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

[Technical Field]

[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. [Background technology]

[0002] Many test kits have been proposed for detecting various targets, such as antibodies, antigens, proteins, biomarkers, chemical molecules, nucleic acids, etc. 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 an analyte and a simple test strip structure, two usually 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 is bound are used, the insoluble carrier particles are trapped by the capture substance immobilized on the membrane to accumulate, and the membrane is irradiated with light to detect light emitted from the site where the insoluble carrier particles have accumulated or from surrounding sites other than the site where the insoluble carrier particles have accumulated, and a light-reflecting material is provided on the side of the membrane opposite to the side where the light is irradiated. 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 proposes a test kit for detecting and / or quantifying a target biological or chemical substance in a liquid sample by a capillary phenomenon test using photoluminescent inorganic nanoparticles of the formula A1 - xLn xVO 4 (1 - y ) (PO4 ) y (II), where 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; 0 < x < 1, 0 ≤ y < 1, and the method detects luminescence with a luminescence lifetime of less than 100 ms of the nanoparticles after one-photon absorption by exciting the matrix at a wavelength of 320 nm or less.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, in the test kits according to the above-mentioned proposals, the chromatographic results cannot be clearly visually confirmed, so a detection device for reading is required, and there is a problem that detection confirmation cannot be easily performed due to the need for a complicated device.

[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. [Means for solving the problem]

[0006] As a result of intensive research to solve the above-mentioned problems, the inventors have found 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 using any special equipment. Based on this finding, further research has led to the discovery that the above-mentioned object can be achieved, and has thus completed the present invention. That is, the present invention provides the following inventions. 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 contains 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. A test kit characterized by: 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 when the intensity of the excitation light is taken as 1. 4. The test kit according to 1 further comprises a fluorescent color level sample, and quantification is possible by visually checking the color level of the fluorescent color level sample and the fluorescent color of the chromatograph. 5. The fluorescent substance is a polymer particle in which the rare earth complex is encapsulated. 2. The test kit described in 2. 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). [Effects of the Invention]

[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) that is 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 more sensitive detection than conventional labels such as gold colloid (reduction of background light due to the Stokes shift). Furthermore, even higher sensitivity is expected in conditions where there is no external light, such as a darkroom, or where light is blocked. [Brief explanation of the drawings]

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

[0009] 1 Test kit, 10 Chromatography main body, 11 Sample pad, 13 Conjugate pad, 15 Membrane, 17 Absorption pad, 20 Light source DETAILED DESCRIPTION OF THE INVENTION

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

[0011] [Detection target] The detection targets in the detection kit of this embodiment include antibodies, antigens, proteins, nucleic acids, and other biomarkers that can be test targets, as well as other compounds. [Chromatography unit] The chromatography body 10 includes a membrane 15, which is a component for separating and visualizing components, the main functions of chromatography; a conjugate pad 13 with a detection label 30 (see FIG. 2 ) attached thereto, and an absorbent pad 17 for absorbing excess liquid, mounted on each of the membrane's longitudinal ends; and a sample pad 11 mounted 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 a conventional manner. The shapes and thicknesses of these components can be formed in the same manner as components used in conventional immunochromatography. In this embodiment, all components are rectangular, and any commonly used thickness can be used without particular restrictions. The materials for these components can be any materials normally used for this type of chromatography without any particular restrictions, and in this embodiment, the membrane 15 is made of a nonwoven fabric such as a polyester nonwoven fabric, and the conjugate pad 13, absorbent pad 17, and sample pad 11 are made of a nonwoven fabric made of cellulose fiber. The conjugate pad may also be made of glass fiber. Although not shown, the chromatography unit of this embodiment may be provided with a case or cover for covering the above-mentioned components. Also, components that are advantageous for visual observation, such as a fluorescent filter and a bandpass filter, may be provided. In this embodiment, two types of lines are shown, but the present invention is not limited to this, and three or more types of lines may be provided. Moreover, instead of the rectangular shape as in this embodiment, various shapes such as a circular shape can be adopted. As the material for forming the membrane, in addition to the nonwoven fabric mentioned above, any material that is normally used to form this type of membrane, such as a metal, can be used without any particular limitation.

[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, it becomes possible to detect it visually using a light source such as a commercially available fluorescent lamp, as described below, without using a special detection device. In conventional immunochromatography, emphasis has been placed on the sensitivity of luminescence, and even in research reports using fluorescent substances, only those aimed at increasing sensitivity, and no reports have focused on the Stokes shift in fluorescence (the difference between the maximum excitation wavelength and the maximum emission wavelength (light emitted from the fluorescent substance)). The inventors have found that if this Stokes shift is narrow, background occurs, making visual confirmation difficult, and have discovered that by widening the Stokes shift, sufficient visual detection and confirmation are possible without the use of special equipment. Therefore, the wavelength of the absorbed light and the wavelength of the emitted light are important, and it is important to use a fluorescent substance with a wide Stokes shift. The fluorescent brightness of the above fluorescent substance (brightness at the wavelength of the emitted fluorescence) is preferably 0.8 or more when the brightness of the excitation light (light at the maximum excitation wavelength) is taken as 1, and more preferably in the range of 0.9 to 1.5 in order to visually confirm 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. Among these, one or more compounds selected from the group consisting of europium complexes, terbium complexes, samarium complexes, and dysprosium complexes are particularly preferred for use as the fluorescent substance. Among 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: Sodium [4'-(4'-amino-4-biphenylyl)-2,2':6',2"-terpyridine-6,6"-diylbis(methyliminodiacetate)]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 M level) A so-called fluorescence enhancement solution containing β-NTA-TOPO-Triton X-100 must be added. Eu of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid 3+ Complex (BCPDA-Eu 3+ ) is used as a labeling agent 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 benzophenone with salicylic acid derivatives Two fluorescent labeling agents: tris(bipyridine cryptate)-Eu 3+ (Fluorescence energy transfer donor dye, TBP-Eu 3+ ) and allophycocyanin (a fluorescent energy transfer acceptor dye, cross-linked allophycocyanin, a pigment protein with a molecular weight of 104 kD, a maximum fluorescence emission wavelength of 665 nm, and a fluorescence quantum yield of approximately 0.7, abbreviated as XL665). BCPDA-Eu3+ Labeled polyvinylamine-biotin streptavidin complex 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} and Eu 3+ Complex with Those encapsulated in particles, such as polystyrene latex nanoparticles containing europium fluorescent complexes The following can be mentioned: The following commercially available products can also be used. Estapor (registered trademark) europium microsphere series (Merck) Europium Conjugation Kit (ab269889, 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, but it is preferable to adjust the amount so that the amount of the fluorescent substance per target substance is in the range of 1 to 20, and more preferably 8 to 12. This adjustment is determined depending on the expected amount of target substance to be detected (calculated from the expected content of target substance relative to the amount of sample collected). The fluorescent substance can be the above-mentioned complex or the like as it is. Furthermore, it is preferable to use the above-mentioned complex encapsulated in particles, etc., from the viewpoints of membrane dispersibility and visual visibility of the fluorescence. For example, it is preferable to use the above-mentioned commercially available product Estapor (registered trademark) Europium Microsphere Series (manufactured by Merck, see Examples). The mixing ratio of the detection antibody to the fluorescent substance is not particularly limited, but when particles (polymer particles, etc.) containing the complex are used, the ratio of detection antibody to particle (mg / g) is preferably 20 to 80, more preferably 40 to 80, and most preferably 40 to 60.

[0015] As the detection antibody used in the detection label, any antibody that is normally used in the detection label in this type of immunochromatography can be used without any particular limitation. For example, anti-H5 HA monoclonal antibody (5H7) can be mentioned.

[0016] 〔light source〕 The light source used in this embodiment is not particularly limited as long as it can irradiate 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 any particular limitations, and specific examples include the following. Alonefire SV16 Compact 5W Ultraviolet Blacklight 365nm Wavelength USB Rechargeable UV LED Light; Portable, compact black light keychain for glowing original puzzles (UV-LED375-nano) manufactured by Nichia Chemical, product name "Single-bulb LED Black Light"

[0017] [Other components] In addition to the above-mentioned chromatographic unit and light source, the detection kit of the present invention can be used in combination with various other components within the scope of the present invention. [Fluorescent color level sample] The other components may 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 chromatographically fluorescing the detection target at multiple concentrations, collating the fluorescent membranes (whose fluorescent intensity varies depending on the concentration), creating fluorescent color samples corresponding to the multiple concentrations, and then assembling these samples. That is, the fluorescent color level sample includes fluorescent color samples (multiple color samples with different fluorescent intensities depending on the concentration) corresponding to the multiple detection target concentrations. The number and level of concentration difference of the multiple concentrations to be prepared is determined based on the detection target, but it is usually preferable to have about 10 to 20 different concentrations, as this facilitates quantitative determination. By providing a fluorescent color level sample in this manner, quantification is possible by visually checking the color level of the fluorescent color level sample and the fluorescent color of the chromatography and confirming the concentration of the fluorescent color level sample with the color level closest to the fluorescent color of the chromatography, and a quantifiable detection kit can be constructed. As described above, the present invention makes it possible to realize "quantification" by "visual inspection" by using a substance that generates fluorescence rather than a substance that emits light, 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 chromatograph body and a light source. The chromatographic body of this embodiment can be obtained as follows. The sample pad, conjugate pad, membrane and absorbent pad are formed in the usual manner and superimposed in the manner shown in FIG. Next, the 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 by this type of chromatography, such as SARS-CoV-2 nucleoprotein antibody, anti-HA antibody, or anti-mouse antibody, without any particular restrictions. 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 the 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 multiple light-emission photographs can then be compiled to create a fluorescent color level sample.

[0019] [Usage and effects] The detection kit of this embodiment can be used as shown in Fig. 2. In Fig. 2, the fluorescent substance 30 is shown schematically, and the sample and the detection target are also shown schematically. 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. At this time, the target substance 33 to be detected in the sample 31 reacts with a detection label (not shown for simplicity of the drawing) in which the fluorescent substance 30 carried by the conjugate pad 13 is bound to a detection antibody, and migrates to the membrane 15 in a state bound to the detection label. The complex between the fluorescent substance 30 and the target substance 33 reacts with the antibody that constitutes the test line 15a and remains at the test line 15a. Then, detection can be performed by irradiating light of a predetermined wavelength from a light source 20. In this case, a fluorescent color level sample is prepared, and the concentration of the detection target 33 can be visually measured by comparing the color with that of the fluorescent color level sample. When carrying out this detection operation, the desired effects of the present invention can be more effectively achieved by carrying out the operation in a dark place, such as by using a dark box (using the detection kit of the present invention).

[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, and it is possible to detect even when the target substance is small, such as in the early stages of infection. Furthermore, since it cannot be confirmed unless irradiated 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. [Example]

[0022] Example 1 Using Estapor (registered trademark) microspheres (K1-050, F1-Eu-010) as a fluorescent substance, a detection kit shown in FIG. 1 was obtained according to the above-mentioned manufacturing method. The raw materials and light sources used are shown below. Sample pad: Product name "CF4" (manufactured by Cytiva) Conjugate pad: Product name "33glass" (manufactured by Cytiva) Membrane: Product 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: Alonefire SV16 Compact 5W UV Black Light Wavelength 365nm The obtained detection kit was used to detect H5 subtype avian influenza antigen (hemagglutinin). 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) and the test was performed. The results are shown in Figure 3. Figure 3 also shows a sample that was simply colored. As is clear from the results shown in Figure 3, the detection kit of the present invention is highly sensitive, being able to adequately detect antigens even at low concentrations, such as 1 ng / mL in the antigen solution. Furthermore, when light is irradiated from a light source, luminescence (red luminescence) is observed on the test line, which can be easily distinguished visually, allowing detection to be performed. Although not shown, no display is made unless light is irradiated from the light source, making it easy to use even in cases where confidentiality is required. Furthermore, because the luminescence level differs 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 the fluorescent substance, 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 (MERCK) Absorbent pad: Trade 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) Developing solution: 50mM Tris-HCl, 0.05% Tween20, 0.5% Casein-Na, 10mMGuanidine 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 ( 10mM Tris, pH 8.0+2% (w / v) BSA ) Micro BCA™ Protein Assay Kit (23235, Thermo Scientific) [Light source] Alonefire SV16 Compact 5W UV Black Light Wavelength 365nm 〔method〕 MERCK's Application Note (Microsphere Coupling ― Based on the Two-step EDC / Sulfo NHS Covalent Coupling Procedure for Estapor® Carboxyl-modified Dyed Microspheres character The detection antibody / particle (particle as fluorescent substance) was then added at 0, 20, 40, 60, 80, and 100 mg / g. inSix types of detection labels were obtained by binding particles as fluorescent substances to the detection antibodies. 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. The immobilized antibody was applied to the test line and control line of the membrane, respectively, and then blocked. The resulting detection label was applied to the control at a particle amount of 0.5 to 4 μg / pad. Jugate Pad The test kit shown in Figure 1 was then created. A photograph was taken before the drops were applied (SV16 B Using rack light, iPhone (registered trademark), camera app P 5 times). 80 μL of developing solution (developing solution with antigen is antigen (+), without antigen is antigen (-; antigen amount is 1.25 ng / mL) was dropped thereto, and after 30 minutes, light was shone from a light source and the fluorescence was photographed. The results are shown in Figure 4. The fluorescence intensity is also shown in Figure 6(a). The fluorescence of the detection label and the background of the membrane were confirmed. As is clear from the results shown in Figure 4, at 100, the background was strong It is understood that this is not very desirable from an economical point of view because it emits fluorescence, making it less visible to the naked eye, and does not provide the advantages of increased visibility (see Figure 6(a)). too Visual inspection Test line The visibility is poor, and the 80 is faint Background fluorescence This is inferior in that it emits light that makes it difficult to see with the naked eye, and the fluorescence of the lines becomes blurred. Furthermore, the concentration of the detection label was set at 80 mg / g (detection antibody / particles (particles as fluorescent substance)), and the particle amounts were set at 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. Figure 6(b) also 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 background fluorescence, reducing visual visibility.

[0024] [Table 1]

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

[0026] [Table 2]

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 for visually confirming test results, 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, and the fluorescent substance contains a rare earth complex; the fluorescent substance is a polymer particle containing the rare earth complex, The mixing ratio of the polymer particles is 40 to 60 in terms of detection antibody / particle (mg / g) in the detection label. A test kit characterized by:

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

3. Furthermore, a fluorescent color level sample is provided, and quantitative determination 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.

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