Measurement sample diluent and immunoassay kit

Abstract

To provide a measurement sample diluent capable of suppressing non-specific reactions.SOLUTION: The present invention relates to a measurement sample diluent to be used to immunologically detect an antigen contained in a sample. The measurement sample diluent contains an inorganic salt and has a dispersion coefficient D of 2400 to 3500 as calculated using the following formula (1): (Dispersion Coefficient D)=(inorganic salt concentration (mM) in the measurement sample diluent)×(pH of the measurement sample diluent) ...(1).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a test sample dilution solution. More specifically, the present invention relates to a test sample dilution solution used in virus detection by immunochromatography, and an immunoassay kit including the test sample dilution solution. [Background technology]

[0002] Patent Document 1 discloses a non-specific reaction suppressor that contains an immunoglobulin derived from the same animal species as the animal from which the antibody used in the immunoassay is derived, and a rabbit immunoglobulin.

[0003] Patent Document 2 describes the suppression of non-specific reactions by using an immunoglobulin that is derived from the same producing animal species as the capture substance and has the same subclass. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2010-025866 A [Patent Document 2] JP 2009-133739 A Summary of the Invention [Problem to be solved by the invention]

[0005] Both of the methods described in Patent Documents 1 and 2 aim to suppress non-specific reactions by examining the types of immunoglobulins, but the present inventors have found a problem that such an approach may not be able to completely suppress non-specific reactions. [Means for solving the problem]

[0006] As a result of intensive research to solve the above problems, the inventors discovered that nonspecific reactions can be suppressed when the inorganic salt concentration in the measurement sample dilution solution and the pH of the measurement sample dilution solution satisfy a certain relationship, and thus completed the present invention.

[0007] That is, a typical configuration of the present invention is as follows. [Section 1] A measurement sample dilution solution used for detecting an antigen contained in a specimen by an immunological method, comprising: The measurement sample dilution solution contains an inorganic salt, and The following formula (1): Dispersion coefficient D = inorganic salt concentration in the diluted solution (mM) × pH of the diluted solution … Equation (1) The measurement sample dilution solution has a dispersion coefficient D calculated by the following formula: [Section 2] Item 2. The measurement sample dilution solution according to Item 1, wherein the dispersion coefficient D is 2,400 to 3,000. [Section 3] Item 3. The measurement sample dilution solution according to item 1 or 2, wherein the inorganic salt is at least one selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, and magnesium chloride. [Section 4] Item 4. The measurement sample dilution solution according to any one of Items 1 to 3, further comprising at least one selected from the group consisting of a buffering agent, a surfactant, a blocking agent, and a preservative. [Section 5] Item 5. The measurement sample dilution solution according to Item 4, wherein the surfactant is a nonionic surfactant. [Section 6] 6. The measurement sample dilution solution according to any one of items 1 to 5, wherein the specimen is at least one selected from the group consisting of a nasopharyngeal swab, a nasal swab, and a nasal aspirate. [Section 7] Item 7. The measurement sample dilution solution according to any one of items 1 to 6, wherein the antigen is at least one selected from the group consisting of influenza virus A (Flu-A), influenza virus B (Flu-B), respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). [Section 8] Item 8. The measurement sample dilution solution according to any one of Items 1 to 7, wherein the antigen is influenza virus B. [Section 9] Item 8. The measurement sample dilution solution according to any one of Items 1 to 7, wherein the immunological method is an immunochromatography method. [Section 10] Item 10. An immunochromatography kit comprising the measurement sample dilution solution according to any one of Items 1 to 9, an immunochromatography test piece, a specimen collecting tool, and a filter unit. [Section 11] A step of dissolving or suspending a specimen in the measurement sample dilution solution according to any one of items 1 to 9 to obtain a measurement sample solution or suspension; detecting an antigen contained in a specimen by an immunological method using the obtained measurement sample solution or suspension; A method for detecting an antigen, comprising: Effect of the Invention

[0008] According to the method of the present invention, non-specific reactions can be suppressed by using a measurement sample dilution solution in which the inorganic salt concentration and pH satisfy a specific relationship. [Brief description of the drawings]

[0009] [Figure 1] An example of the configuration of the kit is shown below. [Diagram 2] FIG. 2 is a side view showing an example of an immunochromatographic test piece. [Diagram 3] FIG. 1 is a plan view showing an example of an immunochromatographic test piece. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will be described in more detail below with reference to the embodiments of the present invention, but the present invention is not limited thereto. It should be understood that the terms used in this specification are used in the sense commonly used in the art unless otherwise specified. In addition, all non-patent literature and patent literature described in this specification are incorporated herein by reference. In this specification, "~" means "more than or equal to, less than or equal to", for example, if "X~Y" is described in the specification, it means "more than or equal to X, less than or equal to Y". In addition, in this specification, "and / or" means either one or both, and in this specification, the singular expression should be understood to include the concept of the plural form, unless otherwise specified.

[0011] 1. Measurement sample dilution solution An embodiment of the present invention relates to a measurement sample dilution solution used for detecting an antigen contained in a specimen by an immunological method.

[0012] The measurement sample dilution solution contains an inorganic salt. The inorganic salt is not particularly limited as long as it can achieve the effects of the present invention, and examples thereof include sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and disodium hydrogen phosphate. Preferably, the inorganic salt is at least one selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, and magnesium chloride.

[0013] The inorganic salt concentration in the measurement sample dilution solution is not particularly limited, and is preferably 10 mM to 1000 mM, more preferably 50 mM to 750 mM, and further preferably 100 mM to 500 mM. Specific values ​​of the inorganic salt concentration in the measurement sample dilution solution include, for example, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, and 450 mM, and these specific values ​​may be set as the lower limit or upper limit.

[0014] The measurement sample dilution solution may contain a buffering agent for adjusting the pH. As the buffering agent, any type of buffering agent may be used as long as it has sufficient buffering capacity in the target pH range, and examples thereof include Tris, phosphoric acid, phthalic acid, citric acid, maleic acid, succinic acid, oxalic acid, boric acid, tartaric acid, acetic acid, carbonic acid, and Good's buffer (MES, ADA, PIPES, ACES, collamine hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycine amide, and bicine). Among these, Tris, phosphoric acid, HEPES, tricine, glycine amide, and bicine are preferred, and Tris, phosphoric acid, tricine, glycine amide, and bicine are more preferred, because they have sufficient buffering capacity in the optimal pH range of the antibody used in the present invention, which is around 7.0 to 9.0.

[0015] The concentration of the buffer is not particularly limited, but is preferably 10 to 200 mM, more preferably 20 to 190 mM. If the concentration is low, the buffer may not have sufficient buffering capacity in the target pH range, and the measurement sensitivity may be significantly reduced. On the other hand, if the concentration is high, the detection particles may aggregate, making it impossible to spread downstream, or may spread unevenly, resulting in a significant reduction in measurement accuracy.

[0016] The pH of the measurement sample dilution solution is not particularly limited, and is preferably 0.1 to 13.9, more preferably 1 to 13, and even more preferably 2 to 12. Specific values ​​of the pH of the measurement sample dilution solution include, for example, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, and 13.5, and these specific values ​​may be used as the lower or upper limit.

[0017] In this specification, the dispersion coefficient D means the ratio of the inorganic salt concentration of a diluted measurement sample solution to the pH value of the diluted measurement sample solution, and is represented by the following formula (1). Dispersion coefficient D = inorganic salt concentration in the diluted solution (mM) × pH of the diluted solution … Equation (1) The value of the dispersion coefficient D is 2400-3500, and preferably 2400-3000.

[0018] The measurement sample dilution solution may further contain a surfactant. The surfactant is not particularly limited, and a surfactant known to those skilled in the art, such as a nonionic surfactant, a cationic surfactant, an anionic surfactant, or an amphoteric surfactant, may be used. However, a nonionic surfactant that improves the spreading of the measurement sample and does not affect the immune reaction is preferable. The nonionic surfactant is not particularly limited, and examples thereof include polyoxyethylene alkylphenyl ether (Triton (registered trademark) surfactant, etc.), polyoxyethylene alkyl ether (Brij (registered trademark) surfactant, etc.), polyoxyethylene sorbitan fatty acid ester (Tween (registered trademark) surfactant, etc.), polyoxyethylene fatty acid ester, sorbitan fatty acid ester, alkyl glucoside, sucrose fatty acid ester, etc. In addition, the surfactant may be used alone or in combination of two or more kinds. The concentration of the nonionic surfactant is preferably 0.01 to 5.0% by mass, more preferably 0.05 to 4.0% by mass, and even more preferably 0.1 to 3.0% by mass. If the concentration is lower than 0.01% by mass, downstream spreading may be difficult. Furthermore, the spread may become uneven, which may reduce the measurement accuracy. On the other hand, if the concentration is higher than 5.0 mass%, the detection particles and the antibodies, and / or the membrane and the antibodies, which are physically adsorbed, may become detached, which may prevent a measurement value from being obtained.

[0019] If necessary, the measurement sample dilution solution may contain blocking proteins (Blocking Peptide Fragment (BPF), bovine serum albumin (BSA), casein, etc.), salts (sodium chloride, potassium chloride, calcium chloride, magnesium chloride, aluminum chloride, etc.), and / or antibody stabilizers (monosaccharides, oligosaccharides, polysaccharides, sugar alcohols, glycerol, gluconates, amino acids, albumins, globulins, fibrous proteins, etc.).

[0020] The measurement sample dilution solution may further contain a preservative. The preservative is not particularly limited as long as it does not inhibit the antigen-antibody reaction, and any preservative known to those skilled in the art can be used, but sodium azide, methylparaben, benzylparaben, etc. are preferred, and sodium azide is more preferred. The concentration of the preservative is not particularly limited, but is preferably 0.001 to 3.0 w / v%, more preferably 0.01 to 0.5 w / v%.

[0021] The substance to be measured is not limited as long as it is a substance detectable by immunochromatography utilizing an antigen-antibody reaction. Examples of the substance to be measured include antigens (proteins, lipids, carbohydrates, etc.) derived from infectious disease-causing microorganisms. Preferably, the pathogenic microorganism of an infectious disease is a virus. As a virus, a virus causing an upper respiratory tract infection is preferable. As a virus causing an upper respiratory tract infection, for example, influenza virus A (Flu-A), influenza virus B (Flu-B) (RSV), RS virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be mentioned. More preferred antigens include the nucleocapsid protein (N protein) of influenza virus A, the N protein of influenza virus B, the fusion protein (F protein) of RS virus, and the N protein of severe acute respiratory syndrome coronavirus 2.

[0022] The specimen may be, for example, a nasopharyngeal swab, a nasal swab, a nasal aspirate, a pharyngeal scraping, sputum, a bronchial scraping, a bronchial aspirate, a pleural effusion, an ascites, urine, a blood sample (whole blood, serum, plasma), a gastric juice, or a feces. Among them, a nasopharyngeal swab, a nasal swab, a nasal aspirate, a pharyngeal scraping, sputum, a bronchial scraping, a bronchial aspirate, a pleural effusion, or the like is preferred, and a nasopharyngeal swab, a nasal swab, or a nasal aspirate is more preferred. For example, a specimen collected with a cotton swab or the like may be dissolved or suspended in a measurement sample dilution solution or the like, and used as a measurement sample solution or suspension.

[0023] The immunoassay method that can be used in the present invention is not particularly limited as long as it is a method for measuring an antigen or antibody in a sample by using an immunoassay method in which an immune reaction occurs with an antigen or antibody in the sample in a liquid phase and measurement is performed on a solid phase. Examples of such immunoassay methods include immunochromatography, RIA, EIA, immunoturbidimetry, latex agglutination, metal colloid agglutination, etc., and is preferably immunochromatography.

[0024] The dilution ratio of the measurement sample with the measurement sample dilution solution is not particularly limited, but is preferably 100 to 2000 times, and more preferably 200 to 1500 times. If the dilution ratio of the measurement sample is small, downstream development becomes difficult. In addition, the measurement sample becomes susceptible to the influence of contaminants in the measurement sample, which may reduce the measurement accuracy. On the other hand, if the dilution ratio of the measurement sample is large, the antigen concentration in the measurement sample decreases, which may reduce the measurement sensitivity.

[0025] The measurement sample dilution solution of the present invention is useful for suppressing non-specific reactions in the detection of FluB. In addition, the measurement sample dilution solution of the present invention is useful for detecting a detection target substance with high sensitivity while suppressing non-specific reactions in the simultaneous detection of Flu-A, Flu-B, SARS-CoV-2, and RSV.

[0026] 2. Immunochromatography Kit Another embodiment of the present invention relates to an immunochromatography kit including the above-mentioned measurement sample dilution solution. The immunochromatography kit is a kit for detecting an antigen or an antibody using an immunochromatography method. Components of the immunochromatography kit are not particularly limited, but preferably include an immunochromatography test piece, a specimen collection tool, and a filter unit.

[0027] The shape of the immunochromatographic test piece is not particularly limited and may be a membrane or strip on which a capture antibody is immobilized, but is preferably a membrane on which a capture antibody is immobilized. The specimen collection tool is not particularly limited and may be a swab, a flocked swab, a dropper, a syringe, etc., with a swab being preferred. The filter unit is used to apply a diluted solution of a measurement sample to the immunochromatographic test strip, and although there are no particular limitations on its shape, it is preferable that it is provided with a drop port for a sample.

[0028] 1 is an example of a preferred embodiment of the present invention. The immunochromatography kit 50 includes the above-mentioned measurement sample dilution solution 56, an immunochromatography test strip 100, a specimen collection tool 52, and a filter unit 54.

[0029] The kit 50 includes an immunochromatographic test strip 100, a specimen collecting tool 52, a filter unit 54, and a measurement sample dilution solution 56. The kit 50 is used to detect the measurement target substance described above. In the example of FIG. 1, the immunochromatographic test strip 100 is stored in a folder 30 .

[0030] The specimen collection tool 52 is, for example, a cotton swab. The cotton swab is used to collect throat swabs, nasal discharge, nasal swabs, nasal aspirates, bronchial scrapings, and the like. The measurement specimen dilution liquid 56 is as described above. The measurement specimen dilution liquid 56 is stored in a test tube 58 with a cap 59, for example, of about 1 mL to 2 mL. The test tube 58 has elasticity such that the sides come close to each other when pressed by a human finger. The filter unit 54 has a shape that can be attached to the test tube 58, and a filter is provided inside the filter unit 54.

[0031] In order to prevent clogging of the membrane, false positives, and clogging of the filter itself, the filter may be of not only one type but a combination of filters made of different materials, with different pore sizes, or with different particle retention sizes.

[0032] The pore size or retention particle size of the filter is preferably 10 to 500 μm. When several filters with different pore sizes or retention particle sizes are combined, it is preferable to arrange the filters with larger pore size or retention particle size on the upstream side and the filters with smaller pore size or retention particle size on the downstream side so that the two filters overlap, in order to avoid clogging of the specimen sample during filtration. For example, when combining two filters, it is preferable to use a filter with a larger pore size or retention particle size as the first filter and a filter with a smaller pore size as the second filter. Specifically, the pore size or retention particle size of the first filter is preferably 50 to 500 μm, and the pore size or retention particle size of the first filter is preferably 10 to 50 μm.

[0033] Regarding the material of the filters, for example, when two filters are combined, it is preferable that the filter of the test tube 58 is a foam made of polyvinyl alcohol resin, and the filter on the drip port 55 side is made of cellulose filter paper. The pore size or the retained particle size can be measured using a perm porometer.

[0034] When detecting the substance to be measured, if the specimen collecting tool 52 is a cotton swab, the specimen is collected by the cotton part of the cotton swab. Next, the cotton part of the cotton swab with the specimen attached thereto is inserted into the measurement specimen dilution liquid 56 in the test tube 58 from which the lid 59 has been removed, and the cotton part is stirred and squeezed out. The test tube 58 can be deformed by pressure from a human finger, so that the cotton part of the cotton swab in the test tube 58 can be pressed from the outside of the test tube 58 to squeeze out the attached specimen from the cotton part. The solution remaining after squeezing out the specimen in the test tube 58 becomes the measurement specimen solution. After squeezing out the specimen, the cotton swab is removed, and the filter unit 54 is set in the test tube 58. The filter unit 54 is provided with a drip port 55, and the measurement specimen solution is dripped from the drip port 55 into the measurement specimen dripping section 20.

[0035] The detection antibody is intended to be an antibody capable of binding to the detection particle and to the substance to be measured. The antibody constituting the detection antibody is not limited as long as it binds to the substance to be measured and does not prevent binding to the capture antibody. The detection antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody. The detection antibody may be of any isotype, for example, IgG, IgA, IgD, IgE, IgM, etc., but is preferably IgG. The detection antibody may be a commercially available product or may be produced by a known method. For example, when the substance to be measured is the nucleocapsid protein (N protein) of influenza virus A, the antibody constituting the detection antibody may be a mouse-derived anti-Flu-A N protein monoclonal antibody 1 (3IN5, InA245, manufactured by HyTest). For example, when the substance to be measured is the N protein of influenza virus B, the antibody constituting the detection antibody may be a mouse-derived anti-Flu-B N protein monoclonal antibody 1 (1131, manufactured by ViroStat). For example, when the substance to be measured is the fusion protein (F protein) of RS virus, the antibody constituting the detection antibody can be mouse-derived anti-RSV F protein monoclonal antibody 1 (3ReS21cc, manufactured by HYTEST Co., Ltd.). For example, when the substance to be measured is the N protein of severe acute respiratory syndrome coronavirus 2, mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 (SCV-108, manufactured by Toyobo Co., Ltd.) can be used.

[0036] The detection particles are particles for visualizing the binding between the measurement target substance and the antibody, and examples of the detection particles include cellulose-based colored fine particles, gold colloid particles, and latex particles.

[0037] Since the cellulose-based colored microparticles have a large amount of hydroxyl groups, they can hold many reactive dyes by covalent bonds, and can also maintain stable dispersibility in water even after dyeing. As the cellulose-based colored microparticles, regenerated cellulose, purified cellulose, natural cellulose, etc. can be used, and partially derivatized cellulose can also be used. It is preferable that 20 to 90% by mass of the mass of the cellulose-based colored microparticles is derived from cellulose, more preferably 20 to 80% by mass, and even more preferably 20 to 70% by mass.

[0038] The average particle diameter of the cellulose-based colored microparticles is not particularly limited, but is preferably 100 nm to 1000 nm, more preferably 200 nm to 800 nm. If the average particle diameter is large, downstream development slows down, and the measurement time becomes long. In addition, it is easily captured on the membrane, and the background itself becomes colored, making the color development at the capture antibody solid-phase part and the control line unclear. On the other hand, if the average particle diameter is small, the amount of antibody that can be physically adsorbed or chemically bound decreases, and the measurement sensitivity decreases.

[0039] Examples of cellulose-based colored microparticles include colored cellulose nanobeads (NanoAct (registered trademark)) manufactured by Asahi Kasei Corporation. The color of the cellulose-based colored microparticles is not particularly limited, but examples include red, orange, yellow, green, blue, navy blue, purple, black, and fluorescent. In particular, red and blue are preferred because the OD of NanoAct (registered trademark) Red (RE2) at a particle concentration of 1% by mass is 240, the OD of NanoAct (registered trademark) Blue (BL2) at a particle concentration of 1% by mass is 265, the OD of NanoAct (registered trademark) Green (GR1) at a particle concentration of 1% by mass is 160, and the OD of NanoAct (registered trademark) Black (KR1) at a particle concentration of 1% by mass is 150.

[0040] For example, when detecting a plurality of different types of analyte substances in one immunochromatographic test piece, each detection antibody may be labeled with detection particles of different colors.

[0041] The method of binding the antibody constituting the detection antibody to the cellulose-based colored microparticles is known. The antibody constituting the detection antibody and the cellulose-based colored microparticles can be mixed and left to stand at room temperature to about 37° C. for a certain period of time, thereby binding the two.

[0042] The capture antibody is an antibody that is immobilized on a membrane and captures a complex of the analyte and the detection antibody bound to the cellulose-based colored microparticles (hereinafter also referred to as the "detection antibody-analyte complex"). The capture antibody is not limited as long as it binds to the analyte and does not interfere with the binding to the detection antibody. The capture antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody. The capture antibody may be of any isotype, for example, IgG, IgA, IgD, IgE, IgM, etc., but is preferably IgG. The capture antibody may be a commercially available product or may be produced by a known method. For example, when the analyte is the nucleocapsid protein (N protein) of influenza virus A, the antibody constituting the detection antibody may be a mouse-derived anti-Flu-A N protein monoclonal antibody 2 (100083, manufactured by Medix Biocamica). For example, when the substance to be measured is the N protein of influenza virus B, the antibody constituting the detection antibody can be mouse-derived anti-Flu-B N protein monoclonal antibody 2 (3IF18 / RIF17, R2 / 3, manufactured by HyTest). For example, when the substance to be measured is the F protein of RS virus, the antibody constituting the detection antibody can be mouse-derived anti-RSV F protein monoclonal antibody 2 (C01773M, manufactured by Meridian). For example, when the substance to be measured is the N protein of severe acute respiratory syndrome coronavirus 2, the antibody constituting the detection antibody can be mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 2 (SCV-109, manufactured by Toyobo).

[0043] The antibody constituting the detection antibody and the capture antibody may be the same or different, but it is more preferable that they are different.

[0044] The animals from which the antibodies constituting the detection antibodies and the capture antibodies are obtained are not limited. Examples include rabbits, mice, rats, guinea pigs (guinea pigs), pigs, camels, etc. It is preferable that the animals from which the antibodies constituting the detection antibodies and the capture antibodies are obtained do not include goats and horses.

[0045] The configuration of immunochromatographic test strip 100 will be described with reference to Figures 2 and 3. Figure 2 shows a side view of immunochromatographic test strip 100. Figure 3 shows a plan view of immunochromatographic test strip 100.

[0046] For example, in the immunochromatographic test piece 100, the side where the measurement sample solution is dropped at the start of the test (the left side in FIG. 2 and FIG. 3) is the upstream side. In FIG. 2 and FIG. 3, from the upstream side, a sample pad 1 having a measurement sample dropping portion 20 and into which the measurement sample dropped to the measurement sample dropping portion 20 permeates, a conjugation pad 2 carrying a detection antibody, a membrane 3 on which a capture antibody is immobilized, and an absorption pad 4 absorbing the measurement sample solution that has passed through the membrane 3 are arranged in this order. Here, the term "supporting" refers to the fact that immunoglobulin derived from a goat or horse can move along the flow of the permeated water when the water permeates, and is distinguished from immobilization. The membrane 3 is illustratively provided with a first capture antibody immobilization portion 6, a second capture antibody immobilization portion 7, a third capture antibody immobilization portion 8, a fourth capture antibody immobilization portion 9, and a control capture antibody immobilization portion 10. In each of the capture antibody immobilization parts 6, 7, 8, 9, capture antibodies that bind to different substances to be measured are immobilized at multiple locations. In Fig. 2 and Fig. 3, each of the capture antibody immobilization parts 6, 7, 8, 9 and the control capture antibody immobilization part 10 are represented linearly by dashed lines and diagonal lines, but in an unused immunochromatographic test piece 100, there are no visible boundaries or regions in these parts. When a measurement sample solution is dropped into the measurement sample dropping part 20 and permeates through the sample pad 1, the conjugation pad 2, and the membrane 3 in this order, if there is a substance to be measured in the measurement sample solution, the substance to be measured first reacts with the detection antibody bound to the detection particles carried in the conjugation pad 2 to form a detection antibody-substance to be measured complex. Furthermore, the detection antibody-substance to be measured complex permeates into the membrane 3 according to the permeation flow of the measurement sample solution. When the analyte reacts with any of the capture antibody immobilized portions 6, 7, 8, and 9, the analyte in the detection antibody-analyte complex binds to the capture antibody at that capture antibody immobilized portion and is captured. This reaction concentrates the detection particles bound to the detection antibody, and the capture antibody immobilized portions 6, 7, 8, and 9 corresponding to the analyte are visualized. The color of the visualization depends on the color of the detection particles labeled with each detection antibody.Finally, the detection antibody in the detection antibody-analyte complex is captured by the control capture antibody provided downstream of the capture antibody immobilization sections 6, 7, 8, and 9, concentrating the detection particles, and the control capture antibody immobilization section 10 is visualized. The control capture antibody is an anti-immunoglobulin antibody or the like corresponding to the animal from which the antibody constituting the detection antibody is derived. In addition, when the conjugation pad 2 contains a detection antibody that binds to the analyte derived from a pathogenic microorganism of an infectious disease, and when each of the detection particles is labeled with a different color, the colors of these detection particles may mix and exhibit a color different from that of any of the capture antibody immobilization sections 6, 7, 8, and 9.

[0047] As shown in FIG. 2, the connecting portions 12, 23, and 34 have an interlocking structure in which the projections and recesses interlock with each other.

[0048] 3, reference numeral 12 denotes a junction between the sample pad 1 and the conjugation pad 2. Reference numeral 23 denotes a junction between the conjugation pad 2 and the membrane 3. Reference numeral 34 denotes a junction between the membrane 3 and the absorbent pad 4.

[0049] 2, reference numeral 11 denotes an adhesive sheet. Reference numeral 5 denotes a backing sheet serving as a support. The sample pad 1, the conjugation pad 2, the membrane 3, and the absorbent pad 4 are connected and fixed on the backing sheet 5 by the adhesive sheet 11.

[0050] The immunochromatographic test piece may be in the form of a long, thin strip having a width of 3 to 5 mm (preferably about 4 mm) and a length of 40 to 100 mm (preferably about 60 mm).

[0051] 2 and 3 show immunochromatographic test strip 100 for detecting four types of target substances, the number of target substances detected in one immunochromatographic test strip 100 can be selected from 1, 2, 3, or 4.

[0052] The sample pad 1 is not particularly limited as long as it is made of a material that can rapidly absorb the measurement sample and then spread to the downstream conjugation pad, membrane, and absorption pad. Examples of the material include cellulose filter paper or nonwoven fabric, glass filter paper or nonwoven fabric, polyester filter paper or nonwoven fabric, and polyethylene filter paper or nonwoven fabric. Among these, cellulose filter paper is preferred. The thickness of the sample pad 1 is preferably 0.1 to 2.0 mm, and more preferably 0.2 to 1.0 mm. If the thickness is small, the flow of the measurement sample downstream may become uneven, resulting in a decrease in measurement accuracy. On the other hand, if the thickness is large, downstream spread may be slow and the measurement time may be long. In addition, the amount of measurement sample required for downstream spread increases.

[0053] The membrane 3 is not particularly limited as long as it can spread the measurement sample uniformly with high accuracy, but examples of the membrane include membranes made of cellulose, cellulose derivatives, nitrocellulose, cellulose acetate, polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, or nylon. Among these, membranes made of nitrocellulose are preferred.

[0054] The conjugation pad 2 is not particularly limited as long as it is made of a material capable of holding the complex of the detection particle and the detection antibody in a dry state and rapidly releasing the complex together with the downstream development of the measurement sample. Examples of the material include cellulose filter paper or nonwoven fabric, glass filter paper or nonwoven fabric, polyester filter paper or nonwoven fabric, and polyethylene filter paper or nonwoven fabric. Among these, glass filter paper is preferred. The thickness of the conjugation pad 2 is preferably 0.1 mm to 2.0 mm, more preferably 0.2 mm to 1.0 mm. If the thickness is small, the target amount of the complex may not be held in a dry state. On the other hand, if the thickness is large, downstream development may be slowed down, and the measurement time may be extended. In addition, the amount of measurement sample required for downstream development increases.

[0055] The absorbent pad 4 is not particularly limited as long as it can quickly absorb the measurement sample developed from the upstream and then hold it so that it does not flow back. Examples of the absorbent pad 4 include cellulose filter paper or nonwoven fabric, glass filter paper or nonwoven fabric, polyester filter paper or nonwoven fabric, and polyethylene filter paper or nonwoven fabric. Among these, cellulose filter paper is preferred. The thickness of the absorbent pad 4 is preferably 0.2 to 5.0 mm, more preferably 0.5 to 2.0 mm. If the thickness is small, the measurement sample once absorbed in the absorbent pad may flow back to the membrane side depending on the amount of the measurement sample dropped. On the other hand, if the thickness is large, the size of the immunochromatographic test piece and the housing case covering the immunochromatographic test piece also becomes large, which is not preferable from the viewpoint of POCT. Immunochromatographic test strip 100 may be stored in a folder 30 made of, for example, plastic.

[0056] The immunochromatography kit 50 can be used as follows. First, a specimen, which is a measurement sample solution, or a mixture of a specimen and a buffer is dropped onto the sample pad 1, and the measurement sample solution permeates through the sample pad 1, the conjugation pad 2, and the membrane 3 in that order. If the specimen contains a substance to be measured, the substance to be measured comes into contact with the detection antibody as the measurement sample solution permeates through the conjugation pad 2, and a complex of the substance to be measured and the detection antibody is formed. The complex flows over the membrane 3 in accordance with the permeation flow of the measurement sample solution, and is captured by the solidified capture antibody. As the detection particles are concentrated due to the capture, the complex of the substance to be measured and the detection antibody is visualized.

[0057] 3. Antigen detection methods The present invention also provides a method for detecting a substance to be measured contained in a specimen by an immunological technique using a measurement sample dilution solution. In a preferred embodiment, the method for detecting an antigen includes the steps of dissolving or suspending a specimen in a measurement sample dilution solution to obtain a measurement sample solution or suspension, and detecting the antigen contained in the specimen by an immunological technique using the obtained measurement sample solution or suspension. In a further preferred embodiment, the method for detecting an antigen includes the steps of dissolving or suspending a specimen in the above-mentioned measurement sample dilution solution to obtain a measurement sample solution or suspension, contacting the obtained measurement sample solution or suspension with an immunochromatographic test piece, and detecting the antigen contained in the specimen by immunochromatography. EXAMPLES

[0058] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0059] (1) Preparation of a complex between anti-Flu-B antibody 1 and cellulose-based colored microparticles 100μL of 1.0% by mass red cellulose-based colored microparticles (NanoAct (registered trademark), RE2: Dark Red, average particle size 340nm, manufactured by Asahi Kasei Corporation), 900μL of 10mM Tris buffer (204-07885, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (pH 8.0), and 100μL of 1.0mg / mL mouse-derived anti-Flu-B N protein monoclonal antibody 1 (InfluenzaB virus Monoclonal Antibody, Cat:1131, manufactured by ViroStat) as anti-Flu-B antibody 1 were added to a 15mL centrifuge tube and stirred with a vortex. Then, the mixture was left to stand at 37°C for 120 minutes. Next, 12 mL of a blocking solution (pH 8.0) consisting of 1.0% by mass casein (030-01505, Fujifilm Wako Pure Chemical Industries, Ltd.) and 100 mM borate buffer (021-02195, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was left to stand at 37°C for 60 minutes. Next, using a centrifuge (MX-307, Tommy Seiko Co., Ltd.), centrifugation was performed at 13,000 x g for 15 minutes at 25°C to precipitate the antibody-sensitized cellulose-based colored microparticles, and the supernatant was removed. Next, 12 mL of a washing solution (pH 10.0) consisting of 50 mM borate buffer (021-02195, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was treated with an ultrasonic disperser (UH-50, SMT Co., Ltd.) for 10 seconds. Next, the mixture was centrifuged at 13,000×g for 15 minutes at 25° C. using a centrifuge (MX-307, Tommy Seiko Co., Ltd.), and the antibody-sensitized cellulose-based colored microparticles were precipitated, and the supernatant was removed. Next, 2.0 mL of a coating solution (pH 9.2) consisting of 15% by mass sucrose (196-00015, Fuji Film Wako Pure Chemical Co., Ltd.), 0.2% by mass casein (030-01505, Fuji Film Wako Pure Chemical Co., Ltd.), and 62 mM borate buffer (021-02195, Fuji Film Wako Pure Chemical Co., Ltd.) was added, and the mixture was treated with an ultrasonic disperser (UH-50, SMT Co., Ltd.) for 10 seconds to obtain a complex of anti-Flu-B antibody 1 and cellulose-based colored microparticles.

[0060] (2) Preparation of membrane card for detection of Flu-B N protein As the anti-Flu-B antibody 2, 2.0 mg / mL mouse-derived anti-Flu-B N protein monoclonal antibody 2 (3IF18 / RIF17, R2 / 3, manufactured by HyTest) and 1.0 mg / mL rabbit-derived anti-mouse IgG polyclonal antibody (Mouse IgG-heavy and light chain antibody, A90-117A, manufactured by BETHYL) were prepared as the control line antibody. Next, a dispensing platform (XYZ3060, manufactured by BIODOT) and a BioJet nozzle (BHQHR-XYZ, manufactured by BIODOT) were used to prepare a 60 mm x 300 mm membrane card (Hi-Flow Plus 120 Membrane) consisting of a 20 mm x 300 mm adhesive tape section on the upstream side, a 25 mm x 300 mm membrane section in the center, and a 15 mm x 300 mm adhesive tape section on the downstream side. The anti-Flu-B antibody 2 was applied at an application rate of 1.0 μL / cm to a position 9 mm from the upstream side of the membrane of a membrane card (Millipore Cards, HF120) at a coating amount of 1.0 μL / cm (test line), and the control line antibody was applied at a position 17 mm from the upstream side of the membrane (control line) at an application rate of 1.0 μL / cm. The membrane card was then dried for 30 minutes in a dryer (Tokyo Rika Kikai Co., Ltd. WFO-510) adjusted to 45° C. to form test lines and control lines with a line width of approximately 1 mm, thereby obtaining a membrane card for detecting Flu-B N protein.

[0061] (3) Preparation of conjugation pad for detection of Flu-B N protein A complex of anti-Flu-B antibody 1 and cellulose-based colored microparticles was uniformly applied to the entire surface of a 10 mm x 300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, Millipore) at a coating volume of 15 μL / cm using a dispensing platform (XYZ3060, BIODOT) and an AirJet nozzle (AJQHR-XYZ, BIODOT), and then dried for 30 minutes in a dryer (WFO-510, Tokyo Rikakikai) adjusted to 45°C to obtain a conjugation pad for detecting Flu-B N protein.

[0062] (4) Preparation of immunochromatographic test strip and device for detecting Flu-B N protein The conjugation pad 1 for detecting N protein of Flu-B was attached to the 20 mm × 300 mm adhesive tape part on the upstream side of the membrane card for detecting N protein of Flu-B so as to overlap with the membrane part by 2 mm. Next, a sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, Millipore) of 15 mm × 300 mm was attached to the upstream side so as to overlap with the conjugation pad by 3 mm. Next, an absorption pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, Millipore) of 20 mm × 300 mm was attached to the adhesive tape part of 15 mm × 300 mm on the downstream side of the membrane card for detecting N protein of Flu-B so as to overlap with the membrane part by 5 mm. Next, the specimen was cut into a strip of 4 mm width and 60 mm length using a guillotine cutting module (CM5000, manufactured by BIODOT) to obtain an immunochromatographic test piece for detecting N protein of Flu-B. The obtained immunochromatographic test piece for detecting N protein of Flu-B was stored in a housing case (K007, manufactured by Shengfeng Plastic Co., Ltd.) to obtain an immunochromatographic device for detecting N protein of Flu-B.

[0063] (5) Preparation of immunochromatographic test strips and devices for detecting the N protein of SARS-CoV-2 An immunochromatographic device for detecting the N protein of SARS-CoV-2 was prepared using the same method as in (1) to (4). However, mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-108, manufactured by Toyobo Co., Ltd.) was used as anti-SARS-CoV-2 antibody 1 instead of anti-Flu-B antibody 1, mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 2 (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-109, manufactured by Toyobo Co., Ltd.) was used as anti-SARS-CoV-2 antibody 2 instead of anti-Flu-B antibody 2, and blue cellulose-based colored microparticles (NanoAct (registered trademark), BL2: Dark Navy, average particle size 365 nm, manufactured by Asahi Kasei Co., Ltd.) were used instead of red cellulose-based colored microparticles.

[0064] (6) Preparation of immunochromatographic test strip and device for detecting Flu-A N protein An immunochromatographic device for detecting the N protein of FluA was prepared in the same manner as in (1) to (4). However, instead of the anti-Flu-B antibody 1, a mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody 1 (InfluenzaA nucleoprotein, antibody, Cat: 3IN5, MAbs: InA245, manufactured by HyTest) was used as the anti-Flu-A antibody 1, instead of the anti-Flu-B antibody 2, a mouse-derived anti-Flu-A N protein monoclonal antibody 2 (InfluenzaA Virus (NP) Antibody, Cat: BMRia017, Clone: ​​FLA-859, manufactured by BioMatrix Research Inc.) was used as the anti-Flu-A antibody 2, and instead of the red cellulose-based colored microparticles, blue cellulose-based colored microparticles (NanoAct (registered trademark), BL2: Dark Navy, average particle size 365 nm, manufactured by Asahi Kasei Corporation) were used.

[0065] (7) Preparation of immunochromatographic test strips and devices for detecting RSV F protein An immunochromatographic device for detecting RSV F protein was prepared using the same method as in (1) to (4). However, mouse-derived anti-RSV F protein monoclonal antibody 1 (Cat: 3ReS21cc, manufactured by HYTEST) was used as anti-RSV antibody 1 instead of anti-Flu-B antibody 1, and mouse-derived anti-RSV F protein monoclonal antibody 2 (Cat: C01773M, manufactured by Meridian) was used instead of anti-Flu-B antibody 2.

[0066] (7) Preparation of sample dilution solution 1 L of 100 mM Tris buffer (204-07885, Fujifilm Wako Pure Chemical Industries, Ltd.), 2 g of polyoxyethylene (20) sorbitan monolaurate: Tween (registered trademark) 20 (166-21213, Fujifilm Wako Pure Chemical Industries, Ltd.), and 9 g of polyoxyethylene (10) octylphenyl ether: TritonX (registered trademark)-100 (160-24751, Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a 500 mL glass bottle and dissolved. Sodium chloride (191-01665, Fujifilm Wako Pure Chemical Industries, Ltd.) was added thereto, and the pH was adjusted using sodium hydroxide to prepare a measurement sample dilution solution.

[0067] (8) Assessment of non-specific reactions Nasal swabs were collected from two healthy individuals and suspended in 450 μL of the measurement sample dilution solution. Four drops of the nasal swabs were dropped onto the immunochromatographic device for detecting the N protein of Flu-B, the immunochromatographic device for detecting the N protein of Flu-A, the immunochromatographic device for detecting the N protein of SARS-CoV-2, and the immunochromatographic device for detecting the F protein of RSV through an Immunoarrow SARS-CoV-2 filter. The devices were then left to stand at 25°C for 15 minutes, and the presence or absence of a test line was visually confirmed. The results are shown in Table 1 (Flu-B), Table 2 (Flu-A), Table 3 (SARS-CoV-2), and Table 4 (RSV), respectively.

[0068] [Table 1]

[0069] [Table 2]

[0070] [Table 3]

[0071] [Table 4]

[0072] (9) Evaluation of the sensitivity of the immunochromatographic device for detecting the N protein of Flu-B Purified Influenza B protein (30-AI75, Fitzgerald), which is a commercially available N protein of Flu-B, was diluted to 4ng / mL using the measurement sample dilution solution, and 100μL was dropped onto the immunochromatographic device for detecting the N protein of Flu-B. Then, after leaving it at 25°C for 15 minutes, the reflection absorbance (mAbs) of the test line on the membrane was measured using an immunochromatographic reader (C10060-10, measurement mode: Gold Colloid, Line, Hamamatsu Photonics). In addition, the relative sensitivity to the measured value of the sample prepared using the measurement sample dilution solution was calculated. The obtained results are shown in Table 5.

[0073] [Table 5]

[0074] (10) Evaluation of the sensitivity of the immunochromatographic device for detecting Flu-A N protein Purified Influenza A protein (H1N1 strain) (30-AI50, Fitzgerald), which is a commercially available N protein of Flu-A, was diluted to 16 ng / mL using the measurement sample dilution solution, and 100 μL was dropped onto the immunochromatographic device for detecting the N protein of Flu-A. Then, after leaving it at 25° C. for 15 minutes, the reflection absorbance (mAbs) of the test line on the membrane was measured using an immunochromatographic reader (C10060-10, measurement mode: Latex, Line, Hamamatsu Photonics). In addition, the relative sensitivity to the measured value of the sample prepared using the measurement sample dilution solution was calculated. The obtained results are shown in Table 6.

[0075] [Table 6]

[0076] (11) Evaluation of the sensitivity of immunochromatographic devices for detecting the N protein of SARS-CoV-2 A commercially available SARS-CoV-2 N protein, Recombinant SARS-CoV-2 Nucleocapsid protein (230-30164, manufactured by RayBiotech), was diluted to 0.5 ng / mL using the measurement sample dilution solution, and 100 μL was dropped onto the immunochromatographic device for detecting the SARS-CoV-2 N protein. After leaving the solution at 25 ° C for 15 minutes, the reflection absorbance (mAbs) of the test line on the membrane was measured using an immunochromatographic reader (C10060-10, measurement mode: Latex, Line, manufactured by Hamamatsu Photonics). In addition, the relative sensitivity to the measured value of the sample prepared using the measurement sample dilution solution was calculated. The results obtained are shown in Table 7.

[0077] [Table 7]

[0078] (11) Evaluation of the sensitivity of an immunochromatographic device for detecting RSV F protein A commercially available RSV F protein, RSV Grade 2 Antigen (8175, Meridian Life Science), was diluted to 1 μg / mL using the measurement sample dilution solution, and 100 μL was dropped onto the immunochromatographic device for detecting the RSV F protein. After leaving the solution at 25° C. for 15 minutes, the reflection absorbance (mAbs) of the test line on the membrane was measured using an immunochromatographic reader (C10060-10, measurement mode: Gold Colloid, Line, Hamamatsu Photonics). In addition, the relative sensitivity to the measured value of the sample prepared using the measurement sample dilution solution was calculated. The results are shown in Table 8.

[0079] [Table 8]

[0080] (12) When a measurement sample dilution solution with a dispersion coefficient D of less than 2400 was used, a nonspecific reaction was observed in the immunochromatographic device for detecting FluB N protein. However, when a measurement sample dilution solution with a dispersion coefficient D of 2400 or more was used, it was confirmed that no nonspecific reaction occurred in any of the immunochromatographic devices. On the other hand, although there is concern that increasing the pH or salt concentration may reduce the sensitivity when measuring positive samples, the dispersion coefficient D did not have a significant effect on the sensitivity of Flu-A and Flu-B. For SARS-CoV-2 and RSV, there was a tendency for sensitivity to decrease when a measurement sample dilution solution with a large dispersion coefficient D was used. However, no significant decrease in sensitivity was observed for either when the dispersion coefficient D was less than 3500.

[0081] The measurement sample dilution solution of the present invention is useful for suppressing non-specific reactions in the detection of FluB. In addition, the measurement sample dilution solution of the present invention is useful for detecting a detection target substance with high sensitivity while suppressing non-specific reactions in the simultaneous detection of Flu-A, Flu-B, SARS-CoV-2, and RSV. [Industrial Applicability]

[0082] The present invention is useful in virus testing and the like using immunochromatography. [Explanation of symbols]

[0083] 100: Immunochromatographic test strip 1: Sample Pad 2: Conjugation pad 3: Membrane 4: Absorbent pad 5: Backing sheet 6~9: Capture antibody immobilization part 10: Control Line 11: Adhesive sheet

Claims

1. A measurement sample dilution solution used for detecting an antigen contained in a specimen by an immunological method, comprising: The measurement sample dilution solution contains an inorganic salt, and The following formula (1): Dispersion coefficient D=concentration of inorganic salt in diluted solution of measurement sample (mM)×pH of diluted solution of measurement sample … Equation (1) The measurement sample dilution solution has a dispersion coefficient D calculated by the following formula:

2. The measurement sample dilution solution according to claim 1, wherein the dispersion coefficient D is 2400 to 3000.

3. The measurement sample dilution solution according to claim 1 , wherein the inorganic salt is at least one selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, and magnesium chloride.

4. The measurement sample dilution solution according to claim 1 , further comprising at least one selected from the group consisting of a buffer, a surfactant, a blocking agent and a preservative.

5. The measurement sample dilution solution according to claim 4 , wherein the surfactant is a nonionic surfactant.

6. The measurement sample dilution solution according to claim 1 , wherein the specimen is at least one selected from the group consisting of a nasopharyngeal swab, a nasal swab, and a nasal aspirate.

7. The measurement sample dilution solution according to claim 1, wherein the antigen is at least one selected from the group consisting of influenza virus A (Flu-A), influenza virus B (Flu-B), respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

8. The measurement sample dilution solution according to claim 7 , wherein the antigen is influenza virus B.

9. The measurement sample dilution solution according to claim 1, wherein the immunological technique is an immunochromatography method.

10. An immunochromatography kit comprising the measurement sample dilution solution according to claim 9, an immunochromatography test strip, a specimen collecting tool, and a filter unit.

11. A step of dissolving or suspending a specimen in the measurement sample dilution solution according to claim 1 to obtain a measurement sample solution or suspension; detecting an antigen contained in a specimen by an immunological method using the obtained measurement sample solution or suspension; A method for detecting an antigen, comprising:

Images