JP7683852B2 - Immunochromatographic assay membrane, immunochromatographic assay test strip, and testing method - Google Patents

Description

The present invention relates to a membrane for immunochromatographic assay, a test strip for immunochromatographic assay, and a testing method.

Immunochromatographic assays are widely used as a simple immunoassay method that uses 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.

In such test strips for performing immunochromatographic assays, 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. As 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.

Commonly used labeling substances include enzyme-containing proteins, metal colloids such as gold colloids, and colored latex particles. 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 rapid, highly accurate diagnosis.

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.

In the fluorescent immunochromatographic assay method, a fluorescent immunochromatographic reader equipped with an excitation light irradiation section and a fluorescence detection section 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 unwanted fluorescence from the porous membrane and its support, which are the development site of the immunochromatographic assay, and the autofluorescence from the backing sheet that supports them. 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 trying to detect even smaller amounts of the analyte.

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 the detector as a fluorescence detection means.

Furthermore, Patent Document 3 proposes a method of detecting fluorescence in a wavelength range that reduces the background effects of autofluorescence from a porous membrane and its support, 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.

JP 2009-115822 A JP 2016-191567 A WO2013/151066 publication

However, the technologies of Patent Documents 2 and 3 detect fluorescence in a wavelength range 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 improvements are needed to increase the sensitivity for high-precision rapid diagnosis.

Therefore, the present invention aims to provide an immunochromatographic assay membrane that can produce an immunochromatographic assay test strip that can be detected with high sensitivity, an immunochromatographic assay test strip that can be detected with high sensitivity, and a highly sensitive testing method.

In order to achieve the above object, 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 high sensitivity and 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.

According to the present invention, it is possible to provide an immunochromatographic assay membrane that can produce a highly sensitive immunochromatographic assay test strip, a highly sensitive immunochromatographic assay test strip, and a highly sensitive testing method.

FIG. 1 is a schematic structural diagram of the immunochromatographic assay membrane of the present invention. FIG. 1 is a schematic structural diagram of a test strip for immunochromatographic assay according to the present invention.

The immunochromatographic assay membrane of the present invention will be described in detail below with reference to the drawings.
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.

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 preferably has a fluorescence quantum yield of 18% or less at an excitation wavelength of 365 nm and a lightness L ^* (D65) of 60% or more as measured by a spectrophotometer. The fluorescence quantum yield is measured in a state where the pores of the immunochromatographic assay membrane 10 are filled with water.

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 the immunochromatographic assay membrane. If the fluorescence quantum yield at 365 nm is 18% or less, the autofluorescence is suppressed when the immunochromatographic assay test strip is prepared and tested, and the sensitivity is improved. On the other hand, if the brightness L ^* (D65) is less than 60%, only the fluorescence of the labeling substance on the membrane surface is detected when the immunochromatographic assay test strip is prepared and tested, leading to a decrease in sensitivity.

Therefore, a lightness L ^* (D65) of 60% or more leads to improved sensitivity. The present inventors have found that by using a specific autofluorescence reducing material, the fluorescence quantum yield at a wavelength of 365 nm of an immunochromatographic assay membrane can be reduced to 18% or less, and the lightness can be increased to 60% or more.

The autofluorescence reducing material in the present invention may be either the polymer film support 12 or the porous membrane 14, and 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.

Polymers used in polymer films containing a dye that absorbs ultraviolet or visible light include, for example, polyesters such as polyethylene terephthalate (PET), polyimides, nylon, and the like. In this specification, "absorbing ultraviolet or visible light" specifically means absorbing 50% or more of light with a wavelength of 190 to 830 nm. There are no particular limitations on the dye that absorbs ultraviolet or visible light, and an example of such a dye is a black dye.

In this specification, "black" refers to a color whose CIE color coordinates, measured in accordance with JIS Z8701-1999 using a C light source and a viewing angle of 2 degrees, are in the ranges of -1.0≦a ^* ≦2.5 and -1.0≦b ^* ≦15.0, and whose L ^* value is in the range of 0<L ^* ≦50.

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 m thick, have a CIE color coordinate system in the range of -1.0≦a ^* ≦2.5 and -1.0≦b ^* ≦15.0, and contain a black pigment such that the L ^* value is in the range of 0<L ^* ≦50.

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

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.

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.

In the immunochromatographic assay membrane 10 of the present invention, the thickness of the polymer film support 12 is not particularly limited and can be in the usual range. Considering ease of handling, it is preferable that the thickness is about 50 to 100 μm.

As the porous membrane 14, a membrane based on nitrocellulose, such as nitrocellulose or a nitrocellulose mixture, which is capable of binding proteins and does not react with the detection reagent, immobilization reagent, or analyte used in the immunochromatographic assay, 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.

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

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

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.), Carbon Black #2600, #2400, #2350, #2200, #1000, #990, #980, #970, #960, #950, #850 (manufactured by Mitsubishi Chemical Corporation), etc.

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%, RESORIN BLACK BS (Bayer Japan AG), KAYASET BLACK A-N (Nippon Kayaku Co., Ltd.), DAIWA BLACK MSC (Daiwa Kasei Co., Ltd.), HSB-202 (Mitsubishi Kasei Co., Ltd.), NEPTUNE BLACK X60, and NEOPEN BLACK. X58 (manufactured by BASF Japan Ltd.), Oleosol Fast BLACK RL (manufactured by Taoka Chemical Co., Ltd.), Chuo BLACK 80, Chuo BLACK 80-15 (manufactured by Chuo Synthetic Chemical Industry Co., Ltd.), and the like.

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 it into a smooth form, and then evaporating the solvent.

The porous membrane as an autofluorescence reducing material preferably has a lightness L ^* (D65) of 60% or more, more preferably 70% or more, when measured by a spectrophotometer. The lightness L ^* (D65) when measured by a spectrophotometer is an index of the whiteness of the porous membrane. The present inventors have found that a porous membrane with a lightness L ^* (D65) of 60% or more has a sensitivity suitable for fluorescence analysis without significantly inhibiting the fluorescence from a fluorescent labeling substance when irradiated with excitation light.

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, which 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 for both the polymer film support 12 and the porous membrane 14.

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 standpoint of the spreadability of the labeled antibody and the retention capacity of the capture antibody solution.

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.

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 labeled substance. Examples of such a substance include cationic surfactants, anionic surfactants, zwitterionic surfactants, and nonionic surfactants.

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

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. Among these, alkyl benzene sulfonate salts are preferred, and sodium dodecylbenzene sulfonate (SDBS) is particularly preferred.

Examples of amphoteric surfactants include alkyl betaine compounds, imidazoline compounds, alkyl amine oxides, and bisoxyborate compounds.

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, sorbitan fatty acid esters, etc.

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, the desired effect is usually achieved when the surfactant is incorporated into the immunochromatographic assay membrane 10 in an amount of about 0.5 to 2.0 μg/ ^mm3 .

The immunochromatographic assay test strip of the present invention comprises 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 the actual operation and the observation of the reaction results.

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.

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

The conjugate pad 26 is composed of a porous body made of glass fiber or cellulose, or a nonwoven fabric. The conjugate pad 26 already contains a labeling substance bound to an antibody that binds to the analyte. As the analyte moves through the conjugate pad 26, it binds to the antibody and becomes labeled.

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

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.

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

First, a dispersion of labeled particles adjusted to a prescribed 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, allowing 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, thereby blocking the labeled particles. Blocking is an operation in which particles alone, or particles carrying antibodies or antigens, are coated. Various blocking agents can be used depending on the substance to be tested, the specimen, or the composition of the solution in which it is diluted.

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. To wash the labeled particles after antibody adsorption and blocking, centrifugation is performed to separate the supernatant containing 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, which includes 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.

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.

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.

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 used. In the case of covalent bonding, cyanogen bromide, glutaraldehyde, carbodiimide, etc. are generally used to activate the chromatographic medium 22, but any method can be used.

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

Examples include fine particles of organic polymeric substances such as organic polymer latex particles obtained by emulsion polymerization methods, such as polystyrene, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, and acrolein-ethylene glycol dimethacrylate copolymer; fine particles of gelatin, bentonite, agarose, cross-linked dextran, and inorganic oxides such as silica, silica-alumina, and alumina, and inorganic particles in which functional groups have been introduced into inorganic oxides by silane coupling treatment or the like.

In the present invention, direct immobilization is preferred due to the ease of sensitivity adjustment, etc. In addition, various methods can be used to immobilize the immobilized reagent to 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 development direction of the chromatographic medium 22, numbers, letters, symbols (+, -, etc.), etc.

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. Generally, 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 with one or a combination of two or more surfactants such as Tween 20, Triton X-100, and SDS, if necessary.

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 fluorophore and enzymes are used as the labeling substance. In the present invention, a labeled detection reagent is prepared by sensitizing the detection reagent to a fluorescent labeling substance.

Fluorescent labeling substances are suitable for detecting the presence of analyte with high accuracy and sensitivity, and the fluorescence generated by irradiation with excitation light can be used as a label.

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 include materials used as 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.

In addition, _an insoluble carrier made of a material capable of emitting fluorescence can also be used. Examples of such particles include particles made of _MIMIIO4 or _MAI5O12 ( _M is yttrium (Y), lanthanum (La), or gadolinium (Gd), and _MII is niobium (Nb), phosphorus (P), or vanadium (V) ₎ activated with a rare _earth element such as europium (Eu) or terbium (Tb) _, and quantum dots made of CdSe, CdTe, InP, InN, InAs, CdS, Si, or Ge.

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

As a method for sensitizing a detection reagent to a fluorescent labeling substance, known methods such as physical adsorption and chemical binding can be used. 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 reactive groups, 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 then adding a bovine serum albumin solution or the like to block the particle surface to which the antibody is not bound.

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 that constitutes 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 that constitutes the stationary phase, i.e., in the region between the end of the immunochromatographic assay test strip 20 where the mobile phase is applied and the reaction site.

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

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

The analyte to be detected by the method of the present invention is not particularly limited as long as there exists a substance to which it specifically binds, and examples 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 binding between nucleic acids and nucleic acid-binding proteins.

Therefore, when the analyte has antigenicity, examples of substances that specifically bind to the analyte include polyclonal antibodies or monoclonal antibodies. When the analyte is a sugar, examples of substances that specifically bind to the analyte include lectin proteins.

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-hemolytic streptococcus antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, and glycated albumin.

Examples of specimens containing the substance to be analyzed include biological samples, such as 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.

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.

The present invention will be specifically explained below with reference to the following examples. The materials, amounts used, ratios, processing procedures, etc. shown in the following examples can be changed as appropriate 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.

A membrane for immunochromatographic assay was prepared and its physical properties were measured. The measurement methods used are as follows:

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

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

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 a water absorption test was carried out on each of them, and the average capillary flow time was calculated.

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

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

A polymer film support and a porous membrane were combined to produce membranes for immunochromatographic assays in the examples and comparative examples.

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 dried to obtain a membrane.
Sodium dodecylbenzenesulfonate was added to the obtained porous membrane to prepare the immunochromatographic assay membrane of Example 1.

(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).

(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 as 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.

The physical properties of the immunochromatographic assay membranes of the Examples and Comparative Examples were examined using the methods described above. The results are summarized in Table 1 below, along with the configuration of each immunochromatographic assay membrane.

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, and therefore have a fluorescence quantum yield of at most 12.4% at an excitation wavelength of 365 nm and a lightness L ^* (D65) of 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 lightness L ^* (D65) of less than 60%.

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.

First, antibody-sensitized labeled particles were prepared by the following method.
Using 2-morpholinoethanesulfonic acid (hereinafter referred to as "MES", manufactured by Tokyo Chemical Industry Co., Ltd., M0606), caustic soda, and pure water, a MES buffer solution with a pH of 6.0 and a concentration of 100 mM was prepared. 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.

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 (030-01505, Wako Pure Chemical Industries, Ltd.) was added 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.

Next, 8640.0 μL of borate buffer (50 mM, pH = 10.0) was added and treated with an ultrasonic disperser (UH-50, manufactured by SMT) for 10 seconds to disperse the antibody-bound labeled particles. After sufficient dispersion, centrifugation was performed 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 particles were sufficiently dispersed with an ultrasonic disperser. By the above method, antibody-bound labeled particles (hereinafter also referred to as "detection reagent") were obtained.

[Impregnation and drying of detection reagent into 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 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.

[Sample pad pretreatment]
A regenerated cellulose continuous long fiber nonwoven fabric (Microline S-06, Asahi Kasei Corporation) was immersed in a large excess of PBS buffer solution (66 mM, pH 7.4) containing 6.0 wt% skim milk (190-12865, Fujifilm Wako Pure Chemical Industries, Ltd.), 1.0 wt% BSA (A7906, Sigma-Aldrich Corporation), and 2.0 wt% Tween-20, and after removing 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.

[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 (300DS, manufactured by Musashi Engineering Co., Ltd.), a PBS solution (66 mM, pH 7.4) containing 0.1 wt% anti-influenza virus type A antibody (BMRia046, manufactured by Biomatrix Research Institute Co., Ltd.) was applied to a portion having a height of 15 mm at a rate of 0.1 μL/mm. The membranes were then dried at 50° C. for 24 hours.

[Preparation of immunochromatographic assay test strips]
The capture antibody-coated membrane obtained as described above, an absorption pad (66211, manufactured by Pall), a conjugate pad containing a detection reagent, and a sample pad were attached to a backing card (AR9020, manufactured by Adhesives Research). Then, the resulting membrane 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.

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.

(Coloring 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% by mass TritonX-100, and 0.5% by 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, and 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.

The resulting 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 taken as the color intensity. The analysis results are shown in Table 2.

In the immunochromatographic assay test strips of Examples 4 to 6, which used 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, which used the immunochromatographic assay membrane of Comparative Example 1, no peaks were detected at antigen concentrations of 0.02 μg/mL. In addition, in the immunochromatographic assay test strips of Comparative Examples 5 and 6, which used the immunochromatographic assay membranes of Comparative Examples 2 and 3, no peaks were detected at antigen concentrations of 0.05 μg/mL and 0.02 μg/mL.

It has been confirmed that the immunochromatographic assay test strip of the present invention is capable of highly sensitive detection.

By using the immunochromatographic assay membrane of the present invention, the immunochromatographic assay test strip of the present invention can reduce background noise caused by autofluorescence from the porous membrane and backing sheet, thereby improving the sensitivity in the fluorescent immunochromatographic assay method.

Reference Signs List 10: Immunochromatographic assay membrane 12: Polymer film support 14: Porous membrane 20: Immunochromatographic assay test strip 22: Chromatographic medium 24: Sample pad 26: Conjugate pad 28: Absorbent pad 30: Backing sheet

Claims (5)

A membrane for immunochromatographic assay comprising a polymer film support and a porous membrane laminated on the polymer film support, the porous membrane being made of an autofluorescence reducing material containing a dye that absorbs 50% or more of light having a wavelength of 190 to 830 nm in a proportion of 0.50% or less relative to the mass of the porous membrane . 2. The immunochromatographic assay membrane according to claim 1, which has a fluorescence quantum yield of 18% or less at an excitation wavelength of 365 nm and a lightness L ^* (D65) of 60% or more. 3. The immunochromatographic assay membrane according to claim 1, wherein the material of the polymer film support is polyethylene terephthalate . 4. A test strip for immunochromatographic assay comprising the immunochromatographic assay membrane according to claim 1 as a chromatographic medium . A method for testing an analyte contained in a sample, comprising the steps of:
5. A testing method using the immunochromatographic assay test strip according to claim 4, comprising contacting a specimen with a fluorescent labeling substance .

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