Method of preventing fluorescence fading, lateral flow assay test strip, lateral flow assay device, and lateral flow assay kit

Abstract

To provide a novel method of preventing fluorescence fading, lateral flow assay test strip, lateral flow assay device, and lateral flow assay kit.SOLUTION: A method of preventing fluorescence fading is provided, involving allowing (A) a dry fluorescent substance or fluorescent label and (B) a dry colorant to coexist.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

[0001] The present invention relates to a method for suppressing fluorescence fading, a test strip for lateral flow assay, a device for lateral flow assay, and a kit for lateral flow assay. [Background technology]

[0002] Lateral flow assays (LFAs) are used as diagnostic testing tools to rapidly detect specific biomarkers above the detection threshold.

[0003] High detection sensitivity for specific biomarkers is required for early detection of diseases using LFA. Furthermore, in order to enable LFA to detect not only positive and negative results but also the progression and severity of diseases, etc., specific biomarkers must be semi-quantitative or quantitative.

[0004] As an example of semi-quantitative analysis using an LFA, Patent Document 1 discloses a lateral flow assay having a test zone for determining a target analyte in a test sample and a calibration zone for semi-quantitative determination. Streptavidin-labeled colloidal gold is used as a calibrator, and a predetermined amount of biotin-labeled BSA (calibrator receptor) is non-diffusively attached to the calibration zone (calibration zone). Binding of the calibrator to the calibrator receptor generates a signal of a predetermined intensity in the calibration zone, and by comparing the signal intensity with that of the colloidal gold in the test zone, the presence of the target analyte in the test sample can be measured semi-quantitatively.

[0005] Furthermore, the use of fluorescent substances has been investigated as a method for improving the detection sensitivity in immunochromatography. For example, Patent Document 2 discloses the use of fluorescently labeled silica particles instead of gold colloids as a means for improving the detection sensitivity using immunochromatography. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO9709620 [Patent Document 2] Japanese Patent Publication No. 2022-62284 Summary of the Invention [Problem to be solved by the invention]

[0007] However, when fluorescent substances or fluorescent labels are dried and stored on a chromatograph for use in a lateral flow assay, they are found to be more susceptible to photobleaching (hereinafter simply referred to as "fading") under light exposure conditions than when stored in liquid form. Furthermore, they have found a problem in that the binding between the faded fluorescent substance or faded fluorescent label and a capture agent (e.g., an anti-fluorescent substance antibody) that captures the fluorescent substance or fluorescent label is reduced.

[0008] Therefore, an object of the present invention is to provide a new method for suppressing fluorescence fading, a test strip for lateral flow assay, a device for lateral flow assay, and a kit for lateral flow assay. [Means for solving the problem]

[0009] As a result of intensive research to solve the above problems, the present inventors discovered that the fading of fluorescence can be suppressed by allowing (A) a fluorescent substance or fluorescent label and (B) a coloring material to coexist in a dry state, and thus completed the present invention.

[0010] That is, the present invention relates to the following inventions. <1> A method for suppressing fading of fluorescence, characterized by causing (A) a fluorescent substance or fluorescent label and (B) a coloring material to coexist in a dry state. <2> The (B) coloring material is a coloring material that reflects a part of visible light. <1> A method for suppressing fading of fluorescence according to claim 1. <3> Used in lateral flow assays or immunoassays, <1> A method for suppressing fading of fluorescence according to claim 1. <4> The lateral flow assay is an immunochromatographic assay. <1> A method for suppressing fading of fluorescence according to claim 1. <5> A lateral flow assay test strip having a matrix, comprising: a fluorescent substance or the like retaining portion arranged on the matrix, which retains (A) a fluorescent substance or a fluorescent label and (B) a coloring material in a dry state; A lateral flow assay test strip comprising: <6> A test strip for immunochromatographic assay, <5> A test strip for a lateral flow assay according to claim 1. <7> the matrix has a reference line; <5> A test strip for a lateral flow assay according to claim 1. <8> a capturer that captures the fluorescent substance or the fluorescent label is immobilized on the reference line; <7> A test strip for a lateral flow assay according to claim 1. <9> The matrix has a test line, and a capture body that captures the detection target substance is immobilized on the test line. <5> A test strip for a lateral flow assay according to claim 1. <10> the fluorescent label is an enzyme labeled with a fluorescent substance; <5> A test strip for a lateral flow assay according to claim 1. <11> The enzyme is peroxidase, galactosidase, or phosphatase. <10> A test strip for a lateral flow assay according to claim 1. <12> <5> ~ <11> A lateral flow assay device comprising the lateral flow assay test strip according to any one of the preceding items. <13> <5> ~ <11> A lateral flow assay kit comprising the lateral flow assay test strip according to any one of the above items. [Effects of the Invention]

[0011] The present invention provides a novel method for suppressing fading of fluorescence. The present invention also provides a lateral flow assay test strip, a lateral flow assay device, and a lateral flow assay kit, each of which includes a matrix and a fluorescent substance or other retaining portion that retains (A) a fluorescent substance or fluorescent label and (B) a coloring material in a dry state. [Brief explanation of the drawings]

[0012] [Figure 1-1] FIG. 1 is a perspective view showing an outline of a test strip for lateral flow assay according to a first embodiment. [Figure 1-2] 1 is a cross-sectional view showing an outline of a test piece for lateral flow assay according to Embodiment 1. FIG. [Figure 2] FIG. 1 is a cross-sectional view showing an outline of a test strip for lateral flow assay according to a variation of the first embodiment. [Figure 3-1] FIG. 1 is a perspective view showing an outline of a test strip for lateral flow assay according to a second embodiment. [Figure 3-2] FIG. 1 is a cross-sectional view showing an outline of a test piece for lateral flow assay according to a second embodiment. [Figure 4] FIG. 10 is a cross-sectional view showing an outline of a test strip for lateral flow assay according to a variation of the second embodiment. [Figure 5] This shows the FITC detection results at the semi-quantitative line when a lateral flow assay is performed on a lateral flow assay device after storing it under light-blocking conditions for 3 hours and after illuminating a designated area of ​​the lateral flow assay test strip with an LED light for 3 hours, and also shows an evaluation of the color intensity of the semi-quantitative line. [Figure 6] In a lateral flow assay device, anti-FITC antibodies A to G that capture FITC were immobilized on the semi-quantitative line. The device was stored under specified exposure conditions with and without the coexistence of FITC, a fluorescent substance, and a colorant (Blue No. 1). The results of FITC detection on the semi-quantitative line when a lateral flow assay was performed were shown. [Figure 7]The sample pad of a lateral flow assay device was irradiated with LED light, UV light, or sunlight for a specified period of time under conditions where the fluorescent substance FITC and a colorant (Blue No. 1) were not present on the sample pad, and where they were present on the sample pad. The results of FITC detection at the semi-quantitative line, as well as the color intensity and its evaluation, were then shown when a lateral flow assay was performed. [Figure 8] The sample pad of a lateral flow assay device was irradiated with an LED light for a specified period of time under conditions where the fluorescent substance FITC was not present in combination with various colorants (Blue No. 1, Red No. 2, Yellow No. 4, Green No. 3, Blue No. 2, or phycocyanin) and where they were present in combination. The results show the detection results of FITC at the semi-quantitative line and an evaluation of color intensity when a lateral flow assay was performed. DETAILED DESCRIPTION OF THE INVENTION

[0013] Preferred embodiments of the present invention will be described in detail below, but the present invention is not limited to the following embodiments.

[0014] In this specification, the term "fading" of fluorescence refers to the decomposition or structural change of a fluorescent substance caused by irradiation of the fluorescent substance with light such as fluorescent lamp, sunlight, LED, or UV light, or the weakening of the intensity of fluorescence emitted by the fluorescent substance in response to irradiation with a specific excitation light due to photodecomposition or the like. This is also called photobleaching. Furthermore, "suppressing fading" means suppressing photodecomposition of a fluorescent substance when the fluorescent substance is irradiated with light such as fluorescent lamps, sunlight, LEDs, or UV light, and also means reducing the degree of attenuation of the fluorescence intensity of the fluorescent substance in response to irradiation with a predetermined excitation light before and after light irradiation. Furthermore, when a specific biomarker is semi-quantified based on the amount of fluorescent substance present rather than the fluorescence intensity of the fluorescent substance (as in the case of embodiment 1), the term "suppressing fading of fluorescence" also refers to suppressing structural changes in the fluorescent substance or fluorescent label and suppressing a decrease in the binding affinity between the fluorescent substance or fluorescent label and a substance that binds to the fluorescent substance or fluorescent label (e.g., an anti-fluorescent substance antibody). The substance that binds to the fluorescent label may be a substance that binds to the fluorescent substance contained in the fluorescent label, or a substance that binds to the binding moiety between the fluorescent substance contained in the fluorescent label and the fluorescently labeled substance.

[0015] Furthermore, in this specification, "coexistence in a dried state" means drying a solution containing (A) a fluorescent substance or a fluorescent label and (B) a coloring material, or simultaneously or at different times attaching or applying a solution containing (A) a fluorescent substance or a fluorescent label and a solution containing (B) a coloring material to the same location on a sample pad or a matrix in a fluorescent substance or other holding portion and drying them, or attaching or applying a solution containing both a fluorescent substance and a coloring material and then drying them.

[0016] Furthermore, as used herein, "detection" not only refers to detecting the presence or absence of a specific substance, but also includes quantifying or semi-quantifying the amount of the specific substance. Semi-quantification refers to estimating the approximate concentration of the substance. For example, in an immunochromatographic assay, semi-quantification can be achieved by comparing the color intensity with that of a reference line (including a semi-quantitative line, calibration line, comparison line, etc.) described below.

[0017] As used herein, the term "signal" refers to a signal that can be detected or measured as appropriate, and includes fluorescence, radioactivity, color development, luminescence, and the like.

[0018] In this specification, the term "reference line" also includes a semi-quantitative line, a calibration line, a comparison line, etc. The signal intensity of the reference line can be used to compare with the signal intensity on the test line.

[0019] [1. Methods for suppressing fluorescence fading] The method for suppressing fading of fluorescence according to this embodiment is characterized in that (A) a fluorescent substance or fluorescent label and (B) a coloring material are allowed to coexist in a dry state.

[0020] [1-1. (A) Fluorescent substance or fluorescent label] [1-1-1. Fluorescent materials] The fluorescent substance according to this embodiment is not particularly limited, and any known and commonly used fluorescent substance can be used. Examples of the fluorescent substance include green fluorescent proteins such as GFP, AcGFP1, and ZsGreen1; blue fluorescent proteins such as AmCyan1, Sirius, SECFP, and EBFP; ZsYellow1, Examples of fluorescent dyes include yellow fluorescent proteins such as mBanana, Topaz, EYFP, and Venus; red fluorescent proteins such as R-phycoerythrin, DsRed, AsRed2, tdTomato, mCherry, mStrawberry, and mOrange; and fluorescent dyes such as fluorescein, rhodamine, coumarin, pyrene, benzofurazan, eosin, cyanine, Alexa Fluor (registered trademark), and boron-dipyrromethene (BODIPY). Alexa Fluor (registered trademark) fluorescent dyes mainly include partially sulfonated fluorescent dyes such as fluorescein, rhodamine, coumarin, and cyanine.

[0021] Specific examples of the fluorescent dye include: Fluorescein-based fluorescent dyes such as 5-carboxy-fluorescein, 6-carboxy-fluorescein, 5,6-diocarboxy-fluorescein, 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein (HEX), 6-carboxy-2',4,7,7'-tetrachlorofluorescein (TET), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE), naphthofluorescein, fluorescein isothiocyanate (FITC), and fluorescein amidite (FAM); Rhodamine-based fluorescent dyes such as 5-carboxy-rhodamine, 5-carboxytetramethylrhodamine (TAMRA), 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, rhodamine B, rhodamine B isothiocyanate (RBITC), and X-rhodamine; Coumarin-based fluorescent dyes such as 7-amino-4-methylcoumarin-acetic acid (AMCA), 7-diethylaminocoumarin (DEAC), 7-hydroxycoumarin, 7-alkoxycoumarin, and 7-aminocoumarin; benzoxazine fluorescent dyes such as 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F), and ammonium 4-fluoro-2,1,3-benzoxazole-7-sulfonate (SBD-F); Eosin-based fluorescent dyes such as eosin Y, eosin B, and eosin isothiocyanate (EITC); cyanine fluorescent dyes such as cyanine 3, cyanine 5, cyanine 5.5, cyanine 7, cyanine 7.5, and indocyanine green (ICG); Alexa Fluor® series fluorescent dyes, such as Alexa Fluor 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, and 750; Examples of BODIPY fluorescent dyes include BODIPY FL, BODIPY TMR, BODIPY 493 / 503, 530 / 550, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650, and 650 / 665. These fluorescent dyes may be used alone or in combination of two or more.

[0022] Among the above fluorescent dyes, fluorescein-based fluorescent dyes, rhodamine-based fluorescent dyes, Alexa Fluro (registered trademark)-based fluorescent dyes, and cyanine-based fluorescent dyes are preferred, and FITC, JOE, HEX, FAM, RITC, TAMRA, Alexa Fluor 488, 594, Cy3, and Cy5 are more preferred.

[0023] [1-1-2. Fluorescent Labels] The fluorescent label according to this embodiment can be a predetermined analyte fluorescently labeled with the fluorescent substance. The analyte to be fluorescently labeled is not particularly limited, and may be, for example, a substance that binds to the target substance, the target substance itself (for example, in the case of a competitive method, the target substance and the target substance labeled with a fluorescent substance competitively bind to a target substance capture body described below), or a semi-quantitative substance used for semi-quantitation.

[0024] Examples of the labeled substance to be fluorescently labeled include, for example, an antibody that binds to the target substance if the target substance is an antigen, an antigen that binds to the target substance if the target substance is an antibody, a substrate that binds to or reacts with the target substance if the target substance is an enzyme, an enzyme that binds to or reacts with the target substance if the target substance is a substrate, a lectin that binds to the target substance if the target substance is a sugar chain or a complex carbohydrate, a sugar chain or complex carbohydrate that binds to the target substance if the target substance is a lectin, a nucleic acid having a complementary sequence that hybridizes with the target substance if the target substance is a nucleic acid, a receptor that binds to the target substance if the target substance is a hormone or cytokine, a hormone or cytokine that hybridizes with the target substance if the target substance is a receptor, avidin or streptavidin if the target substance is biotin, biotin if the target substance is avidin or streptavidin, protein A or protein G if the target substance is IgG, IgG if the target substance is protein A or protein G, a protein and a nucleic acid aptamer or peptide aptamer that hybridizes with the target substance if the target substance is a nucleic acid, etc., are sometimes referred to as probes below. The target substance may also be referred to as a biomarker.

[0025] When the substance to be detected is an antigen, the entity to be fluorescently labeled may be, for example, a polyclonal antibody, a monoclonal antibody, a single-chain antibody, or a fragment thereof (F(ab), F(ab'), F(ab')2, F(v) fragment, etc.) that binds to the antigen. Examples of the antigens include antigens derived from influenza virus, coronavirus, adenovirus, respiratory syncytial virus, rotavirus, human papillomavirus, human immunodeficiency virus, hepatitis B virus, Zika virus, dengue virus, etc. (e.g., hepatitis B virus antigen (HBs antigen, hepatitis B virus-related antigen), influenza virus hemagglutinin, etc.); antigens derived from Aspergillus flavus, chlamydia, Treponema pallidum, Streptococcus, Bacillus anthracis, Staphylococcus aureus, Shigella, Escherichia coli, Salmonella enterica, Salmonella typhimurium, Salmonella paratyphimurium, Pseudomonas aeruginosa, Vibrio parahaemolyticus, etc. (e.g., aflatoxin (B1, B2, G1, G2, or M1) of Aspergillus flavus, verotoxin of enterohemorrhagic Escherichia coli, streptococcal streptococcus streptolysin O, etc.); Blood proteins such as immunoglobulin G (IgG), rheumatoid factor, and C-reactive protein (CRP); glycoproteins such as mucins; Hormones such as insulin, pituitary hormones (e.g., growth hormone (GH), adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, and melanocyte-stimulating hormone (MSH)), thyrotropin-releasing hormone (TRH), thyroid hormones (e.g., diiodothyronine, triiodothyronine, etc.), chorionic gonadotropin, calcium metabolism-regulating hormones (e.g., calcitonin, parathormone, etc.), pancreatic hormones, gastrointestinal hormones, vasoactive intestinal peptide, estrogen (e.g., estrone, etc.), and adrenal cortical hormones (e.g., cortisol, etc.); Other biological substances such as serotonin, urokinase, ferritin, substance P, prostaglandins, cholesterol, etc. Tumor markers such as prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), alkaline phosphatase, transaminases, trypsin, pepsinogen, alpha-fetoprotein (AFP), and carcinoembryonic antigen (CEA); Examples include:

[0026] When the substance to be detected is a sugar chain or a glycoconjugate, the object to be fluorescently labeled may be, for example, a lectin such as concanavalin A (ConA), wheat germ agglutinin, galectin, or C-type lectin that binds to the sugar chain or glycoconjugate, or a fragment thereof. Examples of the sugar chain or complex carbohydrate include monosaccharides, polysaccharides, and complex carbohydrates in which these are bound to proteins or lipids.

[0027] Furthermore, the entity to be fluorescently labeled may be an enzyme that binds to or reacts with a specific substrate. The enzyme is not particularly limited, and examples thereof include peroxidase (for example, horseradish peroxidase), galactosidase (for example, β-D-galactosidase), and phosphatase (for example, alkaline phosphatase). Examples of the substrate include: For peroxidase, chromogenic substrates such as 3,3',5,5'-tetramethylbenzidine (TMB), 3,3',4,4'-diaminobenzidine (DAB), 4-chloro-1-naphthol (4CN), 2,2'-azino-di[3-ethylbenzthiazoline]sulfonate (ABTS), and o-phenylenediamine (OPD) are used; and luminescent substrates such as luminol, polyphenols (e.g., pyrogallol, puprogallin, gallic acid, and umbelliferone), and acridine esters are used. For phosphatases, chromogenic substrates such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitroblue tetrazolium chloride (NBT), and p-nitrophenyl phosphate (p-NPP) are used, and luminescent substrates such as 3-(2'-spiroadamantane)-4-methyl-4-(3'-phosphoryloxyphenyl-1,2-dioxetane (AMPPD) disodium salt are used. For galactosidase, chromogenic substrates such as 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (CFDG), 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl), β-D-galactopyranoside (DDAO galactoside), and luminescent substrates such as (3-(2'-spiroadamantane)-4-methoxy-4-(3'-β-D-galactopyranosyloxyphenyl-1,2-dioxetane (AMPGD)) are used. Examples include:

[0028] When semi-quantifying a specific biomarker based on the amount of a fluorescent substance (described below) present rather than the fluorescence intensity of the fluorescent substance, it is preferable to use an enzyme labeled with a fluorescent substance as the fluorescent label, and the enzyme activity can be detected by a signal such as colorimetry (color development) or chemiluminescence.

[0029] The method for labeling an entity to be labeled with a fluorescent substance is not particularly limited, and a covalent or non-covalent bond between the fluorescent substance and the entity to be labeled can be formed using any known or commonly used method. Examples of methods for labeling an entity to be labeled with a fluorescent substance include the glutaraldehyde method, the periodate method, the maleimide method, the pyridyl disulfide method, avidin-biotin binding methods in which one end is biotinylated and the other end is avidinylated, and methods using various crosslinkers (e.g., N-succinimidyl-4-maleimidobutyrate (GMBS), N-succinimidyl-6-maleimidohexanoate, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, etc.). When labeling using the above method, functional groups present in antigens, antibodies, enzymes, sugar chains, complex carbohydrates, etc. may be used, or functional groups such as thiol groups, amino groups, carboxy groups, and hydroxyl groups may be introduced in advance before using the above method.

[0030] [1-2.(B) Color material] The colorant according to this embodiment is not particularly limited, and any known or commonly used colorant can be used. The colorant may be, for example, a dye, a pigment, or, in some cases, a protein-based dye, or a combination of these. The dye may be, for example, a direct dye, an acid dye, a disperse dye, a cationic dye, or the like. The pigment may also be an organic pigment, an inorganic pigment, or the like.

[0031] Examples of blue dyes or pigments include Blue No. 1 (CI 42090, Acid Blue 9, Brilliant Blue FCF), Blue No. 2 (CI 73015, Acid Blue 74, Indigo Carmine), Blue No. 201 (CI 73000, Vat Blue 1, Indigo), Blue No. 202 (CI 42052, Acid Blue 5, Patent Blue NA), Blue No. 203 (CI 42052, Acid Blue 5, Patent Blue CA), Blue No. 204 (CI 69825, Vat Blue 6, Carvansuren Blue), Blue No. 205 (CI 42090, Acid Blue 9, Alphazurine FG), Blue No. 403 (CI 61520, Solvent Blue 63, Sudan Blue B), and Blue No. 404 (CI 74160, Pigment Blue 15, Phthalocyanine Blue).

[0032] Examples of red dyes or pigments include Red No. 102 (CI 16255, Acid Red 18, New Coccine), Red No. 104 (1) (CI 45410, Acid Red 92, Phloxine B), Red No. 105 (1) (CI 45440, Acid Red 94, Rose Bengal), Red No. 106 (CI 45100, Acid Red 52, Acid Red), Red No. 2 (CI 16185, Acid Red 27, Amaranth), Red No. 201 (CI 15850, Pigment Red 57-1, Lithol Rubin B), Red No. 202 (CI 15850, Pigment Red 57, Lithol Rubin BCA), Red No. 203 (CI 15585, Pigment Red 53, Lake Red C), and Red No. 204 (CI 15585, Pigment Red 53(Ba), Lake Red CBA), Red 205 (CI 15630, Pigment Red 49(Na), Risol Red), Red 206 (CI 15630, Pigment Red 49(Ca), Risol Red CA), Red 207 (CI 15630, Pigment Red 49(Ba), Risol Red BA), Red 208 (CI 15630, Pigment Red 49(Sr), Risol Red SR), Red 213 (CI 45170, Basic Violet 10, Rhodamine B), Red 214 (CI 45170, Solvent Red 49, Rhodamine B Acetate), Red 215 (CI 45170, Solvent Red 49, Rhodamine B Stearate), Red 218 (CI 45410, Solvent Red 48, Tetrachlorotetrabromofluorescein), Red 219 (CI 15800, Pigment Red 64, Brilliant Lake Red R), Red 220 (CI 15880, Pigment Red 63 (Ca), Deep Maroon), Red 221 (CI 12120, Pigment Red 3, Toluidine Red), Red 223 (CI 45380, Solvent Red 43, Tetrabromofluorescein), Red 225 (CI 26100, Solvent Red 23, Sudan III), Red 226 (CI73360, Vat Red 1, Herringdon Pink CN), Red 227 (CI 17200, Acid Red 33, Fast Acid Magenta), Red 228 (CI 12085, Pigment Red 4, Palmerton Red), Red 230 (1) (CI 45380, Acid Red 87, Eosin YS), Red 230 (2) (CI 45380, Acid Red 87, Eosin YSK), Red 231 (CI 45410, Acid Red 92, Phloxine BK), Red 232 (CI 45440, Acid Red 94, Rose Bengal K), Red 3 (CI 45430, Acid Red 51, Erythrosine), Red 401 (CI 45190, Acid Violet) 9, Violamin R), Red 404 (CI 12315, Pigment Red 22, Brilliant Fast Scarlet), Red 405 (CI 15865, Pigment Red 48, Permanent Red F5R), Red 501 (CI 26105, Solvent Red 24, Scarlet Red NF), Red 502 (CI 16155, Food Red 6, Ponceau 3R), Red 503 (CI 16150, Acid Red 26, Ponceau R), Red 504 (CI 14700, Food Red 1, Ponceau SX), Red 505 (CI 12140, Solvent Orange 7, Oil Red XO), and Red 506 (CI 15620, Acid Red 88, Fast Red S).

[0033] Examples of yellow dyes or pigments include Yellow No. 201 (CI 45350:1, Acid Yellow 73, Fluorescein), Yellow No. 202 (1) (CI 45350, Acid Yellow 73, Uranine), Yellow No. 202 (2) (CI 45350, Acid Yellow 73, Uranine K), Yellow No. 203 (CI 47005, Acid Yellow 3, Quinoline Yellow WS), Yellow No. 204 (CI 47000, Solvent Yellow 33, Quinoline Yellow SS), Yellow No. 205 (CI 21090, Pigment Yellow 12, Benzidine Yellow G), Yellow No. 4 (CI 19140, Acid Yellow 23, Tartrazine), Yellow No. 401 (CI 11680, Pigment Yellow 1, Hansa Yellow), and Yellow No. 402 (CI 18950, Acid Yellow 40, Pola Yellow 5G), Yellow 403 (1) (CI 10316, Acid Yellow 1, Naphthol Yellow S), Yellow 404 (CI 11380, Solvent Yellow 5, Yellow AB), Yellow 405 (CI 11390, Solvent Yellow 6, Yellow OB), Yellow 406 (CI 13065, Acid Yellow 36, Metanil Yellow), Yellow 407 (CI 18820, Acid Yellow 11, Fast Light Yellow 3G), Yellow 5 (CI 15985, Food Yellow 3, Sunset Yellow FCF), etc.

[0034] Examples of green dyes or pigments include Green No. 201 (CI 61570, Acid Green 25, Alizarin Cyanine Green F), Green No. 202 (CI 61565, Solvent Green 3, Quinizarin Green SS), Green No. 204 (CI 59040, Solvent Green 7, Pyranine Concentrate), Green No. 205 (CI 42095, Acid Green 5, Light Green SF Yellow), Green No. 3 (CI 42053, Food Green 3, Fast Green FCF), Green No. 401 (CI 10020, Acid Green 1, Naphthol Green B), and Green No. 402 (CI 42085, Acid Green 3, Guinea Green B).

[0035] Examples of orange dyes or pigments include Orange No. 201 (CI 45370, Solvent Red 72, dibromofluorescein), Orange No. 203 (CI 12075, Pigment Orange 5, permanent orange), Orange No. 204 (CI 21110, Pigment Orange 13, benzidine orange G), Orange No. 205 (CI 15510, Acid Orange 7, orange II), Orange No. 206 (CI 45425, Solvent Red 73, diiodofluorescein), Orange No. 207 (CI 45425, Acid Red 95, erythrosine yellow NA), Orange No. 401 (CI 11725, Pigment Orange 1, Hansa orange), and Orange No. 402 (CI 14600, acid orange). 20, Orange I), Orange No. 403 (CI 12100, Solvent Orange 2, Orange SS), etc.

[0036] Examples of protein pigments include chlorophyll-protein complexes, carotenoid-protein complexes, and phycobiliin-protein complexes (phycobiliproteins). Phycobiliproteins include allophycocyanin, phycocyanin, phycoerythrocyanin, phycoerythrin, etc. Phycocyanin may be either C-phycocyanin or R-phycocyanin.

[0037] The coloring material used in this embodiment is preferably a coloring material (a coloring material having a color that can be visually recognized by the human eye) that reflects a portion of visible light (light with a wavelength of approximately 380 nm to 750 nm). Coloring materials reflect visible light of a specific wavelength, allowing humans to recognize the color from the reflected visible light. Visible light is light with a wavelength of approximately 380 nm to 750 nm. Coloring materials that reflect visible light include red, orange, yellow, green, blue, and purple, and each color reflects visible light of the following specific wavelengths. For example, red reflects visible light of 620 to 750 nm, orange reflects 590 to 620 nm, yellow reflects 570 to 590 nm, green reflects 495 to 570 nm, blue reflects 450 to 495 nm, and purple reflects visible light of 380 to 450 nm. In the present invention, preferred are a red colorant that reflects visible light in the range of 620 to 750 nm, an orange colorant that reflects visible light in the range of 590 to 620 nm, a yellow colorant that reflects visible light in the range of 570 to 590 nm, a green colorant that reflects visible light in the range of 495 to 570 nm, a blue colorant that reflects visible light in the range of 450 to 495 nm, and a purple colorant that reflects visible light in the range of 380 to 450 nm. Furthermore, in this embodiment, when the fluorescent substance is a fluorescein-based fluorescent dye, a rhodamine-based fluorescent dye, an Alexa Fluro (registered trademark)-based fluorescent dye, or a cyanine-based fluorescent dye, it is more preferable to use a red colorant that reflects visible light in the range of 620 to 750 nm, a yellow colorant that reflects visible light in the range of 570 to 590 nm, a green colorant that reflects visible light in the range of 495 to 570 nm, and a blue colorant that reflects visible light in the range of 450 to 495 nm.

[0038] When measuring the fluorescence intensity of a fluorescent substance, the (optimal) excitation wavelength λ(ex) can be that described in the catalog of the fluorescent substance, and the (optimal) fluorescence wavelength (ex) can be that described in the catalog of the fluorescent substance.

[0039] It is presumed that by allowing a colorant to coexist with a fluorescent substance or a fluorescent substance of a fluorescent label, the colorant absorbs or reflects light of a wavelength that excites the fluorescent substance, thereby improving the storage stability of the fluorescent substance and suppressing the degree of decrease in fluorescence intensity. It is also presumed that improving the storage stability of the fluorescent substance can suppress fading of the fluorescent substance or fluorescent label, which can prevent a decrease in binding ability with a capturer (e.g., an anti-fluorescent substance antibody) that captures the fluorescent substance or fluorescent label.

[0040] When the method for suppressing fluorescence fading according to this embodiment is used in a lateral flow assay, the fluorescent substance or fluorescent label and the coloring material can coexist during storage and preservation, and the fluorescent substance or fluorescent label and the coloring material are separated during the assay, eliminating the problem of the coloring material absorbing light of wavelengths that excite or are emitted by the fluorescent substance. Therefore, the method for suppressing fluorescence bleaching according to this embodiment is particularly effective when used in a lateral flow assay.

[0041] Therefore, by allowing the above colorant to coexist with a fluorescent substance or fluorescent label, the fluorescent substance or fluorescent label can be preserved or stored for a long period of time, and furthermore, the fluorescence intensity of the fluorescent substance can be utilized when detecting, quantifying, or semi-quantifying a specific biomarker. In another embodiment, a specific biomarker may be semi-quantified based on the amount of fluorescent substance present, rather than the fluorescence intensity of the fluorescent substance. One example is using a fluorescent label in which a fluorescent substance is bound to a labeling substance other than a fluorescent substance (e.g., an enzyme) as the labeled substance. More specifically, in a lateral flow assay, a fluorescent substance bound to an enzyme or other fluorescent label may be captured on a semi-quantitative line or the like on which an antibody that captures the fluorescent substance is immobilized, and the enzyme is then reacted with a predetermined substrate to produce color or light, and the intensity of the color or light emission may be used as a measure of semi-quantitation. For example, a complex of an antibody labeled with the same enzyme as above and a specific biomarker may be captured on a test line or the like on which an antibody that captures the biomarker is immobilized, and the enzyme is then reacted with a predetermined substrate to produce color or light, and the intensity of the color or light emission may be compared with the intensity of the color or light emission on the semi-quantitative line or the like.

[0042] In this embodiment, the molar ratio of the components (A) and (B) when they are allowed to coexist is not particularly limited and can be appropriately set depending on the fluorescent substance, the fluorescent properties of the fluorescent label, the absorption coefficient of the colorant at a predetermined wavelength, etc. The molar ratio of the components (A) and (B) is preferably component (A):component (B)=1-10:1-100,000, and more preferably 1-10:1-10,000.

[0043] [1-3.Application] The method for suppressing fluorescence bleaching according to this embodiment is not particularly limited and can be used in any assay that uses a fluorescent substance, such as an immunoassay, a lateral flow assay, or a flow-through assay.

[0044] Examples of the immunoassay include ELISA (which may be a direct method, an indirect method, a competitive method, or a non-competitive method), fluorescent Western blotting, and the like. Examples of the lateral flow assay include immunochromatographic assay (which may be either a competitive method or a non-competitive method), nucleic acid chromatographic assay, and the like.

[0045] The method for suppressing fluorescence fading according to this embodiment is particularly effective when used in lateral flow assays, because it can suppress fluorescence fading of the fluorescent substance even when the fluorescent substance is stored and preserved in a dry state on a matrix. Therefore, the method for suppressing fluorescence bleaching according to this embodiment can be suitably used in lateral flow assays.

[0046] That is, by using the method for suppressing fluorescence fading according to the present embodiment, fluorescence fading due to light-induced decomposition or structural changes of a fluorescent substance or fluorescent label can be suppressed. This enables long-term storage and preservation of a lateral flow assay test strip containing the fluorescent substance or fluorescent label, or a device or kit containing the same. Furthermore, the method for suppressing fluorescence fading according to the present invention can prevent long-term deterioration of the performance of a fluorescent substance in a lateral flow assay, etc.

[0047] Furthermore, the method for suppressing fluorescence bleaching according to this embodiment can suppress structural changes in the fluorescent substance or fluorescent label and suppress a decrease in the binding affinity between the fluorescent substance or fluorescent label and a substance that binds to the fluorescent substance or fluorescent label (e.g., an anti-fluorescent substance antibody), even when a specific biomarker is semi-quantified based on the amount of the fluorescent substance rather than the fluorescence intensity of the fluorescent substance (as in Embodiment 1), and is therefore suitable for semi-quantitative lateral flow assays and semi-quantitative immunochromatographic assays. The substance that binds to the fluorescent label may be a substance that binds to the fluorescent substance contained in the fluorescent label, or a substance that binds to the binding moiety between the fluorescent substance contained in the fluorescent label and the fluorescently labeled substance.

[0048] [2. Lateral flow assay test strips] The lateral flow assay test strip according to this embodiment is characterized by having a matrix and a fluorescent substance or the like retaining portion arranged on the matrix, which retains (A) a fluorescent substance or fluorescent label and (B) a coloring material in a dry state.

[0049] The lateral flow assay test strip according to this embodiment may have a sample pad, an absorbent pad, etc. in addition to the matrix.

[0050] Figure 1-1 is a perspective view showing an outline of one embodiment (Embodiment 1) of a lateral flow assay test strip for semi-quantitating a specific biomarker based on the amount of fluorescent substance present, rather than the fluorescence intensity of the fluorescent substance, and Figure 1-2 is a cross-sectional view of Figure 1-1. In the lateral flow assay test strip 1 shown in Figures 1-1 and 1-2, the sample pad 20 simultaneously serves as a supply section for the sample 70, a fluorescent substance holding section that holds a fluorescent label (an enzyme labeled with a fluorescent substance) and a coloring material, and a probe holding section that holds a probe (e.g., an antibody) labeled with the enzyme. Furthermore, the matrix 30 may optionally have a test line 31 having a capture body (detection target substance capture body) that captures the substance to be detected, a reference line 32 having a capture body (fluorescent substance capture body) that captures a fluorescent substance or a fluorescent substance contained in a fluorescent label, a control line 33 having a capture body (enzyme capture body) that captures an enzyme, and a substrate line 34, and may further have an absorbent pad 40, a developer pad 50, and a developer liquid tank 60.

[0051] As shown in Fig. 2, a sample supply section 21, a fluorescent substance holding section, and a probe holding section may be provided on a matrix instead of a sample pad 20. In Fig. 2, the sample supply section 21 simultaneously serves as the fluorescent substance holding section and the probe holding section. As shown in Fig. 2, a fluorescent substance holding section can be provided on the matrix by directly immobilizing a fluorescent label (an enzyme labeled with a fluorescent substance) and a coloring material on the matrix.

[0052] FIG. 3-1 is a perspective view showing an outline of one embodiment (Embodiment 2) of a lateral flow assay test strip that detects a target substance using the fluorescence intensity of a fluorescent substance, and FIG. 3-2 is a cross-sectional view of FIG. 3-1. In the lateral flow assay test strip 1 shown in FIGS. 3-1 and 3-2, the sample pad 22 simultaneously serves as a supply section for the sample 70 and a fluorescent substance holding section that holds a fluorescent label (a fluorescent substance and a target substance (e.g., an antibody)) and a colorant. Furthermore, the matrix 30 may optionally have a test line 31 having a capturer (target substance capturer) that captures the target substance, a control line 33 having a capturer (fluorescent substance capturer) that captures the fluorescent substance or the fluorescent substance contained in the fluorescent label, and a substrate line 34. The matrix 30 may further have an absorbent pad 40, a developer pad 50, and a developer tank 60.

[0053] As shown in Figure 2, which shows one embodiment of the lateral flow assay test strip described above, a sample supply section 23 and a fluorescent substance or other holding section may be provided on the matrix instead of the sample pad 22. In Figure 4, the sample supply section 23 also serves as the fluorescent substance or other holding section. As shown in Figure 4, a fluorescent label (a labeled substance (e.g., an antibody) labeled with a fluorescent substance) and a coloring material are directly immobilized on the matrix, thereby providing a fluorescent substance or other holding section on the matrix.

[0054] The lateral flow assay test strip may also be provided with a plastic case that can fix or house the matrix, absorbent pad, etc., or that has a press-in section. The size and spacing of these components can be adjusted as appropriate depending on the type of sample, substance to be detected, the purpose of the assay, etc. In the following description of the lateral flow assay test strip, the developer pad 50 side shown in Figures 1-1 to 4 will be referred to as the upstream side, and the absorbent pad 40 side as the downstream side.

[0055] The "matrix," "absorbent pad," and "other" will be explained below in turn.

[0056] [2-1. Matrix] The lateral flow assay test strip according to this embodiment includes a matrix (matrix 30 in FIGS. 1-1 to 4) that develops the developer. The matrix according to this embodiment is an insoluble carrier that functions as a lateral flow medium and stationary phase. The matrix may include, as the main body of the lateral flow assay test strip, a sample supply section, a section for retaining a fluorescent substance or the like, and / or a probe retaining section, as well as, as necessary, a test line, a reference line, a control line, and / or a substrate line, as described below. The matrix may have one or more test lines, and may or may not have one or more reference and / or control lines.

[0057] The matrix according to this embodiment is not particularly limited, and any material conventionally used as a medium for lateral flow assays can be used as appropriate. The matrix material is preferably a porous material, such as a nitrocellulose membrane, a nitrocellulose mixed ester membrane, a cellulose membrane, an acetylcellulose membrane, a polysulfone membrane, a polyethersulfone membrane, a nylon membrane, glass fiber, a nonwoven fabric, a cloth, or a laminate of two or more of these. Among these, a nitrocellulose membrane or a nitrocellulose mixed ester membrane is preferred as the matrix material. The shape, thickness, and size of the matrix are not particularly limited, and can be adjusted appropriately depending on the type of fluorescent substance, fluorescent label, colorant, etc., the matrix material, etc.

[0058] <Sample supply section> In the lateral flow assay test strip according to this embodiment, the sample supply section may also serve as the fluorescent substance holding section or probe holding section described below, or may be located upstream of the fluorescent substance holding section or probe holding section. The sample supply unit may be provided on a matrix, or may be provided on the matrix as a sample pad. The material of the sample pad is not particularly limited, and known and commonly used materials can be used. Examples of the sample pad material include porous materials such as glass fiber, cellulose filter paper, nonwoven fabric, polyurethane, polyacetate, cellulose acetate, nylon, and cotton cloth. The shape, thickness, and size of the sample pad are not particularly limited, and can be adjusted appropriately depending on the type of sample and target substance, the material of the sample pad, and the like.

[0059] Examples of samples used in this embodiment include samples derived from animals (e.g., primates such as humans, chimpanzees, cynomolgus monkeys, spider monkeys, macaques, and rhesus monkeys; rodents such as mice, rats, woodchucks, ferrets, rabbits, and hamsters; ungulates such as cows, pigs, horses, deer, goats, sheep, and wild boars; carnivores such as dogs, cats, and ferrets; and birds such as chickens and pigeons), plants, microorganisms, and other living organisms, or foods.

[0060] Examples of the above-mentioned biologically-derived substances include biologically-derived tissues (e.g., skin, muscle, bone, thyroid gland, brain, tongue, pharynx, esophagus, stomach, small intestine, duodenum, large intestine, liver, kidney, pancreas, gallbladder, lung, uterus, ovaries, breast, bladder, prostate, mucous membrane, hair, etc.), body fluids (blood, serum, plasma, cerebrospinal fluid, lymph, tissue fluid, body cavity fluid, saliva, swab, nasal discharge, urine, feces, etc.), extracts thereof, and dilutions thereof. Examples of the food-derived substances include beef, pork, chicken, beans, wheat, buckwheat flour, milk, eggs, shellfish, extracts thereof, and dilutions thereof.

[0061] When the sample contains a substance to be detected, the substance to be detected can bind to or react with a fluorescent label such as an antibody labeled with a fluorescent substance to form a complex or the like. Examples of the detection target substance include the above-mentioned antigens, antibodies, enzymes, lectins, sugar chains or complex carbohydrates, nucleic acids, and the like.

[0062] The solution to be mixed with the sample as a diluent and / or developer is not particularly limited, and any known, commonly used solution can be used. Examples of the solution include water (e.g., distilled water, sterilized water, pure water, etc.), buffer solutions, organic solvents (dimethyl sulfoxide, dimethylformamide, methanol, isopropanol, etc.), etc. Examples of buffer solutions include phosphate buffer, phosphate-buffered saline (PBS), acetate buffer, citrate buffer, tartrate buffer, carbonate buffer, borate buffer, glycine buffer, 2-morpholinoethanesulfonic acid (MES) buffer, trishydroxymethylaminomethane (Tris) buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) buffer, and 2-[4-(2-hydroxyethyl)-1-piperazinylethanesulfonic acid (HEPES) buffer, and these may contain added stabilizing proteins such as BSA, serum, surfactants, etc.

[0063] <Fluorescent substance holder> In the lateral flow assay test strip according to this embodiment, the fluorescent substance or other retaining portion serves as a retaining portion for the fluorescent substance or fluorescent label and the coloring material. The fluorescent substance-holding portion may be provided directly on the matrix, or may be provided on the matrix as a fluorescent substance-holding pad. Examples of materials for the fluorescent substance-holding pad include porous materials such as glass fiber, cellulose filter paper, nonwoven fabric, polyurethane, polyacetate, cellulose acetate, nylon, and cotton cloth. The shape, thickness, and size of the fluorescent substance-holding pad are not particularly limited and can be adjusted appropriately depending on the type of fluorescent substance, fluorescent label, colorant, etc., and the material of the fluorescent substance-holding pad.

[0064] The fluorescent substance holding section may be provided separately from the sample supply section and downstream thereof, or the sample supply section and the fluorescent substance holding section may simultaneously serve as both sections. Furthermore, the sample pad may also serve as a pad for holding the fluorescent substance or the like.

[0065] By making a coloring material coexist in the fluorescent substance or fluorescent label in the fluorescent substance holding portion, it is possible to suppress fading of the fluorescence of the fluorescent substance or fluorescent label.

[0066] The method for retaining the fluorescent substance or fluorescent label and the coloring material in the fluorescent substance-retaining portion is not particularly limited, and any known or commonly used method can be used. Examples of methods for retaining the fluorescent substance or fluorescent label and the coloring material in the fluorescent substance-retaining portion include a method in which a solution containing the fluorescent substance or fluorescent label and the coloring material is directly applied to a matrix in a linear or dotted pattern and then dried to retain them in a dissolvable manner, a method in which a solution containing the fluorescent substance or fluorescent label and a solution containing the coloring material are directly applied to a matrix in a linear or dotted pattern and then dried to retain them in a dissolvable manner, a method in which a solution containing the fluorescent substance or fluorescent label and the coloring material is applied to a fluorescent substance-retaining pad in a linear or dotted pattern and then dried, and then the fluorescent substance-retaining pad is placed on the matrix, and a method in which a fluorescent substance-retaining pad is immersed in a solution containing the fluorescent substance or fluorescent label and the coloring material, the solution is applied to the entire surface of the pad, and then dried, and then the fluorescent substance-retaining pad is placed on the matrix.

[0067] The mass ratio of component (A) and component (B) when they are held together is not particularly limited and can be appropriately set depending on the types of fluorescent substance, fluorescent label, colorant, etc., the purpose of the assay, etc. The mass ratio of component (A) to component (B) is, for example, preferably component (A):component (B) = 1-100:5-500,000, more preferably 1-50:10-100,000, even more preferably 1-10:100-50,000, and even more preferably 1:200-10,000.

[0068] The concentration of component (B) is preferably 5 mg / mL or more, more preferably 10 mg / mL or more, more preferably 20 mg / mL or more, and even more preferably 25 mg / mL or more, from the viewpoint of enhancing the effect of protecting the fluorescent substance or fluorescent label from photodecomposition, etc. Furthermore, the concentration of component (B) is preferably 60 mg / mL or less, more preferably 50 mg / mL or less, even more preferably 40 mg / mL or less, and even more preferably 35 mg / mL or less, from the viewpoint of preventing an increase in viscosity due to the colorant component. For this reason, the concentration of component (B) is preferably 5 to 60 mg / mL, more preferably 10 to 50 mg / mL, even more preferably 20 to 40 mg / mL, and even more preferably 25 to 35 mg / mL.

[0069] <Test Line> In this embodiment, the test line has a capture body (detection target substance capture body) immobilized thereon that captures the detection target substance, and serves as a detection section for the detection target substance. The detection target substance capture body is not particularly limited as long as it is a substance that can bind, preferably specifically bind, to the detection target substance. Examples of the detection target substance capture body include the same substances as those exemplified above as the target to be fluorescently labeled.

[0070] In one embodiment (Embodiment 1) of a lateral flow assay test strip for semi-quantitating a specific biomarker based on the amount of fluorescent substance present, rather than the fluorescence intensity of the fluorescent substance, when a sandwich method, for example, is used as a non-competitive detection method at the test line, a complex of a probe (e.g., an antibody, etc.) labeled with a non-fluorescent labeling substance (e.g., an enzyme, gold colloid particles, colored latex, etc.) and the target substance is captured by a target substance capture body immobilized on the test line. Therefore, the target substance can be detected by detecting or measuring a signal derived from the non-fluorescent labeling substance on the test line. In the sandwich method, the probe and the target substance capture body may be different, or they may be the same as long as they do not inhibit each other's binding to the target substance.

[0071] Furthermore, when a competitive method is used as a detection method for the test line, for example, the presence or absence, concentration, etc. of the target substance can be detected or measured by causing a target substance to be labeled in advance with a non-fluorescent labeling substance (e.g., an enzyme, gold colloid particles, colored latex, etc.) to react competitively with an unlabeled target substance in the sample against a target substance capture body.

[0072] In one embodiment (Embodiment 2) of a lateral flow assay test strip for detecting a target substance using the fluorescence intensity of a fluorescent substance, for example, when a sandwich method is used as a non-competitive method for detection at a test line, a complex of a fluorescently labeled probe (e.g., an antibody, etc.) and the target substance is captured by a target substance capture body immobilized on the test line. Therefore, the target substance can be detected by detecting or measuring a signal derived from the fluorescent substance on the test line. In the sandwich method, the probe and the target substance capture body may be different, or they may be the same as long as they do not inhibit each other's binding to the target substance.

[0073] Furthermore, when a competitive method is used as a detection method for the test line, for example, the presence or absence, concentration, etc. of the target substance can be detected or measured by causing a target substance labeled in advance with a fluorescent substance and an unlabeled target substance in the sample to react competitively with a target substance capture body.

[0074] The method for immobilizing the target substance capture body on the matrix is ​​not particularly limited, and any known or commonly used method can be used. Chemical or physical methods can be used to immobilize the target substance capture body on the matrix. When the matrix is ​​a nitrocellulose membrane or a nitrocellulose mixed ester membrane, immobilization can be performed by physical adsorption. After immobilizing the target substance capture body on the matrix, blocking can be performed by any known or commonly used method.

[0075] The shape of the test line is not particularly limited, and can be, for example, a straight line perpendicular to the long side of the lateral flow assay test strip, or any other shape such as a dot, a circle, a polygon, etc. When the test line is straight, the width of the line is preferably 0.1 to 3.0 mm, more preferably 0.2 to 2.5 mm, and even more preferably 0.5 to 1.5 mm.

[0076] <Control Line> In this embodiment, the control line that the matrix may have has a substance (e.g., a capture body that captures an enzyme in the case of Embodiment 1, or a capture body that captures a fluorescent substance in the case of Embodiment 2) that can bind to a part of the labeling substance (e.g., a fluorescent substance, an enzyme, colloidal gold particles, colored latex, etc.) immobilized thereon, and serves as a detection section for the labeling substance. By providing a control line, it is possible to confirm, for example, that a probe (e.g., an antibody, etc.) labeled with a labeling substance in a non-competitive method, or a detection target substance labeled with a labeling substance in a competitive method, has developed by capillary action on the lateral flow assay test strip and reached the control line, i.e., that the assay was performed normally.

[0077] The control line is preferably located downstream of the test line and / or reference line.

[0078] The method for immobilizing the capturer of the labeled substance on the matrix is ​​not particularly limited, and any known or commonly used method can be used. Chemical or physical methods can be used to immobilize the capturer of the labeled substance on the matrix. When the matrix is ​​a nitrocellulose membrane or a nitrocellulose mixed ester membrane, immobilization can be performed by physical adsorption. After immobilizing the capturer of the labeled substance on the matrix, blocking can be performed by a known or commonly used method.

[0079] The shape of the control line is not particularly limited, and can be, for example, a straight line perpendicular to the long side of the lateral flow assay test strip, or any other shape such as a dot, a circle, a polygon, etc. When the control line is straight, the width of the line is preferably 0.1 to 3.0 mm, more preferably 0.2 to 2.5 mm, and even more preferably 0.5 to 1.5 mm.

[0080] <Reference line> In this embodiment, reference lines (e.g., semi-quantitative lines, calibration lines, comparison lines, etc.) may be provided on the matrix. The reference lines have immobilized thereon a substance (hereinafter also referred to as a "reference substance capture body"; this includes fluorescent substance capture substances, etc.) that can capture a substance other than the detection target substance (reference substance (also referred to as a semi-quantitative substance, calibration substance, comparison substance, etc. depending on the role of the reference line)), and serve as a detection portion for the reference substance. It is preferable that the labeling substance for labeling the reference substance is the same as the labeling substance for detecting the detection target substance.

[0081] For example, in the case of the above-described first embodiment, a fluorescent substance can be used as the semi-quantitative substance. In this case, the fluorescent substance is preferably labeled with a non-fluorescent labeling substance (e.g., an enzyme, gold colloid particles, colored latex, etc.). When a fluorescent substance is used as the semi-quantitative substance, a capturer that captures a fluorescent label labeled with a fluorescent substance or a non-fluorescent labeling substance is preferably immobilized on the reference line (e.g., the semi-quantitative line). The capturer that captures the fluorescent label may be a capturer that recognizes the fluorescent substance contained in the fluorescent label, or a capturer that recognizes the binding site between the fluorescent substance contained in the fluorescent label and the fluorescently labeled substance. Furthermore, for example, in the case of the above-mentioned second embodiment, a semi-quantitative substance labeled with a fluorescent substance can be used. Semi-quantitation is possible by comparing the intensity of color development, luminescence, etc. of the test line and the semi-quantitative line. Multiple reference lines may be provided.

[0082] In particular, the method for suppressing fluorescence fading according to this embodiment can improve the accuracy of measurements such as semi-quantitation, calibration, comparison, etc. More specifically, when semi-quantitating a specific detection target substance from the amount of a fluorescent substance contained in a fluorescent label, the binding between the fluorescent substance or fluorescent label and a reference substance capturer (e.g., a fluorescent substance capturer) on the matrix is ​​maintained even under light exposure conditions, so that a signal of a predetermined intensity corresponding to the concentration of the fluorescent substance or fluorescent label can be maintained, and by comparing this with the signal intensity in the test zone, the amount of the detection target substance in the sample can be measured semi-quantitatively.

[0083] Although there are no particular limitations on the reference line, it is preferable that the reference line be located downstream of the test line and upstream of the control line.

[0084] The method for immobilizing a reference substance capture body (e.g., a fluorescent substance capture body, etc.) on a matrix is ​​not particularly limited, and any known or commonly used method can be used. Chemical or physical methods can be used to immobilize a reference substance capture body on a matrix. When the matrix is ​​a nitrocellulose membrane or a nitrocellulose mixed ester membrane, immobilization can be performed by physical adsorption. After immobilizing the reference substance capture body on the matrix, blocking can be performed by a known or commonly used method.

[0085] The shape of the reference line is not particularly limited, and can be, for example, a straight line perpendicular to the long side of the lateral flow assay test strip, or any other shape such as a dot, a circle, a polygon, etc. When the reference line is straight, the width of the line is preferably 0.1 to 3.0 mm, more preferably 0.2 to 2.5 mm, and even more preferably 0.5 to 1.5 mm.

[0086] <Blocking, etc.> In this embodiment, the matrix may be subjected to a blocking treatment. Blocking refers to coating the matrix with a protein and / or polymer solution to prevent nonspecific adsorption onto the matrix in a lateral flow assay. Examples of proteins and / or polymers include proteins such as gelatin, casein, BSA (bovine serum albumin), and skim milk; and polymers such as PVP (polyvinylpyrrolidone) and PVA (polyvinyl alcohol). After the blocking treatment, the matrix may be washed with a surfactant such as TWEEN (registered trademark) 20, TRITON (registered trademark) X-100, or SDS, if necessary.

[0087] The matrix may further contain a substance that promotes capillary action. Examples of such substances include sugars, amino acid derivatives, fatty acid esters, surfactants, alcohols, etc. Examples of sugars include sucrose, maltose, lactose, etc. Examples of surfactants include anionic surfactants such as sodium dodecylbenzenesulfonate (SDBS), sodium dodecylsulfonate (SDS), C12-C18 alkylsodium sulfonate, and dialkylsodium sulfosuccinate.

[0088] <Substrate line> When an enzyme is used as the non-fluorescent labeling substance, the lateral flow assay test strip may be provided with a substrate line on the matrix that holds the substrate.

[0089] The substrate line is preferably located upstream of the test line, reference line, and control line.

[0090] The substrate is not particularly limited as long as it reacts with the predetermined enzyme, and any known or commonly used substrate can be used. Examples of the substrate include substrates that react with the above-mentioned peroxidase, galactosidase, phosphatase, etc.

[0091] The method for retaining the substrate in the matrix is ​​not particularly limited, and any known or commonly used method can be used. For example, the method for retaining the substrate in the matrix includes applying a solution of the substrate in an aqueous solution in a linear pattern, followed by drying. When the substrate is dissolved in an aqueous solution, the aqueous solution may contain a signal enhancer, stabilizer, or dissolution regulator for the substrate.

[0092] [2-2. Absorbent Pads] The lateral flow assay test strip according to this embodiment may have an absorbent pad, and a known, commonly used one can be used. The absorbent pad serves to absorb the sample, the developer, and any substances not captured by the test line or control line. The material of the absorbent pad is not particularly limited as long as it is an absorbent material, and examples thereof include cellulose filter paper, highly absorbent filter paper, nonwoven fabric, cloth, cellulose acetate, and the like.

[0093] The absorbent pad is preferably placed downstream of the matrix, and its shape, thickness, size, etc. are not particularly limited, and can be adjusted appropriately depending on the material of the absorbent pad and the migration speed appropriate for the detection and quantification of the sample or target substance.

[0094] [2-3.Other] A developer tank storing a developer may be provided upstream of the matrix. Furthermore, by providing a pusher with a protrusion (a member that can break the developer tank by pushing the pusher) or the like in advance near the developer tank, the developer in the developer tank can be easily supplied onto the matrix. Furthermore, a developer pad may be provided so as to cover the developer tank and the upper end of the matrix.

[0095] The developing solution is not particularly limited, and any known or commonly used solution can be used. Examples of the developing solution that can be used include water, physiological saline, and buffer solutions (e.g., Tris buffer, phosphate buffer, HEPES buffer, and 2-amino-2-methyl-1-propanol (AMP) buffer). 2-amino-2-methyl-1-propanol (AMP) buffer is preferred. Furthermore, the developing solution may contain various additives, such as a pH adjuster, a surfactant, a preservative, and an inorganic salt, as needed.

[0096] The developing liquid pad is not particularly limited, and any known or commonly used developing liquid pad can be used. Examples of the material for the developing liquid pad include porous materials such as glass fiber, cellulose filter paper, glass fiber filter paper, nonwoven fabric, polyurethane, polyacetate, cellulose acetate, nylon, and cotton cloth. [Example]

[0097] EXAMPLES The present invention will be specifically explained below by showing examples, but the present invention is not limited to these examples.

[0098] [Test Example 1. Evaluation of the effect of suppressing fading of fluorescence by allowing a fluorescent substance and a coloring material to coexist] The fluorescent substances used were FITC-labeled alkaline phosphatase (ALP), Alexa Fluor (registered trademark) 488, and Alexa Fluor (registered trademark) 594. The coloring materials used were Blue No. 1, Red No. 2, Yellow No. 4, and Green No. 3 (all manufactured by Tokyo Chemical Industry Co., Ltd.). The porous sheets were stored in a light-blocking environment for 24 hours, or exposed to LED light for 24 hours, in both cases where the fluorescent substance and the coloring material were present on the porous sheet, and the effect of the coloring material on the fading of the fluorescence was evaluated by extracting the fluorescent substance from the porous sheet and measuring the fluorescence value of the extract.

[0099] <Preparation of extract solution (fluorescent substance: ALP labeled with FITC)> 3.04 μL of 110 μg / mL FITC-labeled alkaline phosphatase (ALP) was attached to a porous sheet (Bell-Eta F(A)B, manufactured by AION Co., Ltd.) measuring 3.65 mm in width and 15.0 mm in length, and then dried. The dried porous sheet was stored in a light-blocking environment for 24 hours or irradiated with an LED light (DK-S90CWH / DK-S90CBK, Gentos Co., Ltd.) for 24 hours to prepare samples. The FITC-labeled ALP-attached portion of the porous sheet was then cut with scissors, and 15 porous sheets were cut and placed in a squeeze tube along with 400 μL of ultrapure water. The squeeze tube was kneaded for approximately 30 seconds and then left to stand at room temperature in a light-blocking environment for 1 hour to obtain an extract of FITC-labeled ALP. The extract prepared under the conditions of 24-hour storage in a light-blocking environment was designated Extract 1-1-1. The extract prepared under the conditions of 24-hour LED light irradiation was designated Extract 1-1-2. In addition, 3.04 μL of a mixture containing 110 μg / mL FITC-labeled ALP and 5.0 mg / mL of Blue No. 1, Red No. 2, Yellow No. 4, or Green No. 3 as colorants was applied to a porous sheet, and extracts were obtained using the same method. Extracts prepared under conditions of 24-hour storage in a light-blocking environment were designated Extracts 1-2-1 (Blue No. 1), 1-3-1 (Red No. 2), 1-4-1 (Yellow No. 4), and 1-5-1 (Green No. 3), respectively. Extracts prepared under conditions of 24-hour LED light irradiation were designated Extracts 1-2-2 (Blue No. 1), 1-3-2 (Red No. 2), 1-4-2 (Yellow No. 4), and 1-5-2 (Green No. 3), respectively.

[0100] <Preparation of extract (fluorescent substance: Alexa Fluor 488)> Extracts were prepared in the same manner as above, except that instead of FITC-labeled ALP, 3.04 μL of a 110 μg / mL solution of Alexa Fluor 488 or 3.04 μL of a solution containing 110 μg / mL of Alexa Fluor 488 and 5.0 mg / mL of each of the above colorants was applied to the porous sheet. The extracts prepared under conditions of 24 hours of storage in a light-blocking environment were designated Extracts 2-1-1 to 2-5-1, respectively. The extracts prepared under conditions of 24 hours of LED light irradiation were designated Extracts 2-1-2 to 2-5-2, respectively.

[0101] <Preparation of extract (fluorescent substance: Alexa Fluor 594)> Extracts were prepared in the same manner as above, except that instead of FITC-labeled ALP, 3.04 μL of a 110 μg / mL solution of Alexa Fluor 594 or 3.04 μL of a solution containing 110 μg / mL Alexa Fluor 594 and 5.0 mg / mL of each of the above colorants was applied to the porous sheet. The extracts prepared under conditions of 24 hours of storage in a light-blocking environment were designated Extracts 3-1-1 to 3-5-1, respectively. The extracts prepared under conditions of 24 hours of LED light irradiation were designated Extracts 3-1-2 to 3-5-2, respectively.

[0102] <Measurement and evaluation of the fluorescence value of each fluorescent substance in the extract> The above extracts 1-1-1 to 3-5-2 were dispensed in 80 μL each into three wells of a 96-well plate, and the fluorescence values ​​were measured under the following measurement conditions using a microplate reader (Varioskan Flash, manufactured by ThermoFisher Scientific). FITC: Excitation wavelength 485nm, Emission wavelength 518nm Alexa Fluor 488: Excitation wavelength 494 nm, Emission wavelength 517 nm Alexa Fluor 594: Excitation wavelength 590 nm, Emission wavelength 617 nm

[0103] The fading rate calculated using the fluorescence value of each of the above extracts using the following formula is shown in Table 1. Fading rate (%) = (1 - (fluorescence intensity of extract prepared under 24-hour LED light irradiation) / (fluorescence intensity of extract prepared under 24-hour storage in a dark environment)) x 100

[0104] [Table 1]

[0105] From the results in Table 1, when comparing the fluorescence value of Extract 1-1-1, which was produced by storing a porous sheet holding FITC in a light-blocking environment for 24 hours, with the fluorescence value of Extract 1-1-2, which was produced by irradiating it with LED light for 24 hours, the fading rate was 96.6%, indicating that the FITC-derived fluorescence was significantly faded by irradiation with LED light. In contrast, the fading rate of Extract 1-2-1, which was prepared by allowing FITC-labeled ALP and Blue No. 1 to coexist and storing them in a dark environment for 24 hours, and Extract 1-2-2, which was prepared by irradiating them with LED light for 24 hours, was 3.4%, which shows that the fading of the fluorescent substance FITC was significantly suppressed compared to the results of the control test examples, Extracts 1-1-1 and 1-1-2. Similarly, the fading rates of extracts 1-3-1 and 1-3-2, which were prepared in the presence of Red No. 2, extracts 1-4-1 and 1-4-2, which were prepared in the presence of Yellow No. 4, and extracts 1-5-1 and 1-5-2, which were prepared in the presence of Green No. 3, were all within ±15%, demonstrating the effect of suppressing the fading of FITC-derived fluorescence values.

[0106] Furthermore, the results in Table 1 show that the fluorescence value derived from Alexa Fluor 488 in Extract 2-1-2, which was produced under conditions where a porous sheet holding Alexa Fluor 488 was stored in a light-blocking environment for 24 hours, faded by approximately 40%. In contrast, the fading rates for extracts 2-2-1 and 2-2-2, which were prepared under conditions in which Alexa Fluor 488 and Blue No. 1 coexisted, extracts 2-3-1 and 2-3-2, which were prepared under conditions in which Alexa Fluor 488 and Red No. 2 coexisted, extracts 2-4-1 and 2-4-2, which were prepared under conditions in which Alexa Fluor 488 and Yellow No. 4 coexisted, and extracts 2-5-1 and 2-5-2, which were prepared under conditions in which Alexa Fluor 488 and Green No. 3 coexisted, were all within ±10%, demonstrating that fading of the fluorescence derived from Alexa Fluor 488 due to exposure to LED light was suppressed.

[0107] Furthermore, the results in Table 1 show that the fluorescence value derived from Alexa Fluor 594 in Extract 3-1-2, which was produced under conditions where a porous sheet holding Alexa Fluor 594 was stored in a light-blocking environment for 24 hours, faded by approximately 26% compared to Extract 3-1-1, which was produced under conditions where a porous sheet holding Alexa Fluor 594 was exposed to LED light for 24 hours. In contrast, the fading rates for extracts 3-2-1 and 3-2-2, which were prepared under conditions in which Alexa Fluor 594 and Blue No. 1 coexisted, extracts 3-3-1 and 3-3-2, which were prepared under conditions in which Alexa Fluor 594 and Red No. 2 coexisted, extracts 3-4-1 and 3-4-2, which were prepared under conditions in which Alexa Fluor 594 and Yellow No. 4 coexisted, and extracts 3-5-1 and 3-5-2, which were prepared under conditions in which Alexa Fluor 594 and Green No. 3 coexisted, were all within ±7%, indicating that fading of Alexa Fluor 594 due to exposure to LED light was suppressed.

[0108] [Test Example 2. Evaluation of fading of fluorescent substances in lateral flow assay] <Preparation of Lateral Flow Assay Test Strips and Devices> A 0.55 μL solution of 2.8 mg / mL anti-hepatitis B virus-related antigen (HBcrAg) antibody was linearly applied and dried at a position 16 mm upstream from the end of the absorbent pad on the matrix of a nitrocellulose membrane (Millipore) 3.65 mm wide and 50 mm long, forming a test line for capturing the antigen HBcrAg. Next, 0.55 μL of a 1.0 mg / mL solution of anti-FITC antibody (Anti-FITC mAb Lot. 8F7-1, manufactured by Advanced Life Science Institute, Inc.) was linearly applied and dried at a position 13.5 mm upstream from the end of the absorbent pad side on the matrix, forming a semi-quantitative line that captures the FITC of the FITC-labeled ALP. Furthermore, 0.55 μL of an anti-alkaline phosphatase (ALP) antibody solution containing 57.1 μg / mL of antibody was linearly applied and dried at a position 11.0 mm upstream from the end of the absorbent pad side on the matrix to form a control line for capturing ALP.

[0109] After drying the test line, semi-quantitative line, and control line, blocking was performed using 9.32 μL of a phosphate buffer solution containing 10 g / L sucrose, 650 mg / L SDBS (Dodecyl benzene sulfonic acid), and 1.0 g / L BSA.

[0110] 0.92 μL of a 80 mg / mL solution of BCIP (5-bromo-4-chloro-3-indolyl phosphate) was linearly applied and dried at a position 47 mm upstream from the end of the absorption pad side of the matrix to form a substrate line.

[0111] Next, 3.04 μL of a solution containing 0.03 mg / mL ALP-labeled anti-HBcrAg antibody and 1 μg / mL FITC-labeled ALP was applied to a 3.65 mm wide, 15.0 mm long porous sheet (Bell-Eta F(A)B, manufactured by AION Co., Ltd.) to prepare a sample pad (which also serves as a fluorescent substance and probe holding pad) and dried. The resulting sample pad was placed between the test line and substrate line of the matrix to prepare a test strip for the lateral flow assay.

[0112] The above-mentioned lateral flow assay test strip, developer pad (glass fiber filter paper), and absorbent pad (highly absorbent filter paper) were bonded together and fixed in a plastic case equipped with a developer tank (developer: 2-amino-2-methyl-1-propanol (AMP) buffer solution) and a push-in section to form a lateral flow assay device.

[0113] <Preparation of lateral flow assay devices stored under various light exposure conditions> In the above-mentioned device for lateral flow assay, those stored for 3 hours in a light-shielded environment (devices 1-1-1 and 1-1-2), those in which the test line, semi-quantitative line, and control line were irradiated with LED light for 3 hours (devices 1-2-1 and 1-2-2), those in which only the sample pad was irradiated with LED light for 3 hours (devices 1-3-1 and 1-3-2), and those in which the test line, semi-quantitative line, control line, and sample pad were irradiated with LED light for 3 hours (devices 1-4-1 and 1-4-2) were each prepared in two replicates.

[0114] <Detection of HBcrAg and FITC and evaluation of the color development intensity of the semi-quantitative line> The HBcrAg calibrator (HBcrAg calibrator at 10000 kU / mL) contained in LumiPulse Presto (registered trademark) iTACT (registered trademark) HBcrAg (hepatitis B virus core-related antigen kit, manufactured by Fujirebio Inc.) was diluted 50-fold with healthy human serum (manufactured by TRINA Bioreactives), and using treatment solution 1 with a pH of less than 2 and treatment solution 2 with a pH of 10 contained in ESPLINE (registered trademark) HBcrAg (RUO) (manufactured by Fujirebio Inc.), the above dilution:treatment solution 1:treatment solution 2 = 1:3:1 ratio was used for mixing to prepare a sample. 20 μL of the sample was dropped onto the sample pads of the above-mentioned devices 1-1-1 to 1-4-2 for lateral flow assay. Subsequently, the pushing part was pushed down to develop the developing solution (2-amino-2-methyl-1-propanol (AMP) buffer), and after 30 minutes, the color development of the test line, semi-quantitative line, and control line was visually confirmed. In addition, photographs of the results of the color development of the above test line, semi-quantitative line, and control line, and the results of evaluation according to the following evaluation criteria for the color development intensity of the semi-quantitative line are shown in Figure 5. Evaluation criteria for color development intensity: +: Color development is easily recognizable +w: Color development can be confirmed compared to +, but the color development is weak +ww: Color development can be confirmed, but the color development is very weak

[0115] Figure 5 shows that when devices 1-1-1 and 1-1-2, which were lateral flow assay devices stored in a light-blocking environment for 3 hours, were used, and when devices 1-2-1 and 1-2-2, which were irradiated with LED light for 3 hours on the test line, semi-quantitative line, and control line, were used, the color of the semi-quantitative line was easily visible. In contrast, when using lateral flow assay devices 1-3-1 and 1-3-2 in which the sample pad was irradiated with LED light for 3 hours, and when using devices 1-4-1 and 1-4-2 in which the test line, semi-quantitative line, control line, and sample pad were irradiated with LED light for 3 hours, slight color development of the semi-quantitative line was visible, but the color intensity was very weak. The above results can be attributed to the fact that when FITC-labeled ALP attached and dried on the sample pad was irradiated with LED light, the FITC underwent structural changes due to photodecomposition, etc., resulting in a decrease in the amount of FITC-labeled ALP captured by the anti-FITC antibody in the semi-quantitative line.

[0116] Test Example 3: Evaluation of the effect of suppressing fluorescence fading when a fluorescent substance and a coloring material are coexistent in a lateral flow assay <Preparation of Lateral Flow Assay Test Strips and Devices> On the matrix of a 3.65 mm wide, 50 mm long nitrocellulose membrane (Millipore), 15.0 mm upstream from the end of the absorption pad side, 0.1 mg / mL of anti-FITC antibody E (MBL, Anti-FITC mAb), 0.1 mg / mL of anti-FITC antibody G (GeneTex, FITC Antibody), 0.5 mg / mL of anti-FITC antibody A (Advanced Life Science Institute, Anti-FITC mAb Lot. 2A7-1), 0.5 mg / mL of anti-FITC antibody B (Advanced Life Science Institute, Anti-FITC mAb Lot. 3D1-1), 0.5 mg / mL of anti-FITC antibody C (Advanced Life Science Institute, Anti-FITC mAb Lot. 4D5-1), and 1.0 mg / mL of anti-FITC antibody D (Advanced Life Science Institute, Anti-FITC mAb Lot. 4D5-1) were added. A 0.55 μL aliquot of an antibody solution containing 1.0 mg / mL of anti-FITC antibody F (Abgent, Anti-FITC, Mouse-Mono) was applied linearly to each matrix and allowed to dry, forming a semi-quantitative line. A 0.55 μL aliquot of an antibody solution containing 57.1 μg / mL of anti-alkaline phosphatase (ALP) antibody was applied and allowed to dry 11.8 mm upstream from the end of the absorbent pad on the matrix, forming a control line.

[0117] After drying the semi-quantitative line and the control line, blocking was carried out with 9.32 μL of a phosphate buffer solution containing 10 g / L sucrose, 650 mg / L SDBS (Dodecyl benzene sulfonic acid), and 1.0 g / L BSA.

[0118] 0.92 μL of a 80 mg / mL solution of BCIP (5-bromo-4-chloro-3-indolyl phosphate) was linearly applied and dried at a position 47 mm upstream from the end of the absorption pad side of the matrix to form a substrate line.

[0119] Next, 3.04 μL of a solution containing 1 μg / mL of FITC-labeled ALP was applied and dried to a 3.65 mm wide, 15.0 mm long porous sheet (Bell-Eta F(A)B, manufactured by AION Co., Ltd.) to prepare a sample pad (which also serves as a fluorescent substance and probe holding pad). The resulting sample pad was placed between the semi-quantitative line and the substrate line of the matrix to prepare a test strip for the lateral flow assay.

[0120] The above-mentioned lateral flow assay test strip, developer pad (glass fiber filter paper), and absorbent pad (highly absorbent filter paper) were bonded together and fixed in a plastic case equipped with a developer tank (developer: 2-amino-2-methyl-1-propanol (AMP) buffer solution) and a push-in section to form a lateral flow assay device.

[0121] <Preparation of lateral flow assay devices stored under various light exposure conditions> For each of the above lateral flow assay devices, devices were prepared that were stored in a light-blocking environment for 20 hours (devices 2-1-1, 2-2-1, 2-3-1, 2-4-1, 2-5-1, 2-6-1, and 2-7-1), and devices that had the sample pads irradiated with LED light for 20 hours (devices 2-1-2, 2-2-2, 2-3-2, 2-4-2, 2-5-2, 2-6-2, and 2-7-2).

[0122] <Preparation of a lateral flow assay test strip and device containing a fluorescent substance and a coloring material> Lateral flow assay test strips and devices were prepared in the same manner as above, except that 3.04 μL of a solution containing 1 μg / mL FITC-labeled ALP and 5 mg / mL Blue No. 1 (Tokyo Chemical Industry Co., Ltd.) was attached and dried on the sample pad of each of the lateral flow assay test strips and devices.

[0123] <Preparation of a lateral flow assay device containing a fluorescent substance and a coloring material, stored under various exposure conditions> In the lateral flow assay device in which the fluorescent substance and the colorant coexist, those stored for 20 hours in a light-shielded environment (devices 2-1-3, 2-2-3, 2-3-3, 2-4-3, 2-5-3, 2-6-3, and 2-7-3), and those irradiated with LED light on the sample pad for 20 hours (devices 2-1-4, 2-2-4, 2-3-4, 2-4-4, 2-5-4, 2-6-4, and 2-7-4) were each prepared.

[0124] <Detection of FITC, measurement of detection time, and evaluation of color development intensity of control line and semi-quantitative line> Using healthy human serum (manufactured by TRINA Bioreactives) and treatment liquid 1 with a pH of less than 2 and treatment liquid 2 with a pH of 10 contained in ESPLINE (registered trademark) HBcrAg (RUO) (manufactured by Fujirebio Inc.), the above healthy human serum: treatment liquid 1: treatment liquid 2 = 1:3:1 ratio was mixed to prepare a sample. 20 μL of the sample was dropped onto the sample pads of the lateral flow assay devices 2-1-1 to 2-7-4. Next, in devices 2-1-1 to 2-7-4, the pressing part was pressed to develop the developing solution (2-amino-2-methyl-1-propanol (AMP) buffer), and the color development of the semi-quantitative line and the control line 30 minutes after the pressing was visually confirmed. The detection time until the color development at the semi-quantitative line and the control line could be visually confirmed from the above pressing, and the results of evaluating the color development intensity of the semi-quantitative line and the control line 30 minutes after the above pressing according to the color development intensity evaluation criteria of Test Example 3 are shown in Table 2 respectively. In addition, photographs of the semi-quantitative lines and control lines of devices 2-1-1 to 2-7-4 are shown in Fig. 6.

[0125]

Table 2

[0126] From the results in Table 2 and Figure 6, when compared with the color intensity at the semi-quantitative line of devices 2-1-1 to 2-7-1 manufactured under conditions of storage in a dark environment for 20 hours, devices 2-1-2 to 2-7-2, which were manufactured under conditions of irradiating the sample pad with LED light for 20 hours and which do not contain any co-existing coloring materials, specifically devices 2-2-2, 2-3-2, 2-5-2, and 2-7-2, no color development at the semi-quantitative line was observed, device 2-1-2 had very weak color development at the semi-quantitative line, and devices 2-4-2 and 2-6-2 could confirm color development at the semi-quantitative line but the color development intensity was weak. In contrast, when compared with the color intensity of the semi-quantitative lines of devices 2-1-3 to 2-7-3, which were manufactured under conditions of storing in a dark environment for 20 hours and which also contained coloring materials, devices 2-1-4 to 2-7-4, which were manufactured under conditions of irradiating the sample pad with LED light for 20 hours and which also contained coloring materials, did not show a significant decrease in color intensity, and it was found that the color of the semi-quantitative lines was easily recognizable in all cases.

[0127] [Test Example 4. Measurement and evaluation of fading suppression effect under various exposure conditions] <Preparation of Lateral Flow Assay Test Strips and Devices> A test line was formed by linearly applying 0.55 μL of an antibody solution containing 2.8 mg / mL of anti-hepatitis B virus-related antigen (HBcrAg) antibody to a matrix of nitrocellulose membrane (Millipore) 3.65 mm wide and 50 mm long, 16.0 mm upstream from the end of the absorbent pad side, and drying the solution. Next, 0.55 μL of a 1.5 mg / mL antibody solution of anti-FITC antibody (Anti-FITC mAb Lot. 3D1-1, manufactured by Advanced Life Science Institute, Inc.) was linearly applied and dried at a position 13.5 mm upstream from the end of the absorbent pad side on the matrix, forming a semi-quantitative line. Furthermore, 0.55 μL of an antibody solution containing 57.1 μg / mL of anti-alkaline phosphatase (ALP) antibody was linearly applied and dried at a position 11.0 mm upstream from the end of the absorbent pad side on the matrix to form a control line.

[0128] After drying the test line, semi-quantitative line, and control line, blocking was performed using 9.32 μL of a phosphate buffer solution containing 10 g / L sucrose, 650 mg / L SDBS (Dodecyl benzene sulfonic acid), and 1.0 g / L BSA.

[0129] 0.92 μL of a 80 mg / mL solution of BCIP (5-bromo-4-chloro-3-indolyl phosphate) was linearly applied and dried at a position 47 mm upstream from the end of the absorption pad side of the matrix to form a substrate line.

[0130] Next, 3.04 μL of a solution containing 0.03 mg / mL ALP-labeled anti-HBcrAg antibody and 1 μg / mL FITC-labeled ALP was applied to a 3.65 mm wide, 15.0 mm long porous sheet (Bell-Eta F(A)B, manufactured by AION Co., Ltd.) to prepare a sample pad. The resulting sample pad was placed between the test line and substrate line of the matrix to prepare a test strip for the lateral flow assay.

[0131] The above-mentioned lateral flow assay test strip, developer pad (glass fiber filter paper), and absorbent pad (highly absorbent filter paper) were bonded together and fixed to a developer tank (developer: 2-amino-2-methyl-1-propanol (AMP) buffer solution) and a plastic case with a push-in section to form a lateral flow assay device.

[0132] <Preparation of lateral flow assay devices stored under various light exposure conditions> For the sample pads of each of the above lateral flow assay devices, samples were prepared by irradiating with LED light for 0 hours (light-shielded) (device 3-1-1), 6 hours (device 3-1-2), 15 hours (device 3-1-3), and 24 hours (device 3-1-4), with UV light for 0 hours (light-shielded) (device 3-2-1), 2 hours (device 3-2-2), and 4 hours (device 3-2-3), and with sunlight for 0 hours (light-shielded) (device 3-3-1), 2 hours (device 3-3-2), and 4 hours (device 3-3-3).

[0133] <Preparation of Test Specimens and Devices for Lateral Flow Assays with Coexisting Fluorescent Substances and Color Materials> For the sample pads of each of the above lateral flow assay test specimens and devices, lateral flow assay test specimens and devices were prepared in the same manner as above, except that in addition to ALP labeled with 1 μg / mL of FITC, 3.04 μL of a solution containing 5 mg / mL of Brilliant Blue No. 1 (manufactured by Tokyo Chemical Industry Co., Ltd.) was adhered and dried.

[0134] <Preparation of Test Specimens and Devices for Lateral Flow Assays with Coexisting Fluorescent Substances and Color Materials Stored under Various Exposure Conditions> For the sample pads of the above lateral flow assay devices with coexisting fluorescent substances and color materials, samples were prepared by irradiating with LED light for 0 hours (light-shielded) (device 4-1-1), 6 hours (device 4-1-2), 15 hours (device 4-1-3), and 24 hours (device 4-1-4), with UV light for 0 hours (light-shielded) (device 4-2-1), 2 hours (device 4-2-2), and 4 hours (device 4-2-3), and with sunlight for 0 hours (light-shielded) (device 4-3-1), 2 hours (device 4-3-2), and 4 hours (device 4-3-3).

[0135] <Detection of HBcrAg and FITC and Evaluation of Color Development Intensity of Semi-Quantitative Lines> The HBcrAg calibrator (10,000 kU / mL HBcrAg calibrator) included in Lumipulse Presto (registered trademark) iTACT (registered trademark) HBcrAg (hepatitis B virus core-related antigen kit, Fujirebio Inc.) was diluted 50-fold with healthy human serum (TRINA Bioreactives), and mixed with Treatment Solution 1 (pH less than 2) and Treatment Solution 2 (pH 10) included in ESPLINE (registered trademark) HBcrAg (RUO) (Fujirebio Inc.) in a ratio of the above dilution solution:treatment solution 1:treatment solution 2 = 1:3:1 to prepare a sample. 20 μL of sample was dropped onto the sample pad of each of the lateral flow assay devices 3-1-1 to 4-3-3. The indenter was then depressed to allow the developer (2-amino-2-methyl-1-propanol (AMP) buffer solution) to develop. After 30 minutes, the color development of the test line, semi-quantitative line, and control line was visually confirmed. Photographs of the color development results of the test line, semi-quantitative line, and control line, as well as the results of evaluating the color intensity of the semi-quantitative line according to the evaluation criteria for color intensity in Test Example 3, are shown in FIG. 7. The relative color intensity of the semi-quantitative line is shown numerically in Figure 7. A color intensity of 0.5 was considered to be the color intensity evaluated as +w, and stronger colors were assigned a higher numerical value.

[0136] From the results in Figure 7, in devices 3-1-1 to 3-1-4, which are devices that irradiate the sample pad with LED light but do not contain any coloring material, it can be seen that the color intensity of the semi-quantitative line tends to decrease as the irradiation time with LED light increases. In contrast, in devices 4-1-1 to 4-1-4, which illuminate the sample pad with LED light and contain a coloring material, the color intensity of the semi-quantitative line remains almost unchanged even when the LED light irradiation time is extended. In other words, it can be inferred that the coexistence of the fluorescent substance FITC and Blue No. 1 suppresses the photodecomposition of FITC, and therefore the amount of FITC-labeled ALP captured by the anti-FITC antibody in the semi-quantitative line remains almost unchanged by the illumination of the LED light.

[0137] Furthermore, from the results of Figure 7, in devices 3-2-1 to 3-2-3, which are devices that irradiate UV light onto the sample pad but do not allow coloring materials to coexist, it can be seen that the color intensity of the semi-quantitative line tends to decrease as the UV light irradiation time increases. In contrast, in devices 4-2-1 to 4-2-3, which irradiate the sample pad with UV light and allow coloring materials to coexist, it can be seen that the color intensity of the semi-quantitative line does not change significantly even if the irradiation time with UV light is longer.

[0138] Furthermore, from the results of Figure 7, it can be seen that in devices 3-3-1 to 3-3-3, which are devices that irradiate sunlight onto the sample pad but do not allow coloring materials to coexist, the color intensity of the semi-quantitative line tends to decrease as the sunlight irradiation time increases, and in device 3-3-3, the color line can be recognized but the color intensity is weaker. In contrast, in devices 4-3-1 to 4-3-3, which are devices that irradiate sunlight onto the sample pad and allow coloring materials to coexist, it can be seen that the color intensity of the semi-quantitative line does not change significantly even if the sunlight irradiation time is longer.

[0139] [Test Example 5. Evaluation of the effect of suppressing fluorescence fading when various coloring materials are present] <Preparation of Lateral Flow Assay Test Strips and Devices> A 0.55 μL antibody solution containing 0.5 mg / mL of anti-FITC antibody (Advanced Life Science Institute, Inc., Anti-FITC mAb Lot. 3D1-1) was linearly applied and dried to a position 15.0 mm upstream from the end of the absorption pad on the matrix of a 3.65 mm wide, 50 mm long nitrocellulose membrane (Millipore), forming a semi-quantitative line. Furthermore, 0.55 μL of an antibody solution containing 57.1 μg / mL of anti-alkaline phosphatase (ALP) antibody was linearly applied and dried at a position 11.8 mm upstream from the end of the absorbent pad side on the matrix to form a control line.

[0140] After drying the semi-quantitative line and the control line, blocking was carried out using 9.32 μL of a phosphate buffer solution containing 10 g / L sucrose, 650 mg / L SDBS (Dodecyl benzene sulfonic acid), and 1.0 g / L BSA.

[0141] 0.92 μL of an 80 mg / mL solution of BCIP (5-bromo-4-chloro-3-indolyl phosphate) was linearly applied and dried at a position 47 mm upstream from the end of the absorption pad side on the matrix to form a substrate line.

[0142] Next, 3.04 μL of a solution containing 1 μg / mL FITC-labeled ALP was applied to a 3.65 mm wide, 15.0 mm long porous sheet (Bell-Eta F(A)B, manufactured by AION Co., Ltd.) and dried to prepare a sample pad. The resulting sample pad was placed between the semi-quantitative line and the substrate line of the matrix to prepare a test strip for the lateral flow assay.

[0143] The above-mentioned lateral flow assay test strip, developer pad (glass fiber filter paper), and absorbent pad (highly absorbent filter paper) were bonded together and fixed to a developer tank (developer: 2-amino-2-methyl-1-propanol (AMP) buffer solution) and a plastic case with a push-in section to form a lateral flow assay device.

[0144] <Fabrication of Lateral Flow Assay Devices Stored under Various Exposure Conditions> In the above lateral flow assay device, devices stored in a light-shielded environment for 24 hours (device 5-1-1) and devices irradiated with LED light on the sample pad for 24 hours (device 5-1-2) were fabricated respectively.

[0145] <Fabrication of Lateral Flow Assay Test Strips and Devices with Coexisting Fluorescent Substances and Various Color Materials> In the fabrication of the above lateral flow assay test strips and devices, in addition to ALP labeled with 1 μg / mL FITC on the sample pad, 3.04 μL of a solution containing 5 mg / mL of Food Blue No. 1 (manufactured by Tokyo Chemical Industry Co., Ltd.), 5 mg / mL of Food Red No. 2 (manufactured by Tokyo Chemical Industry Co., Ltd.), 5 mg / mL of Food Yellow No. 4 (manufactured by Tokyo Chemical Industry Co., Ltd.), 5 mg / mL of Food Green No. 3 (manufactured by Tokyo Chemical Industry Co., Ltd.), 30 mg / mL of Food Blue No. 2 (manufactured by Tokyo Chemical Industry Co., Ltd.), or 30 mg / mL of phycocyanin (manufactured by Tokyo Chemical Industry Co., Ltd.) was adhered and dried, and lateral flow assay test strips and devices were fabricated in the same manner as above.

[0146] <Fabrication of Lateral Flow Assay Devices with Coexisting Fluorescent Substances and Various Color Materials Stored under Various Exposure Conditions> In the above lateral flow assay device with coexisting fluorescent substances and color materials, devices stored in a light-shielded environment for 24 hours (device 5-2-1 (Food Blue No. 1), 5-3-1 (Food Red No. ......

[0147] <Detection of FITC and Evaluation of Color Development Intensity><I A sample was prepared by mixing healthy human serum (TRINA Bioreactives) with treatment solution 1 (pH less than 2) and treatment solution 2 (pH 10) contained in ESPLINE® HBcrAg (RUO) (Fujirebio Inc.) in a ratio of 1:3:1. 20 μL of the sample was dropped onto the sample pads of lateral flow assay devices 5-1-1 to 5-7-2. Next, the pressing section of each of the devices 5-1-1 to 5-7-2 was pressed down to develop the developing solution (2-amino-2-methyl-1-propanol (AMP) buffer solution), and the color development of the semi-quantitative line and the control line 30 minutes after pressing was visually confirmed. Photographs of the color development results of the semi-quantitative line and the control line 30 minutes after pressing, as well as the results of evaluating the color development intensity of the semi-quantitative line according to the evaluation criteria for color development intensity described below, are shown in Figure 8. Color intensity evaluation criteria: +: Line color is easily recognizable -: Line color cannot be recognized

[0148] The results in Figure 8 show that compared to the color intensity of the semi-quantitative line in Device 5-1-1, which was manufactured under conditions of 24-hour storage in a dark environment, the color of the semi-quantitative line in Device 5-1-2, which was manufactured under conditions of 24-hour illumination of the sample pad with LED light and which does not contain any coexisting coloring material, is not discernible. This is thought to be due to photodecomposition or structural changes of FITC caused by illumination with LED light. In contrast, when compared with the color intensity of the semi-quantitative line in Devices 5-2-1 to 5-7-1, which were fabricated under conditions of 24-hour storage in a dark environment, it can be seen that the color of the semi-quantitative line is easily recognizable in Devices 5-2-2 to 5-7-2, which were fabricated under conditions of 24-hour illumination of the sample pad with an LED light and which also contain coloring materials. This is thought to be due to the suppression of photodecomposition or structural changes of FITC by the coexistence of FITC and each coloring material. [Explanation of symbols]

[0149] 1 Lateral flow assay test strip 20 and 22 Sample pads (also serving as fluorescent substance holding pads and probe holding pads) 21 and 23 Sample supply unit (also serving as fluorescent substance holder and probe holder) 30 Matrix 31 Test Line 32 Reference Line 33 Control Line 34 Substrate Line 40 absorbent pads 50 Spreading Liquid Pad 60 Development fluid tank 70 samples

Claims

1. A method for suppressing fading of fluorescence, characterized by causing (A) a fluorescent substance or fluorescent label and (B) a coloring material to coexist in a dry state.

2. 2. The method for suppressing fluorescent light fading according to claim 1, wherein the coloring material (B) is a coloring material that reflects a portion of visible light.

3. The method for suppressing fluorescence fading according to claim 1, which is used in a lateral flow assay or an immunoassay.

4. The method for suppressing fluorescence bleaching according to claim 1 , wherein the lateral flow assay is an immunochromatographic assay.

5. A lateral flow assay test strip having a matrix, comprising: a fluorescent substance or the like retaining portion arranged on the matrix, which retains (A) a fluorescent substance or a fluorescent label and (B) a coloring material in a dry state; Equipped with Test strips for lateral flow assays.

6. The test strip for lateral flow assay according to claim 5, which is a test strip for immunochromatographic assay.

7. the matrix has a reference line; The test strip for lateral flow assay according to claim 5 .

8. a capturer that captures the fluorescent substance or the fluorescent label is immobilized on the reference line; The lateral flow assay test strip according to claim 7.

9. 6. The lateral flow assay test strip according to claim 5, wherein the matrix has a test line, and a capturer for capturing the target substance is immobilized on the test line.

10. The lateral flow assay test strip according to claim 5, wherein the fluorescent label is an enzyme labeled with a fluorescent substance.

11. The lateral flow assay test strip according to claim 10, wherein the enzyme is peroxidase, galactosidase, or phosphatase.

12. A lateral flow assay device comprising the lateral flow assay test strip according to any one of claims 5 to 11.

13. A lateral flow assay kit comprising the lateral flow assay test strip according to any one of claims 5 to 11.

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