Test kit

Abstract

[Problem] To provide a test kit that does not require a complicated device to perform detection and that allows easier detection confirmation than conventional test kits in which chromatography is used. [Solution] Provided is a test kit including: a sample pad that absorbs a sample; a conjugate pad that has a detection marker supported thereon; a chromatographic body that has a membrane; an absorption pad that prevents backflow of the sample; and a light source that irradiates light of 300 nm to 400 nm. The test kit is characterized in that the detection marker is a fluorescent substance that emits light having a wavelength of at least 600 nm when light having a wavelength of 300 nm to 400 nm is applied.

Description

Test kit

[0001] The present invention relates to a test kit for detecting various targets such as antibodies, antigens, proteins, biomarkers, chemical molecules, and nucleic acids. More specifically, the present invention relates to a test kit that does not require complicated equipment for detection and allows for easier detection and confirmation than conventional chromatography-based test kits.

[0002] Numerous test kits have been proposed for detecting various targets, such as antibodies, antigens, proteins, biomarkers, chemical molecules, and nucleic acids. Among these, test kits using immunochromatography are widely used due to their ease of use, and various types have been proposed. For example, Patent Document 1 proposes an immunochromatographic test strip that achieves both highly sensitive detection of analyte and a simple test strip structure, two generally incompatible goals. Specifically, the proposed immunochromatographic test strip includes a membrane on which a capture substance, which is a ligand that binds to the analyte, is immobilized, and insoluble carrier particles to which a ligand that binds to the analyte are bound are used. The insoluble carrier particles are trapped and accumulated by the capture substance immobilized on the membrane, and the analyte is measured by irradiating the membrane with light and detecting light emitted from the site where the insoluble carrier particles have accumulated or from surrounding areas other than the site where the insoluble carrier particles have accumulated. The immunochromatographic test strip proposed includes a light-reflecting material on the side of the membrane opposite to the side irradiated with light. Furthermore, Patent Document 2 proposes a test kit with excellent detection sensitivity for a substance to be detected. Specifically, the proposed test kit has, on a substrate, a reagent holding section containing a labeled reagent in which a binding substance that specifically binds to the substance to be detected is immobilized on labeled microparticles, and a test section in which a test capture substance that specifically binds to the substance to be detected is immobilized, the labeled microparticles being microparticles made of a noble metal, and the test section containing an upconversion material.In addition, Patent Document 3 discloses a compound represented by the formula A1-xLnxVO4(1-y)(PO4) A test kit is proposed for detecting and / or quantifying a biological or chemical substance of interest in a liquid sample by capillary action testing using photoluminescent inorganic nanoparticles of formula y(II) as a probe, wherein Ln is selected from europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), erbium (Er), ytterbium (Yb), thulium (Tm), praseodymium (Pr), holmium (Ho), and mixtures thereof; A is selected from yttrium (Y), gadolinium (Gd), lanthanum (La), lutetium (Lu), and mixtures thereof, with 0<x<1 and 0≦y<1; the method detects luminescence of the nanoparticles with an emission lifetime of less than 100 ms after one-photon absorption by exciting the matrix at a wavelength of 320 nm or shorter.

[0003] Japanese Patent Application Laid-Open No. 2015-31610 Japanese Patent Application Laid-Open No. 2017-223616 Special Publication No. 2021-530700

[0004] However, with the test kits proposed above, the results of chromatography cannot be clearly confirmed visually, so a detection device must be used for reading, which poses the problem of not being able to easily perform detection and confirmation due to the need for complicated equipment.

[0005] Therefore, an object of the present invention is to provide a test kit that does not require complicated equipment for detection and allows for easier detection and confirmation than conventional chromatography-based test kits.

[0006] The present inventors conducted extensive research to solve the above-mentioned problems and discovered that when a europium complex is used as a target reagent, detection and confirmation can be performed visually using only ultraviolet light (such as a commonly used so-called black light) without the need for any special equipment. Further research based on this discovery revealed that the above-mentioned object can be achieved, leading to the completion of the present invention. Specifically, the present invention provides the following: 1. A test kit comprising a sample pad for absorbing a sample, a conjugate pad carrying a detection label, a chromatographic body having a membrane, and an absorbent pad for preventing backflow of the sample; and a light source for irradiating light of 300 nm to 400 nm, wherein the detection label comprises a fluorescent substance that emits light of a wavelength of 600 nm or more when irradiated with light of a wavelength of 300 nm to 400 nm. 2. The test kit according to 1, wherein the fluorescent substance is a rare earth complex. 3. The test kit according to 1, wherein the fluorescent substance has a fluorescence intensity of 0.8 or more, where the intensity of the excitation light is taken as 1. 4. The test kit according to 1, further comprising a fluorescent color level sample, and quantification is possible by visually confirming the color level of the fluorescent color level sample and the fluorescent color of the chromatograph. 5. The test kit according to 2, wherein the fluorescent substance is a polymer particle encapsulating the rare earth complex. 6. The test kit according to 5, wherein the mixing ratio of the polymer particles is 20 to 80 in terms of detection antibody in the detection label / particles (mg / g).

[0007] The test kit of the present invention does not require complicated equipment for detection and allows for easier detection and confirmation than conventional chromatography-based test kits. Therefore, the test kit of the present invention is cost-effective and useful as a test kit for detecting various targets, such as antibodies, antigens, proteins, biomarkers, chemical molecules, and nucleic acids. Furthermore, the test kit of the present invention has excellent confidentiality because it can only be confirmed by irradiating it with light of a specific wavelength. Furthermore, it is possible to perform detection with higher sensitivity than conventional immunochromatography. Specifically, by using a fluorescent dye such as europium, detection is possible at a fluorescence wavelength (615 nm) far removed from the excitation light wavelength of black light (360-370 nm). This allows for a higher signal-to-noise (S / N) ratio and higher sensitivity than conventional labels such as gold colloids (reduced background light due to the Stokes shift). Furthermore, even higher sensitivity is expected in conditions without external light, such as a darkroom, or when light is blocked.

[0008] FIG. 1 is a schematic diagram showing the overall configuration of the test kit of the present invention. FIG. 2 is an explanatory diagram showing a typical usage mode of the test kit shown in FIG. 1. FIG. 3 is a photograph (a substitute for a drawing) showing the use of the detection kit of the example. FIG. 4 is a photograph (a substitute for a drawing) showing the results of use of the detection kit of Example 2. FIG. 5 is a photograph (a substitute for a drawing) showing the use of the detection kit of Example 2 in another aspect. FIG. 6 is a chart showing the use results of the detection kit of Example 2, where (a) is a chart showing the relationship between the detection antibody and the fluorescence intensity of particles, and (b) is a chart showing the relationship between the particle amount and the fluorescence intensity. FIG. 7 is a photograph (a substitute for a drawing) showing the use results of the detection kit of Example 3. FIG. 8 is a photograph (a substitute for a drawing) showing the use of the detection kit of Example 3 in another aspect. FIG. 9 is a chart showing the use results of the detection kit of Example 3, where (a) is a chart showing the relationship between the detection antibody and the fluorescence intensity of particles, and (b) is a chart showing the relationship between the particle amount and the fluorescence intensity.

[0009] 1 Test kit, 10 Chromatography body, 11 Sample pad, 13 Conjugate pad, 15 Membrane, 17 Absorption pad, 20 Light source

[0010] The present invention will be described in further detail below. As shown in Figure 1, a test kit 1 according to one embodiment of the present invention comprises a sample pad 11 for absorbing a sample, a conjugate pad 13 carrying a detection label, a chromatography unit 10 including an absorption pad 17 and a membrane 15 for absorbing the electrophoresed sample and preventing backflow, and a light source 20 for irradiating light of 300 nm to 400 nm. Here, the "detection label" refers to a mixture of a detection antibody and a fluorescent substance (including both those in which the detection antibody and the fluorescent substance are bound and those in which they are not bound). This will be described in further detail below.

[0011] [Detection Target] The detection target in the detection kit of this embodiment includes antibodies, antigens, proteins, nucleic acids, and other biomarkers that can be used as test targets, as well as other compounds. [Chromatography Unit] The chromatography unit 10 includes a membrane 15, which is a component for separating and visualizing components, the main function of chromatography, a conjugate pad 13 with a detection label 30 (see FIG. 2) attached and an absorbent pad 17 for absorbing excess liquid, placed at each end of the membrane 15 in the longitudinal direction, and a sample pad 11 placed on the end of the conjugate pad 13. Furthermore, a test line 15a and a control line 15b are provided on the membrane 15 in accordance with conventional methods. The shape and thickness of these components can be formed similarly to those used in conventional immunochromatography. In this embodiment, all components are rectangular, and their thickness can be any commonly used thickness without any particular restrictions. The materials for forming these components can be any materials typically used in this type of chromatography without any particular limitations. In this embodiment, the membrane 15 is formed from a nonwoven fabric such as a polyester nonwoven fabric, while the conjugate pad 13, the absorption pad 17, and the sample pad 11 are formed from a nonwoven fabric made of cellulose fiber. The conjugate pad may also be formed from glass fiber. The chromatography body of this embodiment may also be provided with a case or cover, not shown, to cover the above-mentioned components. Components that facilitate visual observation, such as a fluorescent filter or a bandpass filter, may also be provided. While this embodiment shows two types of lines, this is not limiting and three or more types of lines may also be provided. Various shapes, such as a circular shape, may also be used instead of the rectangular shape shown in this embodiment. In addition to the nonwoven fabrics mentioned above, materials typically used to form this type of membrane, such as metals, can also be used without any particular limitations.

[0012] [Detection Label] In this embodiment, the detection label carried by the conjugate pad 13 contains a fluorescent substance that emits light with a wavelength of 600 nm or more, preferably light with a wavelength of 500 to 700 nm (hereinafter sometimes referred to as "fluorescence"), when irradiated with light with a wavelength of 300 to 400 nm (hereinafter sometimes referred to as "absorbed light"). That is, the detection label is a mixture containing a detection antibody and a fluorescent substance. By using a detection label containing such a fluorescent substance, visual detection becomes possible without the need for special detection equipment, using a light source such as a commercially available fluorescent lamp, as described below. Conventional immunochromatography has focused on luminescence sensitivity, and even research reports using fluorescent substances have only aimed to achieve high sensitivity, with no reports focusing on the Stokes shift in fluorescence (the difference between the excitation maximum wavelength and the luminescence (light emitted from the fluorescent substance) maximum wavelength). The inventors have found that a narrow Stokes shift generates background light, making visual confirmation difficult. They have also discovered that a wide Stokes shift allows for sufficient visual detection and confirmation without the use of special equipment. Therefore, the wavelengths of the absorbed light and the emitted light are important, and it is important to use a fluorescent substance with a wide Stokes shift. The fluorescent brightness of the fluorescent substance (brightness at the wavelength of the emitted fluorescence) is preferably 0.8 or more, where the brightness of the excitation light (light at the maximum excitation wavelength) is 1. A range of 0.9 to 1.5 is preferable for visual confirmation of the light from the fluorescent substance.

[0013] Specific examples of the fluorescent substance include rare earth fluorescent complexes, and examples of the rare earth complexes include europium complexes, terbium complexes, samarium complexes, and dysprosium complexes. In particular, one or more compounds selected from the group consisting of europium complexes, terbium complexes, samarium complexes, and dysprosium complexes can be preferably used as the fluorescent substance. Of these, europium complexes are preferred in terms of achieving the desired effects of the present invention, and specific examples of europium complexes include the following compounds: [4'-(4'-amino-4-biphenylyl)-2,2':6',2"-terpyridine-6,6"-diylbis(methyliminodiacetate)]sodium europate(III) (ATBTA-Eu ３＋ ), N １ -(p-Isothiocyanatobenzyl)-ethylenediaminetetraacetic acid Eu ?３＋ Complex (SCN-Ph-EDTA-Eu ３＋ ), ?N １ -(p-isothiocyanatobenzyl)-diethylenetriamine-N １ , N ２ , N ３ , N ３ -Eu tetraacetic acid ?３＋ Complex (SCN-Ph-DTTA-Eu ３＋ ) Eu(β-NTA) ３ (TOPO) ２ (10 －１４ (Detection limit at 100 M level) It is necessary to add a so-called fluorescence enhancement solution containing β-NTA-TOPO-Triton X-100. ３＋ Complex (BCPDA-Eu ３＋ ) is used as a labeling agent. The chlorosulfonylated tetradentate β-diketone compound 4,4'-bis(1",1",1",2",2",3",3"-heptafluoro-4",6"-hexanedione-6"-yl)chlorosulfo-o-terphenyl (BHHCT) BHHCT-Eu ３＋ Complex EDTA-Tb ３＋ Ternary complexes of Eu with salicylic acid derivatives and two fluorescent labels, tris(bipyridine cryptate)-Eu ３＋ (Fluorescence energy transfer donor dye, TBP-Eu ３＋ A combination of BCPDA-Eu and allophycocyanin (acceptor dye for fluorescence energy transfer, cross-linked allophycocyanin, pigment protein with a molecular weight of 104 kD, maximum fluorescence emission wavelength of 665 nm, fluorescence quantum yield of approximately 0.7, abbreviated as XL665). ３＋ Labeled polyvinylamine-biotin streptavidin conjugate. Combination of poly(Glu:Lys)-streptavidin conjugate and europium fluorescent complex-labeled poly(Glu:Lys)-BSA-streptavidin conjugate. 4-[2-(4-isothiocyanatophenyl)ethynyl]-2,6-bis{[N,N-bis(carboxymethyl)amino]methyl}pyridine {4-[2-(4-isothiocyanatophenyl)ethynyl]-2,6-bis{[N,N-bis(carboxymethyl)amino]methyl}pyridine} and Eu ３＋ Examples of such a complex include those in a state where the complex is encapsulated in a particle, such as polystyrene latex nanoparticles containing a europium fluorescent complex. The following commercially available products can also be used: Estapor (registered trademark) Europium Microsphere Series (manufactured by Merck), Europium Conjugation Kit (ab269889, manufactured by Abcam), FS Eu beads (manufactured by Tamagawa Seiki Co., Ltd.), etc.

[0014] The amount of the fluorescent substance mixed can be determined arbitrarily depending on the target substance to be detected. However, it is preferable to adjust the amount so that the fluorescent substance is used in a ratio of 1 to 20, and particularly preferably 8 to 12, of one target substance. This adjustment is determined based on the expected amount of target substance to be detected (calculated from the predicted content of target substance relative to the amount of sample collected). Furthermore, the fluorescent substance can be the above-mentioned complex or the like as is. Furthermore, it is preferable to use the above-mentioned complex encapsulated in particles, etc., from the viewpoints of membrane dispersibility and visual fluorescence visibility. For example, it is preferable to use the above-mentioned commercially available Estapor® Europium Microsphere series (manufactured by Merck, see Examples). The mixing ratio of the detection antibody and the fluorescent substance is not particularly limited. However, when particles (e.g., polymer particles) encapsulating the above-mentioned complex are used, the detection antibody / particle (mg / g) ratio is preferably 20 to 80, more preferably 40 to 80, and most preferably 40 to 60.

[0015] The detection antibody used in the detection label can be any antibody commonly used in detection labels for this type of immunochromatography, without any particular limitation, such as anti-H5 HA monoclonal antibody (5H7).

[0016] [Light Source] The light source used in this embodiment is not particularly limited as long as it can emit light with a wavelength of 300 nm to 400 nm, and commercially available products can be used. Specifically, commercially available so-called 365 nm UV LEDs or 375 nm UV LEDs can be used without particular limitations, and specific examples include the following: Alonefire, product name "SV16 Compact 5 W Ultraviolet Black Light Wavelength 365 nm USB Rechargeable UV LED Light"; Portable Compact Black Light Keychain for Glowing Original Puzzles (UV-LED375-nano); Nichia Corporation, product name "Single-Light LED Black Light"; etc.

[0017] [Other Components] In addition to the above-described chromatograph body and light source, the detection kit of the present invention can be used in combination with various other components within the spirit and scope of the present invention. [Fluorescent Color Level Sample] The above-described other components can include, for example, a fluorescent color level sample. The fluorescent color level sample enables the detection kit of this embodiment to perform not only qualitative analysis but also quantitative analysis. This fluorescent color level sample is constructed by chromatographing fluorescent samples according to multiple concentrations of the detection target, collating the fluorescent membranes (whose fluorescent intensity varies depending on the concentration), and creating fluorescent color samples corresponding to the multiple concentrations. In other words, the fluorescent color level sample includes fluorescent color samples (multiple color samples with different fluorescent intensities depending on the concentration) corresponding to the multiple concentrations of the detection target. The number and level of concentration difference of the multiple concentrations can be determined depending on the detection target, but typically, approximately 10 to 20 types are preferred for quantitative determination. By providing a fluorescent color level sample in this way, quantification is possible by visually checking the color level of the fluorescent color level sample and the fluorescent color of the chromatograph and confirming the concentration of the fluorescent color level sample with the color level closest to the fluorescent color of the chromatograph, and a quantifiable detection kit can be constructed. As described above, the present invention enables "quantification" by "visual inspection" by using a fluorescent substance that generates fluorescence rather than a luminescent substance, preferably a fluorescent substance that absorbs light of a specific wavelength and emits light of other wavelengths in the visible range.

[0018] [Manufacturing Method] The detection kit of this embodiment can be obtained by preparing a chromatographic body and a light source. The chromatographic body of this embodiment can be obtained as follows. A sample pad, a conjugate pad, a membrane, and an absorption pad are formed according to standard methods and then stacked as shown in FIG. 1 . Next, a detection antibody is applied to the membrane in a line shape and dried to form test line 15a and control line 15b. The antibody can be any antibody that can be detected using this type of chromatography, such as a SARS-CoV-2 nucleoprotein antibody, an anti-HA antibody, or an anti-mouse antibody, without any particular limitations. Detection substances other than the above antibodies can also be used. Then, a detection label containing a detection antibody bound to a fluorescent substance such as a europium complex is applied to a conjugate pad and dried to form a conjugate pad carrying the fluorescent substance, thereby forming the chromatographic body. Furthermore, multiple chromatographic units are prepared, and sample solutions adjusted to multiple concentrations are dropped onto each chromatographic unit to perform immunochromatography. After a predetermined time (10 to 30 minutes), light is irradiated from a light source, and the resulting light is photographed to capture multiple images of the emitted light. These photographs of the emitted light can then be compiled to create a fluorescent color level sample.

[0019] [Method of Use and Effects] The detection kit of this embodiment can be used as shown in FIG. 2. In FIG. 2, the fluorescent substance 30 is schematically illustrated, as well as the sample and the target of detection. As shown in FIG. 2, to use the detection kit 1 of this embodiment, first, a sample 31 is dropped onto the sample pad 11. The sample 31 migrates from the conjugate pad 13 to the membrane 15 by capillary action. During this process, the target of detection 33 in the sample 31 reacts with the detection label (not shown for simplicity) bound to the detection antibody via the fluorescent substance 30 carried on the conjugate pad 13, and migrates to the membrane 15 in a bound state to the detection label. The complex of the fluorescent substance 30 and the target of detection 33 then reacts with the antibody constituting the test line 15a and remains at the test line 15a. Detection can then be performed by irradiating the sample with light of a predetermined wavelength from the light source 20. In this case, a fluorescent color level sample may be prepared, and by comparing the color of the fluorescent color level sample with that of the test substance, the concentration of the test substance 33 can be visually measured. By performing this detection operation in a dark place, such as in a dark box (using the detection kit of the present invention), the desired effects of the present invention can be more effectively achieved.

[0020] The detection kit of this embodiment is configured as described above, allowing for visual detection by irradiation with light from a light source. Furthermore, the Stokes shift is wide, there is no background effect, and it is easy to determine visually. Furthermore, since it can be distinguished to a degree that allows for sufficient determination even with small amounts of the target substance, it can be said to have improved sensitivity, allowing detection even when the target substance is small, such as in the early stages of infection. Furthermore, since it can only be confirmed by irradiating it with light of a specific wavelength, it is highly confidential and can be confirmed only when necessary, thereby protecting privacy. Therefore, it is useful not only for simply testing and detecting infectious diseases (diagnostic agents for infectious diseases), but also for pregnancy tests and testing for allergens and drugs.

[0021] The present invention is not limited to the above-described embodiment, and various modifications are possible without departing from the spirit of the present invention.

[0022] Example 1 Using Estapor™ microspheres (K1-050, F1-Eu-010) as a fluorescent substance, a detection kit shown in Figure 1 was obtained according to the above-mentioned manufacturing method. The raw materials and light source used are as follows: Sample pad: trade name "CF4" (manufactured by Cytiva) Conjugate pad: trade name "33glass" (manufactured by Cytiva) Membrane: trade name "FF80 HP" (manufactured by Cytiva) Absorbent pad: trade name "Surewick (registered trademark)" cellulose sample pad (C083 type, manufactured by Merck) Microsphere concentration; detection antibody (Anti-H5 HA monoclonal antibody (5H7)): particle = 1:10, particle equivalent 0.025%, 10 μL Light source: trade name "SV16 compact 5 W ultraviolet black light wavelength 365 nm" manufactured by Alonefire Using the obtained detection kit, H5 subtype avian influenza antigen (hemagglutinin) was detected. For detection, five antigen solutions were prepared at concentrations of 0, 1, 10, 100, and 1000 (unit: ng / mL), and 0.1 mL was dropped onto each test kit (the actual amount used is shown in the figure). The results are shown in Figure 3. Figure 3 also shows a simply colored sample. As is clear from the results shown in Figure 3, the detection kit of the present invention is highly sensitive and can adequately detect antigen solutions at low concentrations, such as 1 ng / mL. Furthermore, when light is irradiated from a light source, luminescence (red luminescence) is observed on the test line, which can be easily distinguished visually and allows detection to be performed. Although not shown, no display is displayed unless light is irradiated from a light source, making it easy to use even in cases where confidentiality is required. Furthermore, since the luminescence level varies depending on the antigen concentration in the antigen solution, simple quantitative detection is also possible by comparing it with a level sample.

[0023] Example 2 A detection kit having the configuration shown in FIG. 1 was obtained in the same manner as in Example 1, except that Estapor® microspheres (product number "F1-Eu-030") were used as a fluorescent substance, and the following raw materials were used and the following method was followed.[Raw materials] Sample pad: Product name "CF3" (manufactured by Cytiva) Conjugate pad: Product name "33glass" (manufactured by Cytiva) Membrane: Product name "Hi-Flow? Plus HF12002" (manufactured by Merck) Absorption pad: Product name "Surewick (registered trademark)" cellulose sample pad (C083 type, manufactured by Merck) Immobilized antibody: Anti-H5 HA monoclonal antibody (8C1), Anti-mouse IgG Detection label: Fluorescent substance "F1-Eu-030", detection antibody: Anti-H5 HA monoclonal antibody (5H7) Developer: 50 mM Tris-HCl, 0.05% Tween 20, 0.5% Casein-Na, 10mM Guanidine Antigen; Influenza A H5N1 (A / Anhui / 1 / 2005) HA Protein, His Tag (Sino Biological) [Other materials] Activation / Coupling Buffer? 50 mM MES, pH 6.0 EDC (F024810, Wako) Sulfo NHS (24510, Thermo Scientific) Ethanolamine (411000, Sigma) Blocking Buffer? 10 mM Tris, pH 8.0+2% (w / v) BSA Micro BCA (registered trademark) Protein Assay Kit (23235, Thermo Scientific) Light source: Alonefire product name "SV16 Compact 5W Ultraviolet Black Light Wavelength 365 nm" Method: MERCK Application Note (Microsphere Coupling - Two-step EDC / Sulfo NHS Covalent Coupling Procedure for Estapor (registered trademark) Carboxyl-modified Dye) According to the method of the present invention, six types of detection labels were obtained by binding the detection antibody to particles (particles as fluorescent substance) at 0, 20, 40, 60, 80, and 100 (mg / g).For each detection label, the amount of detection antibody bound per 0.1 mg of particles was calculated by quantifying the amount of detection antibody remaining in the supernatant after the binding reaction between the detection antibody and the fluorescent substance using the BCA method and subtracting this from the amount of detection antibody added. Data for each detection label is shown in Table 1. In Table 1, the detection antibody is simply referred to as "antibody." The same applies to Table 2. Immobilized antibodies were applied to the test line and control line of the membrane, followed by blocking. The resulting detection label was applied to a conjugate pad at a particle amount of 0.5 to 4 μg / pad and dried to create the test kit shown in Figure 1. A photograph was then taken before the dropwise addition (using an SV16 black light, iPhone (registered trademark), camera app, x5). 80 μL of developing solution (those with antigen in the developing solution are designated as antigen (+), those without antigen are designated as antigen (-); the antigen amount was 1.25 ng / mL) was dropped onto the plate, and after 30 minutes, light was shone from a light source and the fluorescence was photographed. The results are shown in FIG. 4. The fluorescence intensity is also shown in FIG. 6(a). The fluorescence of the detection label and the background of the membrane were confirmed. As is clear from the results shown in FIG. 4, 100 is not very preferable from an economical perspective because the background emits fluorescence, making it poorly visible to the naked eye, and it does not offer the advantages of increased visibility (see FIG. 6(a)). Furthermore, 20 emits fluorescence in the background in the antigen (+), making it poorly visible to the naked eye, and 80 emits faint fluorescence from the background, making it poorly visible to the naked eye, and is also inferior in that the fluorescence of the line is blurred. Furthermore, the concentration of the detection label was set to 80 mg / g (detection antibody / particles (particles as fluorescent substance)), and the amount of particles was set to 0.5, 1, 2, and 4, to confirm the relationship between the amount of particles as fluorescent substance and fluorescence. The results are shown in Figure 5. Furthermore, Figure 6(b) shows the fluorescence intensity. As shown in Figure 6(b), increasing the amount improves the fluorescence intensity, but as shown in Figure 5, increasing the amount causes the background to emit fluorescence, resulting in poor visual visibility.

[0024]

[0025] Example 3 Antigen detection was carried out in the same manner as in Example 2, except that Estapor (registered trademark) microspheres (F1-Eu-050) were used as the fluorescent substance. The results are shown in Table 2 and Figures 7 to 9. As is clear from the results shown in Figure 7, a value of 20 to 80 is good, and a value of 40 to 60 is particularly good for both (-) and (+). As with Example 2, the amount of particles also improved the fluorescence intensity when the amount was increased, but increased amount also caused background fluorescence, resulting in poor visual visibility.

[0026]

Claims

1. A sample pad for absorbing a sample, a conjugate pad carrying a detection label, a chromatographic body having a membrane, and an absorption pad for preventing backflow of the sample; A test kit comprising a light source that irradiates light of 300 nm to 400 nm, the detection label has a fluorescent substance that emits light with a wavelength of 600 nm or more when irradiated with light with a wavelength of 300 nm to 400 nm, the fluorescent substance containing a rare earth complex; The fluorescent substance is a polymer particle having the rare earth complex encapsulated therein. A test kit characterized by:

2. The mixing ratio of the polymer particles is 20 to 80 in terms of detection antibody / particle (mg / g) in the detection label; The test kit according to claim 1.

3. 2. The test kit according to claim 1, wherein the fluorescent substance has a fluorescent brightness of 0.8 or more when the brightness of the excitation light is taken as 1.

4. Furthermore, a fluorescent color level sample is provided, and quantification is possible by visually checking the color level of the fluorescent color level sample and the fluorescent color of the chromatogram. The test kit according to claim 1.