Membrane for immunochromatographic assay, test strip for immunochromatographic assay, and inspection method

Abstract

A membrane (10) for immunochromatographic assays according to the present invention is characterized by comprising a polymer film support (12) and a porous membrane (14) laminated on the polymer film support (12), wherein the polymer film support (12) and / or the porous membrane (14) comprises an autofluorescence reducing material.

Description

[Technical field]

[0001] The present invention relates to a membrane for immunochromatographic assay, a test strip for immunochromatographic assay, and a testing method. [Background technology]

[0002] Immunochromatographic assays are widely used as simple immunoassays that utilize antigen-antibody reactions. Immunochromatographic assays are easy to operate and can be performed in a short time, so they are widely used as point-of-care testing (POCT) for clinical testing and diagnosis in cases where rapid results are required, such as pregnancy diagnosis and influenza infection confirmation.

[0003] In such a test strip for performing an immunochromatographic assay, the analyte is detected qualitatively or quantitatively as follows: A liquid sample is dropped onto one end of the test strip and moves through the strip by capillary action. During this process, binding between the analyte and a labeled antibody that binds to the analyte is promoted. When the sample passes through the area where the capture antibody that binds to the analyte is immobilized, a labeled antibody-antigen complex is captured by an antigen-antibody reaction. The labeled antibody-antigen complex is colored in the capture antibody immobilization area and can be optically detected.

[0004] Commonly used labeling substances include enzyme-containing proteins, metal colloids such as gold colloids, colored latex particles, etc. However, immunochromatographic assays using these labeling substances are inferior in terms of detection sensitivity to gene amplification methods (PCR methods), which are known as highly sensitive detection methods, and there is a demand for higher sensitivity for highly accurate and rapid diagnosis.

[0005] As a means for achieving high sensitivity, a fluorescent immunochromatographic assay method using a fluorescent labeling substance has been proposed (see, for example, Patent Document 1). The fluorescent immunochromatographic assay method is an immunoassay method in which a fluorescently labeled antibody is used as the labeled antibody described above. After the labeled antibody-antigen complex is captured by the capture antibody, excitation light in a wavelength range in which the fluorescent labeling substance emits fluorescence is irradiated, and the fluorescence emitted by the fluorescent labeling substance is detected by a detector, thereby enabling qualitative or quantitative detection of the analyte with high sensitivity.

[0006] In the fluorescent immunochromatographic assay method, a fluorescent immunochromatographic reader equipped with an excitation light irradiation unit and a fluorescence detection unit is generally used. In the fluorescent immunochromatographic assay method using such a fluorescent immunochromatographic reader, irradiation with excitation light generates not only fluorescence from the fluorescent label that formed the antigen-antibody complex, but also unnecessary fluorescence from the porous membrane, which is the development site of the immunochromatographic assay, its support, and the backing sheet that supports them, etc. Such fluorescence from sources other than the antigen-antibody complex becomes background noise and reduces the signal / background ratio (S / B ratio), which is an obstacle when attempting to detect even smaller amounts of the analyte.

[0007] As a measure to improve the signal / background ratio (S / B ratio), for example, Patent Document 2 proposes a method of using a fluorescent labeling substance in a color region different from the autofluorescence and using a color sensor in a detector as a fluorescence detection means.

[0008] Furthermore, Patent Document 3 proposes a method of detecting fluorescence in a wavelength region that reduces the background effects of autofluorescence from a porous membrane, its support, etc., by using a fluorescent substance as a labeling substance that fluoresces when excited by light having a wavelength of 600 nm or more and 800 nm or less. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] JP 2009-115822 A [Patent Document 2] JP 2016-191567 A [Patent Document 3] WO2013 / 151066 publication Summary of the Invention [Problem to be solved by the invention]

[0010] However, the techniques of Patent Documents 2 and 3 detect fluorescence in a wavelength region that avoids the peak wavelength of autofluorescence from the porous membrane and its support, but do not reduce the autofluorescence itself from the porous membrane and its support. There is a limit to the detection sensitivity, and further improvement is necessary to increase the sensitivity for high-precision rapid diagnosis.

[0011] Therefore, an object of the present invention is to provide an immunochromatographic assay membrane that can provide an immunochromatographic assay test strip that enables highly sensitive detection, an immunochromatographic assay test strip that enables highly sensitive detection, and a highly sensitive testing method. [Means for solving the problem]

[0012] In order to achieve the above objective, the inventors conducted extensive research and discovered that by using an immunochromatographic assay membrane equipped with a specific autofluorescence reducing material, an immunochromatographic assay test strip capable of highly sensitive detection can be obtained. That is, the present invention provides a membrane for immunochromatographic assay, comprising a polymer film support and a porous membrane laminated on the polymer film support, wherein at least one of the polymer film support and the porous membrane is made of an autofluorescence reducing material. The present invention also relates to an immunochromatographic assay test strip comprising the above-mentioned immunochromatographic assay membrane as a chromatographic medium. Furthermore, the present invention provides a method for testing an analyte contained in a specimen, which comprises contacting the specimen with a fluorescent label using the above-mentioned immunochromatographic assay test strip. Effect of the Invention

[0013] According to the present invention, it is possible to provide an immunochromatographic assay membrane which can provide an immunochromatographic assay test strip capable of highly sensitive detection, an immunochromatographic assay test strip capable of highly sensitive detection, and a highly sensitive testing method. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic structural diagram of the immunochromatographic assay membrane of the present invention. [Diagram 2] FIG. 1 is a schematic structural diagram of a test strip for immunochromatographic assay according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The immunochromatographic assay membrane of the present invention will be described in detail below with reference to the drawings. Fig. 1 is a side view showing the configuration of the immunochromatographic assay membrane of the present invention. The immunochromatographic assay membrane 10 is a member used as a chromatographic medium in an immunochromatographic assay test strip, and includes a polymer film support 12 and a porous membrane 14 laminated thereon.

[0016] At least one of the polymer film support 12 and the porous membrane 14 is made of an autofluorescence reducing material. Furthermore, the immunochromatographic assay membrane 10 of the present invention has a fluorescence quantum yield of 18% or less at an excitation wavelength of 365 nm and a brightness L measured by a spectrophotometer. * It is preferable that (D65) is 60% or more. The fluorescence quantum yield is measured in a state where the pores of the immunochromatographic assay membrane 10 are filled with water.

[0017] In the fluorescent immunochromatography method, the excitation wavelength of the fluorescent labeling substance generally used is 365 nm. The fluorescence quantum yield at 365 nm is an index of autofluorescence, and the brightness L * (D65) is an index of the whiteness of immunochromatographic assay membranes. If the fluorescence quantum yield at 365 nm is 18% or less, autofluorescence is suppressed when an immunochromatographic assay test strip is prepared and tested, improving sensitivity. * If (D65) is less than 60%, when an immunochromatographic assay test strip is prepared and tested, only the fluorescence of the labeling substance on the membrane surface is detected, leading to a decrease in sensitivity.

[0018] Therefore, the lightness L * The present inventors have found that the fluorescence quantum yield at a wavelength of 365 nm of an immunochromatographic assay membrane can be reduced to 18% or less and the brightness can be increased to 60% or more by using a specific autofluorescence reducing material.

[0019] The autofluorescence reducing material in the present invention may be either the polymer film support 12 or the porous membrane 14, or the autofluorescence reducing material may be used as both the polymer film support 12 and the porous membrane 14. For example, the polymer film support 12 made of a polymer film containing a dye that absorbs ultraviolet light or visible light is an autofluorescence reducing material.

[0020] Polymers used in polymer films containing a dye that absorbs ultraviolet or visible light include, for example, polyesters such as polyethylene terephthalate (PET), polyimides, nylons, etc. In this specification, "absorbing ultraviolet or visible light" specifically means absorbing 50% or more of light with a wavelength of 190 to 830 nm. The dye that absorbs ultraviolet or visible light is not particularly limited, and examples thereof include black dyes.

[0021] In this specification, "black" refers to a color in which the CIE color coordinates, measured in accordance with JIS Z 8701-1999, are -1.0≦a * ≦2.5 and -1.0≦b * ≦15.0, L * For the value, 0 <L * This refers to a color tone in the range of ≦50.

[0022] Black pigment-containing polymer films are commercially available, for example, as Lumirror #25-X30, #38-X30, #50-X30, #75-X30, #100-X30, #125-X30, #188-X30, #250-X30, and #25-X36 (manufactured by Toray Industries, Inc.), Mordohar PIB025, PIB050, PIB075, PIB100, and PIB125 (manufactured by Asahi Chemical Industry Co., Ltd.). Commercially available black pigment-containing polymer films are usually about 25 to 250 mm thick and have a CIE color coordinate system of -1.0≦a * ≦2.5 and -1.0≦b * ≦15.0, L * For the value, 0 <L * Black pigment is included to ensure a value in the range of ≦50.

[0023] The dye that absorbs ultraviolet light or visible light is not limited to a black dye, and for example, a mixture of a yellow dye, a cyan dye, and a magenta dye may also be used.

[0024] When an autofluorescence reducing material is used as the porous membrane 14 laminated on the polymer film support 12, the polymer film support 12 does not necessarily have to be an autofluorescence suppressing material. For example, a polymer film that does not contain a dye that absorbs ultraviolet light or visible light can be used as the polymer film support 12.

[0025] To laminate the porous membrane 14 on the polymer film support 12, the polymer film support 12 and the porous membrane 14 may be adhered to each other with an adhesive tape, or the membrane may be laminated by casting a membrane-forming solution onto the polymer film support 12 using the method described below.

[0026] In the immunochromatographic assay membrane 10 of the present invention, the thickness of the polymer film support 12 is not particularly limited and may be within a normal range. In consideration of ease of handling, the thickness is preferably about 50 to 100 μm.

[0027] As the porous membrane 14, a membrane mainly made of nitrocellulose, such as nitrocellulose or a nitrocellulose mixture, which is capable of binding proteins and has no reactivity with detection reagents, immobilization reagents, and analyte substances used in immunochromatographic assays, can be used. Furthermore, membranes made of other materials, such as cellulose ester membranes, nylon membranes, polyethylene membranes, and polypropylene membranes, may also be used.

[0028] By incorporating a dye that absorbs ultraviolet light or visible light into the porous membrane 14 as described above, it is possible to make it into an autofluorescence reducing material. A typical example of a dye that absorbs ultraviolet light or visible light is a black dye. In the case of a black dye (Savinyl Black RLSN), the content in the porous membrane is preferably about 0.50 mass % or less, and more preferably about 0.30 mass % or less. Depending on the amount of black dye added, the color of the porous membrane changes from white to gray to black.

[0029] The type of black pigment is not particularly limited and can be appropriately selected depending on the purpose. For example, dyes and pigments can be used. Examples of pigments include Pigment Black 7, 28, and 26.

[0030] Examples of commercially available products include Chromofine Black A-1103 (manufactured by Dainichiseika Color & Chemicals Mfg. Co., Ltd.), Colortex Black 702, U905 (manufactured by Sanyo Pigment Co., Ltd.), and Carbon Black #2600, #2400, #2350, #2200, #1000, #990, #980, #970, #960, #950, and #850 (manufactured by Mitsubishi Chemical Corporation).

[0031] Examples of dyes include Savinyl Black RLSN (Clariant Japan Co., Ltd.), VALIFAST BLACK (Orient Chemical Industry Co., Ltd.), MS BLACK VPC (Mitsui Chemicals, Inc.), AIZEN SOT BLACK-1, AIZEN SOT BLACK-5 (Hodogaya Chemical Co., Ltd.), RESORIN BLACK GSN 200%, RESOLIN BLACK BS (Bayer Japan KK), KAYASET BLACK AN (Nippon Kayaku Co., Ltd.), DAIWA BLACK MSC (Daiwa Kasei Co., Ltd.), HSB-202 (Mitsubishi Kasei Co., Ltd.), NEPTUNE BLACK X60, NEOPEN BLACK X58 (BASF Japan Ltd.), Oleosol Fast BLACK RL (Taoka Chemical Co., Ltd.), Chuo BLACK 80, Chuo BLACK 80-15 (Chuo Synthetic Chemical Co., Ltd.), and the like.

[0032] A porous membrane containing a dye that absorbs ultraviolet or visible light can be produced, for example, by adding a black dye to a solvent in which a hydrophobic material has been dissolved to produce a membrane-forming solution, casting the solution in a smooth form, and then evaporating the solvent.

[0033] The porous membrane used as an autofluorescence reducing material has a lightness L * (D65) is preferably 60% or more, and more preferably 70% or more. * (D65) is an index of the whiteness of the porous membrane. * It was found that porous membranes with a (D65) of 60% or more have a sensitivity suitable for fluorescence analysis without significantly inhibiting the fluorescence from fluorescently labeled substances when irradiated with excitation light.

[0034] When an autofluorescence reducing material is used as the polymer film support 12 that supports the porous membrane 14, the porous membrane 14 does not necessarily have to be an autofluorescence reducing material. For example, the porous membrane itself that does not contain a dye that absorbs ultraviolet light or visible light may be laminated on the polymer film support 12. Autofluorescence reducing materials may also be used as both the polymer film support 12 and the porous membrane 14.

[0035] The thickness of the porous membrane 14 in the immunochromatographic assay membrane 10 of the present invention is preferably 100 μm or more, and more preferably 110 to 160 μm, from the viewpoints of the spreadability of the labeled antibody and the retention capacity of the capture antibody solution.

[0036] From the viewpoint of high-precision rapid diagnosis, the immunochromatographic assay membrane 10 of the present invention preferably has a capillary flow time of 300 sec / 4 cm or less, more preferably 250 sec / 4 cm or less, and particularly preferably 180 sec / 4 cm or less.

[0037] Furthermore, it is preferable that the immunochromatographic assay membrane 10 of the present invention contains a substance that promotes capillary action. Such a substance is preferably a substance that reduces the surface tension of the membrane surface and provides hydrophilicity, and does not affect the movement of the analyte in the immunochromatographic assay or the color development of the labeling substance. Examples of such a substance include cationic surfactants, anionic surfactants, amphoteric surfactants, and nonionic surfactants.

[0038] Examples of cationic surfactants include higher amine halogen acid salts, alkylpyridinium halides, and quaternary ammonium salts.

[0039] Examples of anionic surfactants include higher fatty acid alkali salts, polyoxyethylene alkyl ether sulfonate salts, polyoxyethylene alkyl ether phosphonate salts, alkyl sulfate salts, alkyl benzene sulfate salts, alkyl sulfonate salts, alkyl aryl sulfonate salts, and sulfosuccinate salts, etc. Among these, alkyl benzene sulfonate salts are preferred, and sodium dodecylbenzene sulfonate (SDBS) is particularly preferred.

[0040] Examples of amphoteric surfactants include alkylbetaine compounds, imidazoline compounds, alkylamine oxides, and bisoxyborate compounds.

[0041] Examples of nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene alkyl allyl ethers, glycerin, glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, and sorbitan fatty acid esters.

[0042] The above-mentioned surfactant can be incorporated into the immunochromatographic assay membrane 10 by a technique such as dipping. Although it depends on the type of surfactant used, it is usually 0.5 to 2.0 μg / mm 3 The desired effect can be achieved if the immunochromatographic assay membrane 10 contains the above amount.

[0043] The immunochromatographic assay test strip of the present invention includes an immunochromatographic assay membrane 10 as a chromatographic medium. The immunochromatographic assay strip is used to detect a liquid sample that may contain a substance to be detected. The shape and width of the immunochromatographic assay strip are not particularly limited and can be appropriately selected in terms of actual operation and observation of reaction results.

[0044] A schematic diagram of an example of an immunochromatographic assay strip is shown in FIG. 2, the immunochromatographic assay strip 20 comprises an elongated rectangular chromatographic medium 22, a sample pad 24 provided at one end of the chromatographic medium 22 via a conjugate pad 26, and an absorbent pad 28 provided at the other end of the chromatographic medium 22, and these members are supported by a backing sheet 30. The chromatographic medium 22 is made of the immunochromatographic assay membrane 10 of the present invention.

[0045] The sample pad 24 can be made of any material, so long as it is a porous body or nonwoven fabric that is normally used in immunochromatographic assays. For example, a porous body made of glass fiber or cellulose that absorbs and retains a sample is used for the sample pad 24. When performing an immunochromatographic assay, a sample is dropped onto the sample pad 24.

[0046] The conjugate pad 26 is made of a porous material such as glass fiber or cellulose, or a nonwoven fabric. The conjugate pad 26 contains a labeling substance bound to an antibody that binds to the analyte. When the analyte moves through the conjugate pad 26, it binds to the antibody and is labeled.

[0047] The absorbent pad 28 is made of a material capable of absorbing the developing solution, and may be a porous material, nonwoven fabric, or the like that is used in conventional immunochromatographic assays. For example, the absorbent pad 28 may be made of cellulose filter paper.

[0048] The backing sheet 30 can be made of any sheet-like material having an adhesive layer to support the chromatographic medium 22, the sample pad 24, the conjugate pad 26, and the absorbent pad 28. For example, a sheet made of polyester or polystyrene can be used. The immunochromatographic assay test strip 20 constructed as above can be used as is, or, if necessary, housed in a housing molded from plastic.

[0049] The immunochromatographic assay test strip 20 can be prepared, for example, by the following method.

[0050] First, a dispersion of labeled particles adjusted to a predetermined concentration is prepared, a buffer solution and an antibody are added, and the mixture is stirred for a certain period of time while adjusting the temperature, to allow the antibody to be adsorbed onto the labeled particles. After stirring for a certain period of time, a blocking agent is added and the mixture is stirred for a certain period of time while adjusting the temperature, to block the labeled particles. Blocking is an operation for coating particles alone or particles carrying antibodies or antigens. Various blocking agents can be used depending on the composition of the test substance, specimen, or solution used to dilute the specimen.

[0051] Casein is particularly preferred for blocking labeled particles. In this case, casein coats the antibody in the labeled particles carrying the antibody and the surface of the labeled particles. In order to wash the labeled particles after antibody adsorption and blocking, centrifugation is performed to separate the supernatant containing the excess antibody and blocking agent from the precipitated particles, and the supernatant is removed by decantation. A liquid such as a buffer solution is added to the precipitated particles, and a dispersion treatment is performed using ultrasound or the like as necessary. This series of washing operations, including centrifugation for sedimentation, removal of the supernatant, and addition of liquid, is performed as many times as necessary to prepare a dispersion containing a predetermined concentration of particles that have been antibody adsorbed and blocked.

[0052] Proteins, surfactants, and sugars such as sucrose and trehalose are added to the obtained dispersion as necessary, and a certain amount of the obtained solution is applied to a polyethylene conjugate pad and dried to prepare a detection reagent-containing portion. In addition, buffer solutions, surfactants, proteins, reagents for trapping impurities in the specimen sample, preservatives, antibacterial agents, antioxidants, moisture absorbents, etc. are applied to a regenerated cellulose continuous long fiber nonwoven fabric as necessary, and the fabric is dried to prepare a sample pad. In addition, a chromatography medium made of a nitrocellulose porous membrane with antibodies immobilized at predetermined positions and an absorption pad made of cellulose filter paper for absorbing the specimen are prepared. These are immobilized on a mount having adhesive sites called a backing sheet, and then cut to a specified size to obtain a test strip for immunochromatographic assay.

[0053] A testing method using the immunochromatographic assay test strip 20 of the present invention will be described below. In the immunochromatographic assay test strip 20 of the present invention, a reaction site is formed on the chromatographic medium 22 where a substance that specifically binds to the analyte, such as an antibody, is immobilized at an arbitrary position as an immobilized reagent. The immobilized reagent can be directly immobilized on the chromatographic medium 22 by physical or chemical means. Alternatively, the immobilized reagent may be physically or chemically bound to fine particles such as latex particles, and the fine particles may be captured on the chromatographic medium 22 to indirectly immobilize the reagent.

[0054] To directly immobilize the immobilized reagent, physical adsorption can be used. The immobilized reagent may also be immobilized on the chromatographic medium 22 by covalent bonding. In the case of a nitrocellulose membrane, physical adsorption can be performed. In the case of covalent bonding, cyanogen bromide, glutaraldehyde, carbodiimide, etc. are generally used to activate the chromatographic medium 22, and any method can be used.

[0055] An example of an indirect immobilization method is a method in which insoluble microparticles to which an immobilization reagent is bound are immobilized on the chromatographic medium 22. As the insoluble microparticles, those having a size that allows them to be captured by the chromatographic medium 22 but not to move can be selected, and microparticles having an average particle size of about 5 μm or less are preferable. Various such particles are known that are used in antigen-antibody reactions, and such microparticles can also be used in the present invention.

[0056] Examples of such particles include fine particles of organic polymeric substances such as organic polymer latex particles obtained by emulsion polymerization of polystyrene, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, etc.; fine particles of gelatin, bentonite, agarose, crosslinked dextran, etc.; inorganic oxides such as silica, silica-alumina, alumina, etc., and inorganic oxides having functional groups introduced therein by silane coupling treatment or the like.

[0057] In the present invention, direct immobilization is preferred because of the ease of sensitivity adjustment. Various methods can be used to immobilize the immobilized reagent on the chromatographic medium 22. For example, various techniques can be used, such as a microsyringe, a pen with an adjustable pump, and ink jet printing. The form of the reaction site is not particularly limited, but it can also be immobilized as a circular spot, a line extending perpendicular to the developing direction of the chromatographic medium 22, numbers, letters, symbols (+, -, etc.), etc.

[0058] After the immobilization reagent is immobilized, in order to prevent a decrease in analytical accuracy due to non-specific adsorption, a blocking treatment can be performed on the chromatographic medium 22 by a known method, if necessary. In general, proteins such as bovine serum albumin, skim milk, casein, and gelatin are preferably used for blocking treatment. After the blocking treatment, washing can be performed, if necessary, with one or a combination of two or more surfactants such as Tween 20, Triton X-100, and SDS.

[0059] The detection reagent used in the present invention is a substance that specifically binds to the analyte, such as an antibody, and is labeled with a labeling substance. In immunochromatographic assays, substances such as insoluble carriers and enzymes are generally used to label the detection reagent, but fluorescent labeling substances such as insoluble carriers containing a fluorescent substance and enzymes are used as the labeling substance. In the present invention, the labeled detection reagent is prepared by sensitizing the detection reagent to a fluorescent labeling substance.

[0060] A fluorescent labeling substance is a labeling substance suitable for detecting the presence of an analyte with high accuracy and sensitivity, and the fluorescence generated by irradiation with excitation light can be used as a label.

[0061] As the insoluble carrier, organic latex particles and inorganic colloid particles used in technical fields such as diagnostic drugs can be used. The material of the particles is not limited, but examples thereof include those used as materials of solid phase carriers for binding proteins such as antibodies, antigens, ligands, and receptors in technical fields such as diagnostic drugs. Examples of organic latex particles include particles made of polystyrene, styrene copolymers such as styrene-acrylic acid copolymers, polycarbonate, polymethylene methacrylate (PMMA), polyvinyl toluene, cellulose, etc. Examples of inorganic colloid particles include silica nanoparticles, etc.

[0062] In addition, an insoluble carrier made of a substance capable of emitting fluorescence can also be used. Examples of such particles include M I M II O 4 or M I Al 5 O 12 (M I is yttrium (Y), lanthanum (La) or gadolinium (Gd), M II Examples of the materials include particles made of niobium (Nb), phosphorus (P) or vanadium (V), and quantum dots made of, for example, CdSe, CdTe, InP, InN, InAs, CdS, Si, and Ge.

[0063] The type of fluorophore contained in the fluorescent labeling substance is not limited, and any fluorophore used in the field of diagnostic drugs and the like can be used. Examples of such fluorophore include organic fluorescent dyes having a basic skeleton such as fluorescein, rhodamine, coumarin, Cy dye, Alexa (registered trademark) Fluor, EvoBlue, oxazine, carbopyronin, naphthalene, biphenyl, anthracene, phenanthrene, pyrene, and carbazole, and derivatives of such fluorescent dyes. In addition, europium (Eu 3+ ) chelate and terbium (Tb 3+ Rare earth complexes such as europium (Eu) chelates can also be used. 3+ ) ATBTA-Eu with amino group as chelate 3+ Furthermore, fluorescent proteins such as Green Fluorescent Protein (GFP) can also be used.

[0064] The detection reagent can be sensitized to a fluorescent labeling substance by known methods such as physical adsorption and chemical binding. For example, a detection reagent in which an antibody is sensitized to latex particles made of a synthetic polymer containing a fluorescent substance is prepared by surface-modifying the latex particle surface with a reactive group, adding an antibody and a crosslinking agent to a solution in which the surface-modified latex particles are dispersed to chemically bond the antibody and the crosslinking agent, and then adding a bovine serum albumin solution or the like to block the particle surface to which the antibody is not bound.

[0065] In the actual implementation of the immunochromatographic assay method, the detection reagent labeled with an insoluble carrier can be applied by dispersing it in the developing solution constituting the mobile phase. Alternatively, the detection reagent labeled with an insoluble carrier can be applied by being present on the developing and moving path of the mobile phase in the immunochromatographic assay test strip 20 constituting the stationary phase, that is, in the region between the end of the immunochromatographic assay test strip 20 where the mobile phase is applied and the reaction site.

[0066] When a detection reagent is present on the immunochromatographic assay test strip 20, it is preferable to support the detection reagent so that the detection reagent can be quickly dissolved in the developing solution and can move freely by capillary action. In order to increase the resolubility of the insoluble carrier to which the detection reagent is sensitized, sugars such as saccharose, maltose, lactose, etc., and sugar alcohols such as mannitol, etc. can be added and applied to the supporting portion. These substances may be coated in advance on the supporting portion.

[0067] When the detection reagent is applied and dried on the immunochromatographic assay test strip 20, the detection reagent can be applied and dried directly on the immunochromatographic assay test strip 20 on which the immobilized reagent is immobilized. Alternatively, the detection reagent may be applied and dried on another porous material, such as cellulose filter paper, glass fiber filter paper, or nylon nonwoven fabric, to form a detection reagent holding member, which may then be arranged so as to be connected to the immunochromatographic assay test strip 20 on which the immobilized reagent is immobilized via a capillary.

[0068] The analyte to be detected by the method of the present invention is not particularly limited as long as there exists a substance that specifically binds to it, and examples thereof include proteins, peptides, nucleic acids, sugars (particularly the sugar moieties of glycoproteins and glycolipids), complex carbohydrates, etc. "Specifically bind" means binding based on the affinity possessed by biomolecules. Examples of such binding based on affinity include binding between antigens and antibodies, binding between sugars and lectins, binding between hormones and receptors, binding between enzymes and inhibitors, binding between complementary nucleic acids and between nucleic acids and nucleic acid-binding proteins, etc.

[0069] Therefore, when the analyte has antigenicity, examples of the substance that specifically binds to the analyte include polyclonal antibodies or monoclonal antibodies, and when the analyte is a sugar, examples of the substance that specifically binds to the analyte include lectin proteins.

[0070] Specific examples of substances to be detected include, but are not limited to, carcinoembryonic antigen (CEA), HER2 protein, prostate-specific antigen (PSA), CA19-9, alpha-fetoprotein (AFP), immunosuppressant acidic protein (IPA), CA15-3, CA125, estrogen receptor, progesterone receptor, fecal occult blood, troponin I, troponin T, CK-MB, CRP, human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), syphilis antibody, influenza virus, human hemoglobin, chlamydia antigen, group A beta streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, and glycated albumin.

[0071] Examples of specimens containing the substance to be analyzed include biological samples, i.e., whole blood, serum, plasma, urine, saliva, sputum, nasal or pharyngeal swabs, cerebrospinal fluid, amniotic fluid, nipple secretions, tears, sweat, skin exudates, extracts from tissues, cells and stool, as well as extracts from milk, eggs, wheat, beans, beef, pork, chicken, etc., and foods containing these.

[0072] In the present invention, a developing liquid may be used if necessary. The developing liquid is a liquid that constitutes the mobile phase in the immunochromatographic assay method, and moves on the immunochromatographic assay test strip 20, which is the stationary phase, together with the sample containing the substance to be detected and the labeled detection reagent. Any developing liquid may be used as long as it is of this type. EXAMPLES

[0073] The present invention will be specifically described below with reference to examples. The materials, amounts, ratios, processing procedures, etc. shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the specific examples shown below.

[0074] A membrane for immunochromatographic assay was prepared and its physical properties were measured by the following measurement methods.

[0075] (Thickness) The thickness of the immunochromatographic assay membrane was measured at six points using a contact-type film thickness meter (Mitutoyo Corporation) and the average was calculated. The contact terminal used was a cylindrical terminal with a bottom diameter of 1 cm. The measurement force was 0.4 N or less.

[0076] (Capillary flow time) The capillary flow time of the immunochromatographic assay membrane was measured by the following water absorption test.

[0077] A test piece measuring 10 mm in width and 50 mm in length was cut out, and water (pure water) was drawn up from one end of the test piece in the longitudinal direction by capillary action, and the time it took for the water to be drawn up to a height of 40 mm was measured. Six test pieces were prepared, and the water absorption test was carried out on each of them, and the average capillary flow time was calculated.

[0078] (Fluorescence quantum yield) The fluorescence quantum yield of the immunochromatographic assay membrane was determined using Quantaurus-QY manufactured by Hamamatsu Photonics K.K. at an excitation wavelength of 365 nm.

[0079] (brightness) The surface of the immunochromatographic assay membrane was measured at two points using a spectrophotometer (Konica Minolta Japan, Inc.) with a 6 mm target mask, and the brightness L * The average value of (D65) was calculated.

[0080] A polymer film support and a porous membrane were combined to prepare membranes for immunochromatographic assays in Examples and Comparative Examples.

[0081] Example 1 Nitrocellulose resin 4.6% by mass, cellulose acetate resin 0.2% by mass, acetone 43.7% by mass, propanol 46.1% by mass, isopropyl alcohol 2.0% by mass, and water 3.4% by mass were placed in a tank and stirred to obtain a uniform membrane-forming solution. Then, in an atmosphere adjusted to a temperature of 33°C and a relative humidity of 55%RH, the membrane-forming solution was cast onto a black pigment-containing PET film (thickness: 100 μm) heated to 33°C, and the solvent was evaporated to solidify the membrane-forming solution. Then, the membrane was obtained by drying. Sodium dodecylbenzenesulfonate was added to the obtained porous membrane to prepare the immunochromatographic assay membrane of Example 1.

[0082] Comparative Example 1 An immunochromatographic assay membrane of Comparative Example 1 was prepared in the same manner as in Example 1, except that the polymer film support was changed to a colorless and transparent (not containing a black pigment) PET film (thickness: 100 μm).

[0083] (Examples 2 to 3, Comparative Examples 2 to 3) A porous membrane containing a black pigment was produced in the same manner as in Example 1, except that the acetone in the membrane-forming solution was changed to a black pigment (Savinyl Black RLSN) acetone solution and the polymer film support was changed to a colorless and transparent (not containing black pigment) PET film (thickness: 100 μm). The concentration of the black pigment in the porous membrane is shown in Table 1 below. Sodium dodecylbenzenesulfonate was added to each porous membrane in the same manner as in Example 1 to prepare immunochromatographic assay membranes of Examples 2 and 3 and Comparative Examples 2 and 3.

[0084] The physical properties of the immunochromatographic assay membranes of the Examples and Comparative Examples were examined by the above-mentioned methods. The results are summarized in Table 1 below, together with the configuration of each immunochromatographic assay membrane.

[0085] [Table 1]

[0086] The immunochromatographic assay membranes of Examples 1 to 3 are provided with an autofluorescence reducing material as a polymer film support or a porous membrane. Therefore, the immunochromatographic assay membranes of Examples 1 to 3 have a fluorescence quantum yield of 12.4% at an excitation wavelength of 365 nm and a brightness L * (D65) is 60% or more. On the other hand, the immunochromatographic assay membrane of Comparative Example 1 does not include an autofluorescence reducing material. Therefore, the fluorescence quantum yield at an excitation wavelength of 365 nm reaches 19%. The immunochromatographic assay membrane of Comparative Example 2 has a fluorescence quantum yield of 18% or more at an excitation wavelength of 365 nm, and the immunochromatographic assay membrane of Comparative Example 3 has a brightness L * (D65) is less than 60%.

[0087] Immunochromatographic assay test strips were prepared using the immunochromatographic assay membranes of Examples 1 to 3 and Comparative Examples 1 to 3, and were evaluated. The substance to be analyzed in the immunochromatographic assay test strips was an influenza antigen. All operations, unless otherwise specified, were performed in an environment at a temperature of 23°C and a relative humidity of 55%RH.

[0088] First, antibody-sensitized labeled particles were prepared by the following method. A 100 mM MES buffer solution with a pH of 6.0 was prepared using 2-morpholinoethanesulfonic acid (hereinafter referred to as "MES", manufactured by Tokyo Chemical Industry Co., Ltd., M0606), caustic soda, and pure water. 693.3 μL of the obtained MES buffer solution, 26.7 μL of a 2.7 wt% dispersion of labeled particles, 9.0 μL of a 4.0 wt% solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter referred to as "EDC", manufactured by Tokyo Chemical Industry Co., Ltd., D1601), and 18.0 μL of a 4.0 wt% solution of N-hydroxysuccinimide (hereinafter referred to as "NHS", manufactured by Tokyo Chemical Industry Co., Ltd., B0249) were placed in a container and allowed to stand at room temperature.

[0089] After 15 minutes, the supernatant was removed by centrifugation to remove unreacted EDC and NHS. 720.0 μL of MES buffer was added to disperse the microparticles, and then anti-influenza virus type A antibody (BMRia042, Biomatrix Research Institute) was added to the labeled particles at 10.0 wt% and reacted at 37 ° C for 2 hours. 8640.0 μL of blocking solution (100 mM borate buffer, pH = 8.5) containing 1.0 wt% casein (Wako Pure Chemical Industries, Ltd., 030-01505) was added thereto and allowed to stand for 1 hour in a thermostatic chamber at 37 ° C. After 1 hour, centrifugation was performed at 20,000 g for 30 minutes using a centrifuge (6200, Kubota Shoji Co., Ltd.) and a centrifuge rotor (AF-5008C, Kubota Shoji Co., Ltd.), and the antibody-bound labeled particles were precipitated, and the supernatant was discarded.

[0090] Next, 8640.0 μL of borate buffer (50 mM, pH=10.0) was added, and the mixture was treated with an ultrasonic disperser (UH-50, manufactured by SMT Co., Ltd.) for 10 seconds to disperse the antibody-bound labeled particles. After sufficient dispersion, the mixture was centrifuged at 20,000 g for 20 minutes, and the supernatant was discarded. Borate buffer (50 mM, pH=10.0) was added so that the concentration of the antibody-bound labeled particles was 0.076 wt%, and the mixture was sufficiently dispersed with an ultrasonic disperser. By the above method, antibody-bound labeled particles (hereinafter also referred to as "detection reagent") were obtained.

[0091] [Impregnation and drying of detection reagent onto conjugate pad] A polyethylene conjugate pad (Pall, 6613) was immersed in a large excess of 0.05% by weight Tween-20 (registered trademark, Sigma-Aldrich, T2700) and the excess liquid was removed. After drying at 50°C for 60 minutes, it was cut into a shape with a height of 10 mm and a length of 300 mm. Next, 795 μL of detection reagent was evenly applied using a micropipette and dried at 37°C for 30 minutes.

[0092] [Pretreatment of sample pad] A regenerated cellulose continuous long fiber nonwoven fabric (Asahi Kasei Microline S-06) was immersed in a large excess of PBS buffer (66 mM, pH 7.4) containing 6.0 wt% skim milk (Fujifilm Wako Pure Chemical Industries, 190-12865), 1.0 wt% BSA (Sigma-Aldrich A7906), and 2.0 wt% Tween-20, and after removing the excess liquid, it was dried at 50°C for 120 minutes. Then, it was cut into a shape of 22 mm in height and 210 mm in length.

[0093] [Preparation of capture antibody-coated membrane] The immunochromatographic assay membranes of Examples 1 to 3 and Comparative Examples 1 to 3 were cut to a shape of 25 mm in width and 150 mm in length. Using a liquid applicator (Musashi Engineering, 300DS), a PBS solution (66 mM, pH 7.4) containing 0.1 wt% anti-influenza virus type A antibody (BMRia046, Biomatrix Research Institute) was applied to a portion of 15 mm in height at a rate of 0.1 μL / mm. The membranes were then dried at 50° C. for 24 hours.

[0094] [Preparation of immunochromatographic assay test strips] The capture antibody-coated membrane obtained as described above, an absorbent pad (Pall, 66211), a conjugate pad containing a detection reagent, and a sample pad were attached to a backing card (Adhesives Research, AR9020). Then, the resulting strip was cut to a width of 5 mm using a cutter to obtain immunochromatographic assay test strips of Examples 4 to 6 and Comparative Examples 4 to 6, each having a width of 5 mm and a height of 60 mm.

[0095] The immunochromatographic assay test strips of Examples 4 to 6 and Comparative Examples 4 to 6 were tested by the following method to evaluate their performance.

[0096] (Color Intensity) Influenza A H1N1 subtype antigen (manufactured by HyTest) was diluted to a predetermined concentration with 200 mM Tris buffer (pH 8.0) containing 200 mM sodium chloride, 1.5 mass% TritonX-100, and 0.5 mass% Tween-20 to prepare a sample. The antigen concentrations were 0.05 μg / mL and 0.02 μg / mL. 100 μL of the sample was dropped onto the sample pad of the immunochromatographic assay test strip of Examples 4 to 6 and Comparative Examples 4 to 6 and developed. 10 minutes after dropping, a digital photograph was taken at an excitation light intensity of 256 / 256 using a reader (manufactured by Musashi Optical Systems Co., Ltd.) equipped with an LED light source (OPDR-110-60UV375P manufactured by Optics FA Co., Ltd.) with an excitation light peak wavelength of around 365 nm.

[0097] The obtained digital photograph was subjected to image analysis to determine the color intensity. Specifically, using image analysis software (ImageJ, National Institutes of Health, USA), a 3 mm wide × 12.5 mm long region including the detection area was graphed with the horizontal axis representing the longitudinal position and the vertical axis representing brightness from 0 to 255. The peak area value of the detection area was obtained, and this peak area value was regarded as the color intensity. The analysis results are shown in Table 2.

[0098] [Table 2]

[0099] In the immunochromatographic assay test strips of Examples 4 to 6 using the immunochromatographic assay membranes of Examples 1 to 3, peaks were detected at both antigen concentrations of 0.05 μg / mL and 0.02 μg / mL. In contrast, in the immunochromatographic assay test strip of Comparative Example 4 using the immunochromatographic assay membrane of Comparative Example 1, no peak was detected at an antigen concentration of 0.02 μg / mL. In addition, in the immunochromatographic assay test strips of Comparative Example 5 and Comparative Example 6 using the immunochromatographic assay membranes of Comparative Example 2 and Comparative Example 3, no peak was detected at either antigen concentration of 0.05 μg / mL or 0.02 μg / mL.

[0100] It was confirmed that the immunochromatographic assay test strip of the present invention is capable of highly sensitive detection.

[0101] By using the immunochromatographic assay membrane of the present invention, the immunochromatographic assay test strip of the present invention can reduce background noise due to autofluorescence from the porous membrane or backing sheet, thereby improving the sensitivity in the fluorescent immunochromatographic assay method. [Explanation of symbols]

[0102] 10... Immunochromatographic assay membrane 12... Polymer film support 14…Porous membrane 20...Test strip for immunochromatographic assay 22...Chromatographic medium 24...Sample pad 26...Conjugate pad 28...Absorbent pad 30…Backing sheet

Claims

1. An immunochromatographic assay membrane comprising a polymer film support and a porous membrane laminated on the polymer film support, wherein the porous membrane is made of a self-fluorescence reducing material containing a dye that absorbs 50% or more of light with a wavelength of 190 to 830 nm at a ratio of 0.50% or less based on the mass of the porous membrane.

2. The fluorescence quantum yield at an excitation wavelength of 365 nm is 18% or less, and the lightness L ＊ (D65)is 60% or more, and the membrane for immunochromatographic assay according to claim 1, characterized in that.

3. The immunochromatographic assay membrane according to claim 1 or 2, wherein the material of the polymer film support is polyethylene terephthalate.

4. An immunochromatographic assay test strip comprising the immunochromatographic assay membrane according to any one of claims 1 to 3 as a chromatographic medium.

5. A method for examining an analyte substance contained in a specimen, The method for examination comprising contacting the specimen with a fluorescent labeling substance using the immunochromatographic assay test strip according to claim 4.

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