Immunochromatographic measurement method, immunochromatographic measurement auxiliary liquid, immunochromatographic chip, and immunochromatographic measurement kit

Abstract

The present invention provides a high-sensitivity immunochromatography measuring method in which a labeling substance for labeling a subject to be detected in a specimen undergoing immunochromatography is quantified by being identified with precision, high resolution, and increased contrast by using an electron microscope.　An immunochromatography measuring method according to one embodiment of the present invention is characterized in that the measurement is performed using an electron microscope after applying an auxiliary liquid other than the specimen in the immunochromatography.

Description

[Technical field]

[0001] The present invention relates to an immunochromatographic measurement method, an auxiliary liquid for immunochromatographic measurement, an immunochromatographic chip, and an immunochromatographic measurement kit. [Background technology]

[0002] In order to prevent the spread of infectious diseases caused by pathogens such as bacteria and viruses, early detection and treatment of infections are important, and highly sensitive detection of pathogens is required. Conventionally, culture methods, PCR methods, LAMP methods, ELISA methods, immunochromatography methods, etc. have been used to confirm the diagnosis of infectious diseases.

[0003] The culture method requires a very long time to determine the results, and only the pathogens expected in advance can be detected. The PCR method can also only detect the pathogens expected in advance, and a negative result does not necessarily deny the presence of the pathogen. The PCR method enables high-sensitivity detection by repeating DNA amplification, but since the amplification reaction needs to be repeated, there is a risk of false positive results due to contamination from the positive control. In addition, the PCR method is more complicated to operate than immunochromatography, and takes a long time (about 5 to 6 hours) to determine the results. The LAMP method (loop-mediated isothermal amplification method) can perform the DNA amplification reaction to detection in one step by using a primer design different from that of the PCR method, but like the PCR method, the operation is more complicated than immunochromatography. The ELISA method can detect pathogens (antigens) in a sample using antibodies against the pathogen, and is faster than the PCR method and LAMP method, but there is a possibility of false positive results due to non-specific antigen-antibody reactions. All of these techniques still have the risk of false positive or false negative results.

[0004] Immunochromatography is currently being implemented in society as a diagnostic aid for various diseases, mainly influenza viruses. Its principle is based on antigen-antibody reactions, and it is widely used in medical settings due to its simplicity and effectiveness. The problem with immunochromatography is that its sensitivity is not as high as that of the PCR method, and it is not possible to diagnose the presence or absence of infection unless the virus (pathogen) has multiplied to a certain extent in the patient's body. In addition, while immunochromatography can determine whether a test is positive or negative with the naked eye, it has the disadvantage that it is difficult to quantify the results (for example, to measure the number of viruses).

[0005] In order to compensate for the problems and drawbacks of immunochromatography described above, various methods have been proposed. For example, in the case of a system that combines immunochromatography with a small densitometry analyzer, quantification is possible by using densitometry, but the measurement sensitivity of the detection part (test line) is often not good, and there are difficulties in terms of increasing sensitivity. In addition, there are reports that the use of densitometry may be inappropriate depending on the type of sample (see Patent Document 1).

[0006] Patent Document 2 proposes amplifying the signal of the gold label by using gold fine particles containing a predetermined amount of silver as a detection label. Although the high sensitivity achieved by silver sensitization as described in Patent Document 2 is more sensitive than the conventional method, the silver sensitization step is involved, which makes the result judgment unstable and dependent on the sensitization reaction. Furthermore, signal amplification involving a sensitization step, not limited to silver, merely means that the antigen-antibody reaction on the immunochromatography chip can be easily confirmed by the naked eye, and does not achieve the high sensitivity expected in practical use. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2000-338106 A [Patent Document 2] JP 2009-192224 A Summary of the Invention [Problem to be solved by the invention]

[0008] In view of the above circumstances, an object of the present invention is to provide a highly sensitive immunochromatographic measurement method in which a labeling substance that labels an object to be detected in a sample is quantified by accurately identifying the labeling substance with high resolution and increased contrast using an electron microscope. Another object of the present invention is to provide an auxiliary liquid for immunochromatography measurement used in the measurement of an immunochromatography chip using an electron microscope. Another object of the present invention is to provide an immunochromatography chip for use in the above-mentioned immunochromatographic measurement method. Another object of the present invention is to provide an immunochromatography assay kit which contains the above auxiliary liquid as a constituent element and, if necessary, further contains the above immunochromatography chip as a constituent element. [Means for solving the problem]

[0009] Many of the immunochromatography chips currently in general use use a labeled antibody, which is an antibody for a detection target bound to a labeling substance, and a capture antibody that recognizes an epitope different from that of the labeled antibody, to form an antigen-antibody reaction product consisting of the labeled antibody, the detection target (antigen), and the capture antibody, and the color generated by the accumulation of the labeling substance bound to the labeled antibody is confirmed with the naked eye or the like. The present inventors have found that when the detection unit of such an immunochromatography chip is subjected to measurement with an electron microscope, the image becomes unclear, as shown in Comparative Example 1 in the Examples described later, and it is difficult to clearly identify the carrier (e.g., nitrocellulose membrane) to which the capture antibody is fixed and the antigen-antibody reaction product (labeling substance) on the carrier in the image. This is considered to be due to the fact that the carrier and the antigen-antibody reaction product are charged by continuous irradiation of an electron beam from the electron source of the electron microscope, and heat is generated in the carrier and the antigen-antibody reaction product, which causes a structural change and destabilizes the carrier and the antigen-antibody reaction product, resulting in a loss of image clarity.

[0010] Therefore, the inventors came up with the idea of ​​applying an auxiliary liquid other than the specimen to structurally stabilize the sample (detection target) under measurement conditions using an electron microscope, thereby improving the clarity of the electron microscope image (obtaining an electron microscope image with high contrast).As a result of extensive research, they have completed the present invention.

[0011] That is, the present invention includes the following aspects. (1) A measurement method using immunochromatography in which an auxiliary liquid other than the sample is applied and then measurement is performed using an electron microscope. (2) The method according to (1), wherein the auxiliary liquid has electrical conductivity to prevent the generation of static electricity and heat, which contribute to image clarity under conditions of measurement using an electron microscope. (3) The method according to (1) or (2), wherein the auxiliary liquid has a property of polymerizing to form a film under conditions for measurement using an electron microscope. (4) The method according to any one of (1) to (3), which is an immunochromatography method using a labeled antibody carrying metal nanoparticles as a labeling substance and a capture antibody having the property of binding to a complex of the labeled antibody and an object to be detected. (5) The method according to (4), wherein the capture antibodies are immobilized at intervals on an immunochromatography chip. (6) The method according to any one of (1) to (5), wherein the sample and the auxiliary solution are applied simultaneously. (7) The method according to any one of (1) to (5), wherein the auxiliary liquid is developed after development of the sample. (8) The method according to any one of (1) to (7), further comprising using artificial intelligence to identify a labeling substance in an electron microscope image, thereby identifying an object to be detected. (9) The method according to any one of (1) to (7), wherein the object to be detected is identified by identifying a labeling substance in an electron microscope image using energy dispersive X-ray spectroscopy (EDX). (10) An immunochromatographic measurement auxiliary solution that improves image clarity when measuring immunochromatographic chips using an electron microscope. (11) The auxiliary liquid according to (10), which has a property of being conductive under measurement conditions using an electron microscope. (12) The auxiliary liquid according to (10) or (11), which has a property of polymerizing to form a film under conditions for measurement using an electron microscope. (13) The auxiliary solution according to any one of (10) to (12), comprising, as essential components, at least one compound selected from glycerin and glycerin substitutes; polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, and polysorbate 85, and polysorbate substitutes, and, as an optional component, at least one compound selected from monosaccharides, disaccharides, salts, and buffer solutions. (14) An immunochromatography chip for use in the method according to any one of (1) to (9), comprising a detection unit dedicated to measurement by electron microscopy. (15) An immunochromatography chip as described in (14), in which a labeled antibody carrying metal nanoparticles as a labeling substance is fixed at a predetermined position, and a capture antibody having the property of binding to a complex of the labeled antibody and the object to be detected is fixed to a detection section dedicated to measurement by the electron microscope. (16) The immunochromatography chip according to (15), in which the capture antibodies are immobilized at intervals. (17) The immunochromatography chip according to any one of (14) to (16), further comprising a detection unit for visual inspection. (18) The immunochromatography chip according to any one of (14) to (16), which does not include a detection unit other than the detection unit dedicated to the measurement using an electron microscope. (19) An immunochromatography assay kit comprising, as a component, the auxiliary liquid according to any one of (10) to (13). (20) The kit according to (19), further comprising the immunochromatography chip according to any one of (14) to (18) as a component. Effect of the Invention

[0012] According to the present invention, a highly sensitive immunochromatographic measurement method is provided in which a labeling substance that labels a detection target in a specimen is accurately identified by increasing the contrast under high resolution using an electron microscope, and quantified. Specifically, in the method of the present invention, by applying an auxiliary liquid other than the specimen, the contrast between the background carrier and the target detection target becomes clear in the measurement of the immunochromatography chip using an electron microscope, and the labeling substance of the detection target can be identified as a clear (sharply outlined) particle, so that the result of immunochromatography can be quantified in a short time and easily by measuring the number of the labeling substance. In one aspect of the method of the present invention, the auxiliary liquid has conductivity that prevents the generation of charge and heat that contributes to the clarity of the image under the measurement conditions using an electron microscope, and / or has the property of polymerizing to form a film, so that the above-mentioned effects can be efficiently obtained. In the method of the present invention, a detection target on a chip can be identified using an electron microscope at high magnification and high resolution, so that sensitivity can be increased by about 10 to 100 times compared to conventional qualitative judgment by visual inspection. In other words, in the method of the present invention, even if the concentration of the target substance in the sample is about 10 to 100 times lower than that of conventional samples, it is possible to identify the target substance by subjecting the immunochromatography chip to measurement using an electron microscope to identify the labeling substance of the target substance.

[0013] The present invention also provides an auxiliary liquid for immunochromatography measurement used in measurement of an immunochromatography chip using an electron microscope. Specifically, the auxiliary liquid for immunochromatography measurement of the present invention improves the clarity of an electron microscope image of an immunochromatography chip, and by applying the auxiliary liquid of the present invention, an image with high contrast can be obtained in measurement of an immunochromatography chip using an electron microscope. In one aspect of the auxiliary liquid of the present invention, the auxiliary liquid has a property of being conductive under measurement conditions using an electron microscope and / or has a property of polymerizing to form a film, thereby making it possible to efficiently obtain the above-mentioned effects.

[0014] The present invention also provides an immunochromatography chip used in the immunochromatography measurement method. Specifically, the immunochromatography chip of the present invention is provided with a detection unit dedicated to measurement by an electron microscope. In the immunochromatography chip of the present invention, a high-magnification, high-resolution image is obtained under the measurement conditions of the electron microscope in the detection unit dedicated to measurement by an electron microscope, so that a detection unit other than the detection unit is not necessarily required. That is, in an embodiment in which only a detection unit dedicated to measurement by an electron microscope is provided as a detection unit, a quantified result can be obtained quickly and more simply by subjecting the immunochromatography chip on which the sample is developed to measurement by an electron microscope. On the other hand, in another embodiment of the immunochromatography chip of the present invention, in addition to the detection unit dedicated to measurement by an electron microscope, a detection unit for visual observation is further provided, whereby visual screening and measurement by an electron microscope can be performed in separate detection units. That is, a two-step detection is possible in which, as a first step, a simple screening is performed by visually checking the color development resulting from the labeling substance that labels the detection target in a visual detection section, and then, as a second step, a detection section dedicated to measurement with an electron microscope is used to measure the number of the labeling substance and thereby quantify the amount of the labeling substance. The effects obtained by these aspects will be described in detail below.

[0015] According to the present invention, a highly sensitive immunochromatography measurement kit is provided, which quantifies a labeling substance that labels a detection target in a specimen by accurately identifying the labeling substance with high resolution and increasing the contrast using an electron microscope. Specifically, the kit of the present invention includes the above-mentioned immunochromatography measurement auxiliary liquid as a component. The kit of the present invention may further include the above-mentioned immunochromatography chip as a component, if necessary. The kit of the present invention can accurately identify a labeling substance that labels a detection target in a specimen by accurately increasing the contrast with high resolution, for example, by using metal nanoparticles such as gold nanoparticles or platinum-gold nanoparticles used in conventional immunochromatography as a labeling substance in combination with a scanning electron microscope (SEM) as a signal, and can easily and quickly quantify the results of immunochromatography by measuring the number of metal nanoparticles. In addition, the quantified data can be compared with background data, and a significant difference (for example, a p value of p<0.01 or less in a t-test) can be determined as a positive value, and the kit can be used as a diagnostic aid for various diseases. When the kit of the present invention is used in combination with an SEM, the SEM is not limited to a specific model, and a wide range of models from FE-SEM to tabletop SEM can be used. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic plan view showing an example of the configuration of an immunochromatography chip that can be used in the method of the present invention. [Diagram 2] FIG. 1 is a schematic perspective view showing an example of the configuration of an immunochromatography chip that can be used in the method of the present invention. [Diagram 3] Schematic plan views showing examples of the embodiments of the detection part of the immunochromatography chip shown in FIG. 1 and FIG. 2; (a) an embodiment in which the capture antibody is immobilized in a line shape, and (b) an embodiment in which the range over which the capture antibody is immobilized is smaller than that shown in (a). [Figure 4]Schematic plan views showing examples of the embodiments of the detection part of the immunochromatography chip shown in FIG. 1 and FIG. 2; (a) an embodiment in which capture antibodies are immobilized at intervals, and (b) an embodiment in which the range in which the capture antibodies are immobilized is smaller than that shown in (a). [Diagram 5] Schematic plan views showing examples of the configuration of the detection section of the immunochromatography chip shown in Figures 1 and 2; (a) an embodiment in which two types of capture antibodies are immobilized in a line shape, and the capture antibodies are immobilized aligned in parallel in a direction perpendicular to the development direction; (b) an embodiment in which two types of capture antibodies are immobilized in a line shape, and one capture antibody is immobilized shifted downstream from the other capture antibody. [Figure 6] Schematic plan views showing examples of the configuration of the detection section of the immunochromatography chip shown in FIG. 1 and FIG. 2; (a) an embodiment in which two types of capture antibodies are immobilized at intervals and aligned in parallel in a direction perpendicular to the development direction; (b) an embodiment in which two types of capture antibodies are immobilized at intervals and one capture antibody is immobilized shifted downstream from the other capture antibody. [Figure 7] Schematic plan views showing an example of the configuration of the chip of the present invention; (a) an embodiment in which a detection unit dedicated to measurement by an electron microscope is disposed on both sides of a detection unit for visual inspection, and (b) an embodiment in which a detection unit dedicated to measurement by an electron microscope is disposed upstream of a detection unit for visual inspection. [Figure 8] Schematic plan views showing an example of the configuration of the chip of the present invention; (a) an embodiment in which a visual detection unit is disposed on both sides of a detection unit dedicated to measurement by an electron microscope; (b) an embodiment in which the detection unit dedicated to measurement by an electron microscope in the embodiment shown in (a) is in the form of a continuous line. [Figure 9] Schematic plan views showing an example of the configuration of the chip of the present invention; (a) an embodiment in which detection units dedicated to measurement by an electron microscope are arranged in a continuous line, and (b) an embodiment in which two detection units dedicated to measurement by an electron microscope are arranged in a continuous line and different types of capture antibodies are immobilized on each of them. [Figure 10]SEM images of the detection area (A line) of influenza A virus antigen when a sample kit positive for influenza A virus antigen was measured; (a) Example 1, (b) Comparative Example 1 [Figure 11] (a) An example of an SEM image of a detection area (test line) determined to be positive under the measurement conditions and determination criteria described in the Examples, (b) an example of an SEM image of a background area DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Hereinafter, an embodiment of the present invention will be described in detail. Note that the specific form is not limited to the following embodiment, and the present invention includes design changes and the like within the scope of the gist of the present invention.

[0018] <Immunochromatography measurement method> The immunochromatographic measurement method of the present invention (hereinafter also referred to simply as "the method of the present invention") is characterized in that in immunochromatography, an auxiliary liquid other than the sample (hereinafter also referred to as "immunochromatographic measurement auxiliary liquid") is applied, and then measurement is performed using an electron microscope.

[0019] [Immunochromatography measurement auxiliary solution] It is preferable that the auxiliary liquid for immunochromatography measurement (hereinafter, simply referred to as "auxiliary liquid") has conductivity to prevent the generation of static electricity and heat, which contribute to the clarity of electron microscope images, and / or has the property of polymerizing to form a film. In addition, it is preferable that the auxiliary liquid can wash away impurities from the entire carrier 4 including the detection section t and control section c of the immunochromatography chip 10, which will be described below with reference to Figures 1 and 2, under measurement conditions using an electron microscope, and clarify the labeled substance in the detection section t and control section c. When the auxiliary liquid has at least one or more, and more preferably all, of these characteristics, the contrast between the background carrier (e.g., a nitrocellulose membrane) and the target substance to be detected is clear in the measurement of the immunochromatography chip using an electron microscope, background instability caused by swelling due to heat generation is eliminated, and the labeled substance of the target substance to be detected (e.g., metal nanoparticles such as gold nanoparticles and platinum-gold nanoparticles) can be identified as clear (sharply defined) particles, and the results of immunochromatography can be quantified quickly and easily by measuring the number of these labeled substances.

[0020] That is, the auxiliary liquid for immunochromatography measurement of the present invention improves image clarity in the measurement of an immunochromatography chip using an electron microscope. The auxiliary liquid of the present invention is preferably conductive under the measurement conditions using an electron microscope and / or polymerizes to form a film. More specifically, the auxiliary liquid of the present invention contains, as essential components, at least one compound selected from glycerin and glycerin substitutes; polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, and polysorbate 85, and polysorbate substitutes, and, as optional components, at least one compound selected from monosaccharides, disaccharides, salts, and buffer solutions.

[0021] Glycerin is a trihydric alcohol (so-called polyhydric alcohol), has a hydroxyl group in the molecule, and is a low vapor pressure substance. Glycerin also has viscosity. Substances having these characteristics can be included in the auxiliary liquid of the present invention as alternative components to glycerin. Specifically, examples of glycerin alternatives include polyethylene glycol, polyvinyl alcohol, triglyceride, polyresorcinol, polyphenol, tannic acid, urushiol, and saponin. Glycerin and glycerin alternatives may be used alone or in combination of two or more.

[0022] In this specification, the term "polysorbates" refers to those prepared by reacting sorbitan fatty acid esters (nonionic surfactants) with ethylene oxide. Currently available polysorbates include polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 65 (Tween 65), polysorbate 80 (Tween 80), and polysorbate 85 (Tween 85), but the polysorbates that can be included in the auxiliary liquid of the present invention are not limited to these. In addition, substances classified as nonionic surfactants similar to polysorbates can be included in the auxiliary liquid of the present invention as alternative components to polysorbates. Specifically, examples of polysorbate substitutes include polyoxyethylene alkyl ethers, polyoxyethylene hydrogenated castor oil, polyoxyethylene monofatty acid esters, sucrose fatty acid esters, polyglycerin fatty acid esters, alkyl polyglycosides, N-methyl alkyl glucamides, etc. Polysorbates and polysorbate substitutes may be used alone or in combination of two or more.

[0023] Examples of monosaccharides include glucose and fructose. Disaccharides include, for example, sucrose and trehalose. Examples of the salts include imidazolium salts, pyridinium salts, piperidinium salts, pyrrolidinium salts, and quaternary ammonium salts. Examples of buffer solutions include acetate buffer (acetic acid / sodium acetate buffer), phosphate buffer (phosphoric acid / sodium phosphate buffer), citrate buffer (citric acid / sodium citrate buffer), citrate phosphate buffer (citric acid / sodium phosphate buffer), borate buffer, tartrate buffer, and Tris buffer. These monosaccharides, disaccharides, salts and buffer solutions may be used alone or in combination of two or more.

[0024] The essential component consisting of at least one compound selected from glycerin, a glycerin substitute, polysorbates, and polysorbate substitutes is preferably contained in the auxiliary liquid in an amount of 0.01 to 10 percent by weight, more preferably 0.1 to 2 percent by weight.

[0025] [Immunochromatography] In the method of the present invention, the principle of immunochromatography and the method of detecting the target substance are not particularly limited. In the following, as a representative embodiment of the method of the present invention, immunochromatography using a labeled antibody carrying metal nanoparticles as a labeling substance and a capture antibody having a property of binding to a complex of the labeled antibody and the target substance is described as an example, but it should be noted that the specific aspect of immunochromatography in the method of the present invention is not limited to this.

[0026] In this embodiment, the "detection target" is a substance that has the property of specifically recognizing and binding to the labeled antibody and the capture antibody, i.e., antigenicity. In other words, in this embodiment, the detection target is an antigen. The antigen may have immunogenicity or may lack immunogenicity. In the latter case, the detection target is also called a hapten or an incomplete antigen.

[0027] Examples of antigens include, but are not limited to, pathogens such as viruses, viroids, bacteria, and fungi; extracellular endoplasmic reticulum such as exosomes, microvesicles, and apoptotic bodies; proteins, DNA, and RNA derived from body fluids (blood, serum, saliva, urine, etc.) and hair of test animals; proteins, DNA, and RNA derived from organs, tissues, and cells of test plants; heavy metals such as mercury, arsenic, aluminum, cadmium, lead, nickel, and tin; and allergens such as pollen from trees and flowers, mites, house dust, and food (eggs, wheat, etc.). In particular, the method of the present invention is suitable for cases where measurement by optical microscope is impossible, such as viruses and viroids, since it is measured by electron microscope.

[0028] The type of virus may be either a DNA virus or an RNA virus.

[0029] Examples of DNA viruses include Poxviridae (smallpox virus, monkeypox virus, etc.), Herpesviridae (herpes simplex virus, varicella-zoster virus, cytomegalovirus, EB virus, etc.), Adenoviridae (adenovirus), Papovaviridae (papillomavirus, JC virus, etc.), Parvoviridae (parvovirus), and Hepadnaviridae (hepatitis B virus, etc.).

[0030] Examples of RNA viruses include Arenaviridae (Lassa virus, etc.), Orthomyxoviridae (influenza virus, etc.), Caliciviridae (norovirus, Sapovirus, etc.), Coronaviridae (SARS virus, MERS virus, etc.), Togaviridae (rubella virus, etc.), Nodaviridae (viral nervous necrosis virus, etc.), Paramyxoviridae (mumps virus, measles virus, respiratory syncytial virus, etc.), Picornaviridae (poliovirus, coxsackievirus, echovirus, etc.), Filoviridae (Marburg virus, Ebola virus, etc.), Bunyaviridae (Crimean-Congo hemorrhagic fever virus, severe fever with thrombocytopenia syndrome virus, etc.), Flaviviridae (yellow fever virus, dengue fever virus, hepatitis C virus, hepatitis G virus, etc.), Rhabdoviridae (rabies virus, etc.), Reoviridae, and Retroviridae (human immunodeficiency virus, human T-lymphotropic virus, simian immunodeficiency virus, STLV, etc.).

[0031] Viroids include, for example, the Absinthe Viroidae family and the Pospiviroidae family.

[0032] Examples of bacteria include Staphylococcus aureus, Escherichia coli, Salmonella, Pseudomonas aeruginosa, Vibrio cholerae, Shigella, Bacillus anthracis, Mycobacterium tuberculosis, Clostridium botulinum, Clostridium tetani, and Streptococcus.

[0033] Examples of fungi include tinea, Candida, and Aspergillus.

[0034] The above-mentioned viruses, viroids, bacteria, and fungi may be known or unknown.

[0035] [Immunochromatography chip] 1 and 2 are schematic plan and perspective views, respectively, showing an example of the configuration of an immunochromatography chip (also called a development support, chromatographic medium, etc.) that can be used in the method of the present invention. Note that the immunochromatography chip that can be used in the method of the present invention is not particularly limited to those having the configurations shown in Figs. 1 and 2, as long as it is a chip that can be used in normal immunochromatography.

[0036] The chip 10 shown in Figures 1 and 2 comprises, from upstream to downstream in the development direction (the direction shown by arrow D in Figures 1 and 2), a sample pad 2, a conjugate pad 3, a carrier 4, and an absorbent pad 5. These members are fixed onto a base material (also called a backing sheet, not shown) with an adhesive or an adhesive sheet. A transparent film (not shown) may be applied to the upper surface of the carrier 4, and a functional sheet may be provided between each member.

[0037] A sample is dropped onto the sample pad 2. As the sample pad 2, for example, a glass fiber pad, a cellulose fiber pad, a polyester pad, or the like can be used.

[0038] The conjugate pad 3 has a labeled antibody, which is an antibody against a detection target bound to a labeling substance, immobilized thereon. The conjugate pad 3 can be prepared, for example, by preparing a suspension containing the labeled antibody, applying the suspension to an appropriate absorbent pad (for example, a glass fiber pad, a cellulose fiber pad, a polyester pad, etc.), and then drying the pad. As the labeled substance, a labeled substance used in conventional immunochromatography can be used. For example, metal nanoparticles (metal fine particles), latex fine particles, organic polymer fine particles, inorganic fine particles, color-developing fine particles such as liposomes containing a color developer can be used. More specifically, for example, metal nanoparticles include precious metal nanoparticles such as gold nanoparticles, platinum nanoparticles, platinum-gold nanoparticles, and silver nanoparticles, titanium nanoparticles, iron nanoparticles, nickel nanoparticles, and cadmium nanoparticles. The metal nanoparticles may be colloidal metal nanoparticles having a particle size of 1 nm to 100 nm.

[0039] The carrier 4 may be a material used in conventional immunochromatography, such as a nitrocellulose membrane. The carrier 4 has a detection section t and a control section c. A capture antibody that recognizes an epitope different from that of the labeled antibody immobilized on the conjugate pad 3 and has the property of binding to a complex of the labeled antibody and the detection target is immobilized on the detection section t. An antibody (also called a control antibody) that specifically recognizes the labeled antibody immobilized on the conjugate pad 3 is immobilized on the control section t. Note that, for ease of understanding, the ranges of the detection section t and the control section c are shown by solid lines in Figs. 1 and 2, but this does not mean that a line indicating the boundary between the detection section t and the control section c is drawn on the chip that is actually used.

[0040] The absorbent pad 5 serves to absorb excess specimen after chromatographic development. As the absorbent pad 5, for example, a glass fiber pad, a cellulose fiber pad, a polyester pad, or the like can be used.

[0041] The substance to be detected in the sample dropped onto the sample pad 2 reacts with the labeled antibody immobilized on the conjugate pad 3 to form a complex. This complex spreads over the carrier 4 and is captured by the capture antibody immobilized on the detection section t of the carrier 4, which causes color development derived from the labeled substance of the labeled antibody accumulated at the detection section t. Furthermore, excess labeled antibody is captured by the control antibody immobilized on the control section c of the carrier 4, which causes color development derived from the labeled substance of the labeled antibody accumulated at the control section c. Excess sample that spreads downstream of the control section c of the carrier 4 is absorbed by the absorption pad 5.

[0042] In addition, in conventional immunochromatography chips, the capture antibody and the control antibody are usually immobilized in a line shape perpendicular to the development direction (i.e., parallel to the short side of the carrier). Therefore, the detection part and the control part are also called a detection line (test line) and a control line, respectively. Figure 3(a) is a schematic diagram showing the case where the detection part t of the chip 10 shown in Figures 1 and 2 is in this embodiment.

[0043] On the other hand, as described in detail below, according to the method of the present invention, the labeling substance that labels the detection target in the specimen can be accurately identified by increasing the contrast under high resolution using an electron microscope, so that the range of the detection section and the control section can be made smaller than that of the conventional immunochromatography chip that is based on visual confirmation. FIG. 3(b) is a schematic diagram showing an embodiment in which the range in which the capture antibody is fixed is smaller than that of the embodiment of the detection section t shown in FIG. 3(a). That is, in this embodiment, the amount of capture antibody used can be reduced compared to the embodiment shown in FIG. 3(a), and thus the amount of specimen can be reduced. Alternatively, it is also possible to increase the antibody concentration in the detection section t, that is, the amount of antibody per unit area of ​​the detection section t, by fixing the same amount of capture antibody as in the conventional method in a smaller range. Naturally, it is possible to increase the antibody concentration in the detection section t even if the amount of capture antibody used is reduced compared to the conventional method. The same is true for the control section c, although it is not shown in FIG. 3(b). The small range of the detection unit t and the control unit c means that in measurements using an electron microscope, it is easier to select the measurement site (measurement position) of the detection unit t or the control unit c and to align the measurement site (measurement position) with the field of view of the electron microscope, and this is expected to have the effect of improving the efficiency of measurements and shortening the measurement time, etc. Furthermore, by making the range of the detection unit t and the control unit c smaller, it is also possible to miniaturize the chip 10.

[0044] Furthermore, it should be noted that in the chip 10 that can be used in the method of the present invention, the immobilization form of the capture antibody and the control antibody is not limited to a continuous line shape. Specifically, the capture antibody and the control antibody may be immobilized parallel to the short side of the carrier 4 of the chip 10 at an interval. FIG. 4(a) is a schematic diagram showing the case where the detection part t of the chip 10 shown in FIG. 1 and FIG. 2 is in this embodiment. FIG. 4(a) shows an embodiment in which the capture antibody is immobilized in two lines, and the region where these capture antibodies exist is the detection part t, in other words, an embodiment in which the detection part t is composed of two lines to which the capture antibody is immobilized. In this embodiment, the same effect as that described with reference to FIG. 3(b) can be obtained. In addition, the capture antibody may be immobilized in a range smaller than that shown in FIG. 4(a), as shown in FIG. 4(b), as long as the size is large enough to visually distinguish the presence or absence of color development due to accumulation of the labeling substance.

[0045] Furthermore, the fact that the capture antibodies and the control antibodies can be arranged (immobilized) at intervals on the immunochromatography chip also means that multiple types of capture antibodies and control antibodies can be used. That is, in one embodiment of the chip 10 that can be used in the method of the present invention, multiple types of capture antibodies are immobilized at intervals in the detection section t of the carrier 4, and multiple types of control antibodies are immobilized at intervals in the control section c of the carrier 4. In this embodiment, the multiple types of capture antibodies and the multiple types of control antibodies may be immobilized in an array parallel to the short side of the carrier 4 of the chip 10, or may be immobilized shifted upstream or downstream in the development direction D for each type.

[0046] 5(a) and (b) are schematic diagrams showing the above-mentioned embodiment of the detection unit t of the chip 10 shown in FIG. 1 and FIG. 2. In the embodiment shown in FIG. 5(a), two kinds of capture antibodies (capture antibodies 1 and 2) are fixed in a line shape, and the capture antibodies 1 and 2 are fixed in parallel in a direction perpendicular to the development direction D. The region where the capture antibody 1 exists is the detection unit t1, the region where the capture antibody 2 exists is the detection unit t2, and the detection units t1 and t2 constitute the detection unit t. In the embodiment shown in FIG. 5(b), two kinds of capture antibodies (capture antibodies 1 and 2) are fixed in a line shape, and the capture antibody 2 is fixed shifted downstream of the capture antibody 1 in the development direction D. The region where the capture antibody 1 exists is the detection unit t1, the region where the capture antibody 2 exists is the detection unit t2, and the detection units t1 and t2 constitute the detection unit t. Although the embodiment shown in FIG. 5(a) and (b) shows two kinds of capture antibodies, it is also possible to fix three or more kinds of capture antibodies on a single chip. According to the chip 10 having the embodiment illustrated in Figures 5(a) and (b), multiple types of detection targets can be detected from a single sample by immunochromatography, and the labeling substances that label the detection targets can be identified and quantified using an electron microscope.

[0047] 6(a) and (b) are schematic diagrams showing the embodiment shown in FIG. 5(a) and (b) in which capture antibodies 1 and 2 are immobilized at an interval. In the embodiment shown in FIG. 6(a), two types of capture antibodies (capture antibodies 1 and 2) are immobilized in two lines, and the capture antibodies 1 and 2 are immobilized in parallel in a direction perpendicular to the development direction D. The region where the capture antibody 1 exists is the detection section t1, and the region where the capture antibody 2 exists is the detection section t2, and the detection sections t1 and t2 constitute the detection section t. In the embodiment shown in FIG. 6(b), two types of capture antibodies (capture antibodies 1 and 2) are immobilized in two lines, and the capture antibody 2 is immobilized shifted downstream of the capture antibody 1 in the development direction D. The region where the capture antibody 1 exists is the detection section t1, and the region where the capture antibody 2 exists is the detection section t2, and the detection sections t1 and t2 constitute the detection section t. 6(a) and (b) show an embodiment in which two types of capture antibodies are used, but it is also possible to fix three or more types of capture antibodies on a single chip. According to the chip 10 of the embodiment shown in FIG. 6(a) and (b), multiple types of detection targets can be detected from a single sample by immunochromatography, and the labeling substances that label the detection targets can be identified and quantified using an electron microscope.

[0048] As a method for arranging the capture antibody and the control antibody at intervals on the carrier 4, for example, a method of applying minute (fine) droplets on the order of fL (femtoliter) or pL (picoliter) can be applied. More specifically, for example, a method of applying droplets by attaching them to the outer periphery of an application needle, a method of applying by ejecting a jet flow from the tip of a nozzle using electrostatic force, etc. can be used.

[0049] Furthermore, the chip 10 that can be used in the method of the present invention can be configured to have a detection unit dedicated to measurement by electron microscope, separate from the detection unit for visual inspection. Specific embodiments of the immunochromatography chip of the present invention (hereinafter, also simply referred to as the "chip of the present invention") will be described below with reference to Figures 7(a) and (b) and Figures 8(a) and (b).

[0050] In the embodiment shown in FIG. 7(a), two detection units tE dedicated to measurement by electron microscope are arranged in a line on both sides of the visual detection unit tV (the vertical direction of the paper surface of FIG. 7(a)), and capture antibodies are fixed to the detection units tV and tE, respectively. In FIG. 7(a), the detection unit tV is approximately rectangular, but the shape is not limited to this as long as it is large enough to visually distinguish the presence or absence of color development due to accumulation of the labeling substance. The same applies to FIG. 7(b), FIG. 8(a) and (b) described later.

[0051] In addition, from the viewpoint of ensuring the accuracy of the measurement by the electron microscope, the number of the detection units tE is preferably 2 or more, and more preferably 3 or more. In other words, even if the number of the detection units tE is 3 or less in the chip of the present invention, it is possible to ensure the accuracy of the measurement by the electron microscope.

[0052] In addition, in FIG. 7(a), the line of the detection unit tE is shown with a constant size for ease of understanding, but the size of the detection unit tE may be smaller than that of the detection unit tV. That is, in the chip of the present invention, since the arrangement of the detection unit tV and the detection unit tE is known in advance, in the first step, a simple screening is performed by visually confirming the color development derived from the labeling substance that labels the detection target in the detection unit tV, and then, in the second step, a two-step detection is possible in which the number of the labeling substances is measured by an electron microscope in the detection unit tE to measure and quantify the number of the labeling substances. In other words, in the chip of the present invention, since a high-magnification and high-resolution image is obtained under the measurement conditions of the electron microscope in the detection unit tE, it is sufficient that the detection unit tV can confirm the presence or absence of color development derived from the labeling substance. According to the chip of the present invention having such a configuration, not only can the same effect as that described with reference to FIG. 3(b) be obtained, but also the amount of capture antibody used can be reduced compared to the embodiment shown in FIG. 3(b), so that even greater effects can be expected.

[0053] In the embodiment shown in Fig. 7(b), three detection units tE dedicated to measurement by an electron microscope are arranged in a line at intervals upstream of the visual detection unit tV, and a capture antibody is fixed to each of the detection units tV and tE. As described above with reference to Fig. 7(a), the size of the detection unit tE may be smaller than the detection unit tV, so that when visually checking the presence or absence of color development derived from the labeling substance in the detection unit tV, the effect of the detection unit tE being arranged upstream of the detection unit tV can be ignored.

[0054] In the embodiment shown in Fig. 8(a), two detection units tE dedicated to measurement by an electron microscope are arranged in a line with a space between them, and detection units tV for visual inspection are arranged on both sides of the detection unit tE (in the vertical direction of the paper in Fig. 8(a)), and capture antibodies are fixed to the detection units tV and tE, respectively. Fig. 8(b) shows an embodiment in which the detection unit tE is a continuous line in the embodiment shown in Fig. 8(a).

[0055] The above-mentioned embodiments shown in Fig. 7(b), Fig. 8(a) and (b) can also provide the same effect as that described with reference to Fig. 7(a). It should be noted that the embodiments shown in Fig. 7(a) and (b) and Fig. 8(a) and (b) are intended to illustrate specific embodiments of the chip of the present invention, and are not intended to be limited to a specific embodiment. Although not shown in these drawings, the embodiment of the control part c is not particularly limited, and may be for visual inspection as described above with respect to the detection part t, or may be dedicated to measurement by an electron microscope. In an embodiment in which the control part c is for visual inspection, there is an advantage in that the detection part t can be subjected to measurement by an electron microscope after visually confirming the color development of the control part c due to the development of the sample. On the other hand, in an embodiment in which the control part c is dedicated to measurement by an electron microscope, there is an advantage in that the step of visually confirming the presence or absence of color development due to the accumulation of the labeling substance can be omitted, and the results of immunochromatography can be quantified quickly and more easily by subjecting both the detection part t and the control part c to measurement by an electron microscope.

[0056] Furthermore, in another embodiment of the chip of the present invention, it is possible to configure the chip to have only a detection unit dedicated to measurement by an electron microscope, i.e., in this embodiment, the chip of the present invention does not have a detection unit (such as a detection unit for visual inspection) other than the detection unit dedicated to measurement by an electron microscope.

[0057] 9(a) and (b) are diagrams showing the chip of the present invention in the above-mentioned embodiment. In the embodiment shown in FIG. 9(a), the detection units tE dedicated to measurement by an electron microscope are arranged in a continuous line shape, and these detection units tE constitute the detection unit t. In the embodiment shown in FIG. 9(b), different types of capture antibodies are fixed in the detection units tE1 and tE2 dedicated to measurement by an electron microscope, and the detection units tE1 and tE2 constitute the detection unit t. In addition, in FIG. 9(a) and (b), for ease of understanding, the detection units tE, tE1, and tE2 constituting the detection unit t are shown by solid lines, and a part of them is shown in an enlarged view on the right side. In addition, in FIG. 9(a) and (b), the detection units tE, tE1, and tE2 are in a continuous line shape, but may be in a line shape with a space between them. In addition, although not shown in FIG. 9(a) and (b), the embodiment of the control unit c is not particularly limited, and may be for visual observation as described above, or may be for measurement by an electron microscope only. In particular, by dedicating the control section c to measurement by electron microscopy, the step of visually checking for the presence or absence of color development due to accumulation of the labeling substance can be omitted, and by subjecting both the detection section t and the control section c to measurement by electron microscopy, the results of immunochromatography can be quantified quickly and more easily, which is preferable.

[0058] [Application of auxiliary liquid] In the method of the present invention, the timing of applying the auxiliary liquid is not particularly limited. For example, in the method of the present invention, the specimen and the auxiliary liquid can be applied simultaneously. More specifically, the specimen and the auxiliary liquid may be dropped simultaneously onto the specimen pad 2 of the chip 10. In this case, the auxiliary liquid may be dropped onto the specimen pad 2 immediately after the specimen is dropped onto the specimen pad 2, or the auxiliary liquid may be dropped onto the specimen pad 2 after the specimen is dropped onto the specimen pad 2 and before the color development of the control part c of the carrier 4 is visually confirmed by the development of the specimen. That is, in this specification, "simultaneous" with respect to the application of the specimen and the auxiliary liquid means that the application of the auxiliary liquid is performed between the time when the specimen is dropped onto the specimen pad 2 of the chip 10 and before the color development of the control part c of the carrier 4 is visually confirmed.

[0059] In addition, in the method of the present invention, the auxiliary liquid can be developed after the specimen is developed. More specifically, the specimen is dropped onto the specimen pad 2 of the chip 10, and the auxiliary liquid may be dropped onto the specimen pad 2 after the color development of the control part c of the carrier 4 due to the development of the specimen is visually confirmed. Alternatively, the auxiliary liquid may be applied to the detection part t of the carrier 4 after the specimen is dropped onto the specimen pad 2 of the chip 10, and the color development of the control part c of the carrier 4 due to the development of the specimen is visually confirmed. When applying the auxiliary liquid to the detection part t of the carrier 4, the application means of the auxiliary liquid is not particularly limited, but it is desirable to apply the auxiliary liquid without directly touching the detection part t of the carrier 4. For example, a commonly used pipetter may be used, or an application device capable of controlling the amount of droplets may be used. In addition, when a transparent film or the like is applied to the upper surface of the carrier 4, the auxiliary liquid is applied in a state in which the film or the like is removed.

[0060] In the method of the present invention, the auxiliary liquid may be applied before the development of the specimen. In one aspect of this embodiment, the auxiliary liquid is dropped onto the specimen pad 2 of the chip 10, and the auxiliary liquid is allowed to stand for a predetermined time to develop, after which the specimen is dropped onto the specimen pad 2. Alternatively, in another aspect of this embodiment, the auxiliary liquid is applied in advance to the detection unit t of the chip 10, and the specimen is dropped onto the specimen pad 2 after it has been dried. The above embodiment may be combined with the above-mentioned embodiment in which the specimen and the auxiliary liquid are applied simultaneously, or the embodiment in which the auxiliary liquid is developed after the specimen is developed.

[0061] [Measurement by electron microscope] The immunochromatography chip to which the auxiliary liquid for immunochromatography measurement has been applied as described above is subjected to measurement by an electron microscope. Specifically, the detection part t of the carrier 4 of the chip 10 is subjected to measurement by an electron microscope. At this time, the detection part t (i.e., the material constituting the carrier, for example, a nitrocellulose membrane) and the complex of the detection target in the sample and the labeled antibody captured by the capture antibody fixed to the detection part t (i.e., an antigen-antibody reactant composed of the capture antibody-detection target-labeled antibody) are in a state in which the auxiliary liquid has been applied, in other words, the detection part t and the antigen-antibody reactant are in a state in which they are exposed to the auxiliary liquid.

[0062] Although the detailed mechanism by which the application of the auxiliary liquid improves the clarity of an electron microscope image (contributes to the clarity of an image) is not necessarily clear, the present inventors believe that, under the measurement conditions using an electron microscope, when the detection unit t and the above-mentioned antigen-antibody reactant are exposed to the auxiliary liquid and the detection unit t is irradiated with an electron beam, a thin film is formed on the surface of the detection unit t and the antigen-antibody reactant, and electrical conductivity is imparted to the detection unit t (nitrocellulose membrane) and the entire area of ​​the antigen-antibody reactant. This disperses the energy of the electron beam irradiated to the detection unit t, suppresses the charging (charge-up) of the antigen-antibody reactant and the generation of heat, and also retains the moisture contained in the nitrocellulose membrane and the antigen-antibody reactant. As a result, in the electron microscope image, the contrast between the background carrier (cellulose fiber of the nitrocellulose membrane) and the target detection target becomes clear, and the labeling substance of the detection target (for example, gold nanoparticles, platinum-gold nanoparticles, etc.) can be identified as a clear (sharply outlined) particle. Then, by measuring the number of these labeling substances, the results of immunochromatography can be quantified in a short time and easily.

[0063] The number of labeling substances can be measured visually. This will be specifically demonstrated in the examples described later. The number of labeling substances can also be measured by an image recognition system using machine learning or deep learning, and an automatic analysis system can be used to identify the labeling substances of the detection target in the image using artificial intelligence (AI). It is also possible to identify the detection target by combining a scanning electron microscope (SEM) or a transmission electron microscope (TEM) with energy dispersive X-ray spectroscopy (EDX) and using EDX to identify the labeling substances of the detection target in the image.

[0064] <Immunochromatography Assay Kit> The immunochromatography assay kit of the present invention (hereinafter also simply referred to as "the kit of the present invention") comprises an immunochromatography assay auxiliary liquid as a component.

[0065] In the kit of the present invention, the auxiliary liquid may be the same as that described above in relation to the method of the present invention. The characteristics and specific composition of the auxiliary liquid are as described above, and therefore detailed description thereof will be omitted.

[0066] In one embodiment of the kit of the present invention, a liquid for preparing a specimen (specimen extract) may be included as a component separate from the auxiliary liquid. That is, in this embodiment, a user can prepare a specimen using the specimen extract included in the kit of the present invention, and then apply the auxiliary liquid to perform measurement using an electron microscope.

[0067] The kit of the present invention may further include an immunochromatography chip as a component in addition to the auxiliary liquid. The immunochromatography chip that can be used in the kit of the present invention is not particularly limited as long as it is a chip that can be used for normal immunochromatography. For example, the chip 10 described with reference to Figures 1 and 2 in relation to the method of the present invention can be used. The immunochromatography chip may also be the chip of the present invention described with reference to Figures 7(a) and (b), Figures 8(a) and (b), and Figures 9(a) and (b). The configuration of the chip 10 and the chip of the present invention is as described above, so detailed description will be omitted.

[0068] The kit of the present invention may include, as a component, an instruction manual for using the above-mentioned auxiliary liquid. The instruction manual may include, but is not limited to, a package insert, a package label, a package insert ...

[0069] Hereinafter, the embodiments of the present invention will be described in more detail based on examples. However, the scope of the present invention should not be construed as being limited by the following examples. EXAMPLES

[0070] [Example of measurement using influenza virus antigen detection kit] Using a commercially available influenza A and B virus antigen detection kit (Towns), the substance to be detected was measured by immunochromatography according to the following conditions and procedures.

[0071] With the cooperation of the Laboratory Department of the Hamamatsu University School of Medicine Hospital, a national university corporation, the measurement was performed on two sample kits that had been used in actual tests and were positive for influenza A virus antigens. These sample kits were in a state where the specimens (liquid samples prepared from patients' throat swabs) were chromatographed and the color development of the detection area (A line) for influenza A virus antigen and the control area (C line) could be visually confirmed.

[0072] An aqueous solution containing 1 weight percent polysorbate 20 as an auxiliary liquid (1% aqueous solution of Tween 20) was dropped onto the specimen pad of one of the sample kits, and after chromatographic development, the A line was subjected to measurement by SEM (Example 1). For the other sample kit, no auxiliary liquid was applied, and the A line was subjected to measurement by SEM as it was (Comparative Example 1). For SEM, a HITACHI tabletop microscope Miniscope (registered trademark) TM4000Plus was used.

[0073] FIG. 10(a) is an SEM image of line A of the sample kit of Example 1, and FIG. 10(b) is an SEM image of line A of the sample kit of Comparative Example 1.

[0074] In the sample kit of Example 1 shown in FIG. 10(a), the contrast between the background carrier (cellulose fibers of the nitrocellulose membrane) and the labeled substance (platinum-gold nanoparticles) of the labeled antibody bound to the influenza virus is clear, and the platinum-gold nanoparticles can be confirmed as clear (sharply outlined) particles. The outline of the cellulose fibers in the background can also be clearly confirmed. This suggests that not only the antigen-antibody reaction product formed on the A line, but also the auxiliary liquid has been applied to the carrier, and more specifically, that the auxiliary liquid has passed through the A line (detection section) and developed chromatographically, causing the impurities contained in the nitrocellulose membrane constituting the carrier to flow downstream together with the auxiliary liquid, in other words, that the nitrocellulose membrane of the carrier has been washed by the auxiliary liquid.

[0075] On the other hand, in the sample kit of Comparative Example 1 shown in Fig. 10(b), a bubble-like structural change occurs in the cellulose fiber part in the background, making it difficult to distinguish the platinum-gold nanoparticles that are the labeling substance for influenza viruses. It is noteworthy that in actual SEM measurements, the electron beam is continuously irradiated from the electron source, so the structural change in the cellulose fiber part described above also occurs continuously, making it more difficult to identify and count the platinum-gold nanoparticles that are the labeling substance than in the still image shown in Fig. 10(b).

[0076] These results demonstrate that the method of the present invention makes it possible to identify the labeling substance that labels the target substance in a sample at high magnification and high resolution using an electron microscope, and that the results of immunochromatography can be quantified quickly and easily by measuring the number of labeling substances.

[0077] Next, with the cooperation of the Department of Laboratory Medicine, Hamamatsu University School of Medicine, National University Corporation, 197 sample kits used in actual tests were measured. In these sample kits, the specimen (sample liquid prepared from the patient's throat swab) was chromatographed, and the color of the control part (C line) of the carrier was visually confirmed. An aqueous solution containing 1 weight percent of polysorbate 20 as an auxiliary liquid (1% aqueous solution of Tween 20) was dropped onto the sample pad of each sample kit, and after chromatographic development, the detection parts of influenza A and B virus antigens (A line and B line, respectively) and the background part (any part of the carrier except A line, B line, and C line) were measured by SEM.

[0078] The instruments, measurement conditions and criteria used in the above measurements are as follows: [Equipment and measurement conditions] Scanning electron microscope: HITACHI Tabletop Microscope Miniscope(registered trademark) TM4000Plus Voltage: 10kV, mode 3 or 4 ·Measurement magnification: 1000~1200x Shooting mode: Reflected electron mode [Judgment criteria] First, six fields of view are randomly selected from the background area, the number of platinum-gold nanoparticles in each field of view is counted, and the average number per field of view is calculated. Next, randomly select six fields from lines A and B, count the number of platinum-gold nanoparticles in each field, and calculate the average value per field. If the average value is 5 or more, it is judged to be positive, and the following t-test is performed. The average value of the A line and / or B line determined to be positive above is compared with the average value obtained for the background area, and a p-value of p<0.01 by t-test is determined to be “positive.”

[0079] For the measurement sites (measurement positions) of the A line and the B line, the site where the upstream boundary line of the C line is located in the center of the field of view was set as the reference (position zero), and the site 3.8 to 3.9 mm upstream from position zero was set as the B line measurement site, and the site 6.8 to 6.9 mm upstream from this position zero was set as the A line measurement site. Note that platinum-gold nanoparticles may also be detected in cellulose fibers other than each line (background signal), so as a preliminary experiment, the frequency of this background signal was compared upstream of the A line measurement site, between the A line measurement site and the B line measurement site, and between the B line measurement site and position zero, and the results were almost the same in all cases. Therefore, it was decided to select an arbitrary point from these parts as the measurement site of the background part.

[0080] FIG. 11(a) is an example of an SEM image of a detection area (test line) that was determined to be positive based on the above measurement conditions and criteria. In FIG. 11(a), the bright particles are platinum-gold nanoparticles. As shown in FIG. 11(a), the contrast between the platinum-gold nanoparticles and the cellulose fibers is clear, so the number of platinum-gold nanoparticles can be counted visually. FIG. 11(b) is an example of an SEM image of a background area. As shown in FIG. 11(b), the outline of the cellulose fibers is clear, so the presence or absence of platinum-gold nanoparticles in the background area can be clearly determined.

[0081] As a result of measuring a total of 197 sample kits as described above, in the conventional qualitative judgment by visual inspection, 25 cases / 197 cases (12.7%) were positive for line A, and 0 cases / 197 cases (0%) were positive for line B. In contrast, in the quantitative judgment by visual identification of the labeling substance using an electron microscope, 31 cases / 197 cases (15.7%) were positive for line A, and 2 cases / 197 cases (1.0%) were positive for line B. In comparison with rRT-PCR, in the high sensitivity area with a threshold (Ct value) of 30≦Ct≦38 where the amount of virus is low, 2 / 7 (28.6%) were positive for the naked eye, and 5 / 7 (71.4%) were positive for the electron microscope, achieving overwhelming high sensitivity. These results show that the method of the present invention can measure the detection target with higher sensitivity than the conventional qualitative judgment by visual inspection, and more accurate positive judgment is possible. [Industrial Applicability]

[0082] According to the present invention, the results of immunochromatography can be quantified easily and in a short time, and the accuracy can be improved significantly compared to the quantification achieved by combining with densitometry that has been developed so far. In addition, the instability of the conventional method involving a sensitization reaction using silver or the like can be overcome, and more accurate and highly sensitive measurement results can be obtained. In particular, since the method of the present invention does not require a sensitization reaction, it is advantageous in that artifacts of the sensitization reaction can be reliably excluded. Furthermore, compared with the conventional PCR method and ELISA method, the measurement results can be obtained more accurately in a short time. [Explanation of symbols]

[0083] 10 Immunochromatography chip 2 Sample Pads 3 Conjugate Pad 4. Carrier 5 Absorbent Pads D Deployment direction t, t1, t2 detector tV, tV1, tV2 Visual detection section tE, tE1, tE2 Dedicated detector for measurement using electron microscope c Control section

Claims

1. A method for immunochromatography, comprising the steps of: applying an auxiliary liquid other than a sample to the immunochromatography; and then performing measurement using an electron microscope; The method according to any one of claims 1 to 5, wherein the auxiliary liquid has a property of polymerizing to form a film under conditions for measurement using an electron microscope.

2. 2. The method according to claim 1, wherein the auxiliary liquid has electrical conductivity to prevent the generation of static electricity and heat, which contribute to image clarity under conditions of measurement using an electron microscope.

3. 3. The method according to claim 1, which is an immunochromatography method using a labeled antibody carrying metal nanoparticles as a labeling substance and a capture antibody having the property of binding to a complex of the labeled antibody and the substance to be detected.

4. The method of claim 3, wherein the capture antibodies are immobilized at intervals on an immunochromatography chip.

5. The method according to any one of claims 1 to 4, wherein the sample and the auxiliary liquid are applied simultaneously.

6. The method according to any one of claims 1 to 4, wherein the auxiliary liquid is developed after development of the sample.

7. The method according to any one of claims 1 to 6, wherein the detection target is identified by identifying a labeling substance in an electron microscope image using artificial intelligence.

8. The method according to any one of claims 1 to 6, wherein the detection target is identified by identifying a labeling substance in an electron microscope image using energy dispersive X-ray spectroscopy (EDX).

9. An auxiliary liquid for immunochromatography measurement that improves image clarity in the measurement of an immunochromatography chip using an electron microscope, the auxiliary liquid having the property of polymerizing to form a film under measurement conditions using the electron microscope.

10. 10. The auxiliary liquid according to claim 9, which has a property of being conductive under measurement conditions using an electron microscope.

11. The auxiliary liquid according to claim 9 or 10, comprising as essential components glycerin, and at least one compound selected from the group consisting of polyethylene glycol, polyvinyl alcohol, triglyceride, polyresorcinol, polyphenol, tannic acid, urushiol and saponin; polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80 and polysorbate 85, and nonionic surfactants.

12. An immunochromatography chip for use in the method according to any one of claims 1 to 8, comprising a detection unit dedicated to measurement by an electron microscope.

13. The immunochromatography chip described in claim 12, wherein a labeled antibody carrying metal nanoparticles as a labeling substance is fixed at a predetermined position, and a capture antibody having the property of binding to a complex of the labeled antibody and the object to be detected is fixed to a detection section dedicated to measurement by electron microscope.

14. The immunochromatography chip according to claim 13, wherein the capture antibodies are immobilized at intervals.

15. The immunochromatography chip according to any one of claims 12 to 14, further comprising a visual detection unit.

16. The immunochromatography chip according to any one of claims 12 to 14, which does not include a detection unit other than the detection unit dedicated to measurement by an electron microscope.

17. An immunochromatography assay kit comprising the auxiliary liquid according to any one of claims 9 to 11 as a component.

18. The kit according to claim 17, further comprising the immunochromatography chip according to any one of claims 12 to 16 as a component.

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