing agent (region impregnated with the neutralizing agent) was attached so that the overlap between the C.Pad and the pad impregnated with the neutralizing agent became 4 mm.

[0118] A top laminate sheet (60 mm) which is a PET sheet was attached so as to be aligned with the upper part of the immunochromatographic test piece.

[0119] Finally, on the pad impregnated with the neutralizing reagent, the pad impregnated with the solid acid reagent (the area impregnated with the solid acid reagent) is aligned and attached to the upstream (lower end) of the immunochromatographic test piece in such a manner that the top laminate sheet is interposed between them and the pad impregnated with the solid acid reagent is in contact with the pad impregnated with the neutralizing reagent.

[0120] The structure of the immunochromatographic test piece prepared in this way is shown in FIG. Figure 4 In addition, Figure 5 Mode Figure 1 The distances (mm) between the various parts are shown in the figure. Figure 4 and Figure 5 In the figure, the pad impregnated with the solid acidic agent and the pad impregnated with the neutralizing agent are shown as being separated without contact even at a portion where no top laminate is present between the two pads, but in reality, the two pads are in contact at the portion where no top laminate is present.

[0121] Example 2

[0122] 75 μL of a sample prepared by diluting a 2M sodium nitrite solution containing group A β-hemolytic streptococci suspended therein to 2 times was added dropwise to the immunochromatographic test strip prepared in Example 1 to compare the minimum detection sensitivity.

[0123] As a result, as shown in Table 1, the sensitivity of glass fiber is about 4 to 8 times higher than that of polyester.

[0124] [Table 1]

[0125]

[0126] Example 3

[0127] In 5. Preparation of a solid acid reagent-impregnated pad according to the method of Example 1, a nonwoven fabric made of a hydrophilic material (cotton, glass fiber, rayon) and a hydrophobic material (polyester, polyester / polyethylene blend) is used to prepare an immunochromatographic test piece.

[0128] Example 4

[0129] 75 μL of a 2M sodium nitrite solution in which group A β-hemolytic streptococci were suspended was dropped onto the immunochromatographic test strip prepared in Example 3, and the reaction was observed for 5 minutes.

[0130] As shown in Table 2, compared with the hydrophobic raw materials, the hydrophilic materials all showed a stronger reaction 4 minutes after the droplet was applied.

[0131] [Table 2]

[0132]

[0133] ND: Not Determined (no color was developed on the control line, so it cannot be determined)

[0134] Dropping port: The time it takes for the sample liquid to be completely absorbed by the pad and disappear from the dropping port

[0135] Explanation of symbols

[0136] 1 Support body (including detection area)

[0137] 2 Logo area

[0138] 3 Detection area

[0139] 5 Solid acid reagent area

[0140] 6 Neutralization reagent area

[0141] 7 Absorption band

[0142] 8 Bottom lining

[0143] 9 Top laminate

[0144] 10 Sample pad

[0145] Industrial Availability

[0146] The immunochromatographic device of the present invention can correctly detect infection of group A beta-hemolytic streptococci.

[0147] All publications, patents and patent applications cited in this specification are incorporated into this specification by reference in their original form.

Claims

1. An immunochromatographic test strip, characterized in that: Used to extract and measure sugar chain antigens in samples, this immunochromatographic test piece contains: Sample pad for adding sample mixed with nitrite or acid solution; a marker region comprising a marker antibody that labels an antibody against a sugar chain antigen; and In the detection area, an antibody against the sugar chain antigen is solid-phased. A complex of antibody-sugar chain antigen-labeled antibody is formed in the detection area, and the sugar chain antigen is measured. There is a region impregnated with a neutralizing agent upstream of the marker region, and further upstream of the region impregnated with the neutralizing agent, there is a region impregnated with a solid acidic agent when a sample mixed with nitrite is used, and there is a region impregnated with nitrite when a sample mixed with an acidic solution is used, In the area impregnated with nitrite or solid acid reagent, a hydrophilic raw material is used, and the hydrophilic raw material has a gram weight of 30g / m 2 ~131g / m 2 , and the hydrophilic raw material with a thickness of 0.2mm~1.0mm is a filter paper formed of cotton; Nonwoven fabric formed of nylon; And filter paper or nonwoven fabric that is a mixture of any two or more hydrophilic raw materials selected from the group consisting of glass filter paper formed of glass fiber, filter paper formed of cotton, nonwoven fabric formed of rayon, and nonwoven fabric formed of nylon.

2. The immunochromatographic test piece according to claim 1, wherein There is an area on the sample pad impregnated with a solid acidic reagent or nitrite.

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

4. The immunochromatographic test piece according to claim 1 or 2, wherein The neutralizing agent is tris(hydroxymethyl)aminomethane or sodium hydroxide.

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

6. An immunochromatographic test kit, The method comprises the immunochromatographic test piece according to any one of claims 1 to 5 and a nitrite solution or an acidic solution.

7. A method for determining sugar chain antigens in a sample, Using the immunochromatographic test piece according to any one of claims 1 to 5, measuring sugar chain antigens in a sample by immunochromatography, The method for determining sugar chain antigens in a sample comprises: When the immunochromatographic test piece has a region impregnated with a solid acidic reagent, the sample is mixed with a nitrous acid solution, and when the immunochromatographic test piece has a region impregnated with a nitrite, the sample is mixed with an acidic solution and added to the sample pad of the immunochromatographic test piece. In the area impregnated with the solid acidic reagent or the area 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 area impregnated with the neutralizing agent, the acidic solution containing the sugar chain antigen is neutralized. In the detection area, a complex of antibody-sugar chain antigen-labeled antibody is formed. The method for measuring sugar chain antigens in a sample controls the development speed and / or development direction of the sample on an immunochromatographic test piece by immunochromatography, controls the treatment based on an acidic reagent, nitrite and a neutralizing reagent, and measures the sugar chain antigens in the sample.

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