Immunochromatographic specimens and immunochromatographic kits

Abstract

[Problem] To provide an immunochromatographic test strip that can detect an N protein of SARS-CoV-2 at high sensitivity while suppressing false positives. [Solution] An immunochromatographic test strip according to the present invention comprises (1) a sample pad, (2) a conjugation pad which supports a composite of fine colored cellulose particles and an antibody composition A specifically binding to a nucleocapsid protein (N protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a measurement sample, (3) a membrane to which an antibody composition B specifically binding to the N protein of SARS-CoV-2 in the measurement sample and an antibody composition C specifically binding to the antibody composition A are linearly fixed at respective positions that differ from each other, and (4) an absorption pad, wherein the antibody composition A is a mixture of an antibody A1 and an antibody A2 that are from different origins, and the antibody composition B is a mixture of an antibody B1 and an antibody B2 that are from different origins.

Description

Technical Field

[0001] The present invention relates to an immunochromatographic test strip capable of detecting the nucleocapsid protein (N protein) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) with high sensitivity and suppressing false positives, and a kit containing the immunochromatographic test strip.

Background Art

[0002] SARS-CoV-2 is the causative virus of Coronavirus Disease 2019 (COVID-19) and has been rapidly spreading worldwide since early 2020. Similar to common coronaviruses, SARS-CoV-2 is composed of a nucleocapsid and an envelope surrounding the nucleocapsid. The nucleocapsid contains a viral genome (RNA) and a nucleocapsid protein (N protein) that binds to the viral genome. The envelope contains lipids and spike protein (S protein), membrane protein (M protein), and envelope protein (E protein) that bind to the lipids.

[0003] The N protein is a protein involved in the formation of the virus core, as well as the packaging and transcription of the viral genome, and has a structure in which an N-terminal domain (NTD) and a C-terminal domain (CTD) are bound via a linker having a serine (S) / arginine (R)-rich region. Non-Patent Document 1 describes that the N protein is phosphorylated in large amounts, and the mapping of phosphorylation sites is shown. Since the amino acid sequence of the N protein is conserved among strains, the N protein is used as a diagnostic marker and the like.

[0004] Among the various methods for diagnosing infectious diseases, immunochromatography is one of the most widely used techniques due to its rapid, simple, and inexpensive nature. Immunochromatography is an immunoassay method that utilizes capillary action and is widely used globally for influenza testing and other applications. One method for detecting target substances using immunochromatography is the sandwich method, which utilizes an antigen-antibody reaction. The sandwich method uses two antibodies with different epitopes for the target substance. One antibody is used as a detection antibody sensitized with detection particles such as gold colloid, colored latex particles, or fluorescent particles. The other antibody forms a test line as a capture antibody linearly immobilized on the surface of a porous support. In addition, an antibody that specifically captures the detection antibody is linearly immobilized on the surface of the porous support at a different location from the test line to form a control line. The target substance contained in the sample unfolds from one end (upstream side) of the porous support, moves while forming an immune complex with the detection antibody, and comes into contact with the capture antibody on the test line, where it is captured and colored. Free detection particles that did not form an immune complex with the target substance, along with the sensitized detection antibody, pass through the test line and are captured by the antibody in the control line, causing color development. By visually checking the intensity of these color developments, the presence or absence of the target substance can be determined.

[0005] Generally, immunoassay methods using mouse monoclonal antibodies are used to measure analytes (target substances) contained in samples such as blood and urine. However, in immunoassay methods based on the specific binding of antigen-antibody reactions, the reliability of the measurement values ​​is often compromised by non-specific reactions other than the intended specific antigen-antibody reaction.

[0006] This phenomenon is caused by the reaction of components other than the antigen in the sample with the labeled antibody. Heterophilic antibodies, such as human anti-mouse antibodies (hereinafter referred to as HAMA), present in the sample being tested can cause nonspecific crosslinking between the antibody bound to the solid phase and the labeled antibody to be detected, even in the absence of the analyte, for example, in a standard sandwich ELISA measurement, resulting in a false positive signal. While automation of testing has enabled rapid measurement, false reactions like HAMA have increased, and these nonspecific reactions are often overlooked. Thus, nonspecific reactions have been observed in immunoassay systems, and by reacting even with samples that do not contain the target analyte, samples that should be negative have been judged as positive (see Non-Patent Documents 2 and 3). This phenomenon is also observed in immunochromatography.

[0007] One reason for the presence of HAMA in human-derived samples is the administration of large amounts of mouse monoclonal antibodies to patients as a treatment. The administration of mouse antibodies into the body produces HAMA, and the resulting immune response to heterologous antigens is a problem. In recent years, advances in antibody drugs, such as chimeric antibodies that fuse mouse-derived antigen-binding sites with human-derived constant regions, and advances in humanized antibody production technology have reduced the occurrence of HAMA in the body. However, the number of patients with HAMA continues to increase, and this problem cannot be completely ignored.

[0008] Heterophilic antibodies are known not only against mice, but also against animals such as goats, sheep, and rabbits (goat: HAGA, sheep: HASA, rabbit: HARA).

[0009] If the binding of monoclonal antibodies used for measurement to heterophile antibodies is not prevented, inaccurate measurement results can lead to serious problems such as misdiagnosis. A major problem is that even when monoclonal antibodies with excellent specificity are used in actual measurement systems, their performance cannot be fully utilized (see Patent Document 1).

[0010] Various attempts have been made to obtain accurate measurements while avoiding the nonspecific reactions described above. For example, it has been common practice to pre-treat the sample to be measured by heating or with appropriate reagents, or to add various animal serums, immunoglobulin fractions, albumin, skim milk, surfactants, etc., to the measurement system. In addition, nonspecific reactions caused by the Fc site of the antibody have been avoided by using antibody fragments such as Fab or F(ab')2 in specific reactions (see Patent Documents 2 and 3). Furthermore, monoclonal antibodies that have different reaction specificity from the monoclonal antibody used in the measurement system and do not inhibit the reactions involved in the measurement system have also been added to the measurement system. For example, the use of aggregates derived from monoclonal or polyclonal antibodies has been proposed. These aggregates may be homopolymers of the antibody, antibody fragments, or heteropolymers with macromolecules such as proteins like albumin or polysaccharides like dextran.

[0011] Furthermore, to suppress non-specific reactions, monoclonal antibody-derived substances have been disclosed that are prepared by heat treatment or other methods of the monoclonal antibody used in the specific reaction, thereby losing the specific activity of the original antibody but retaining the activity to suppress non-specific reactions (see Patent Document 4). However, although these methods have some effect in suppressing non-specific reactions, their effect is still insufficient for some samples, and they may also partially inhibit the target antigen-antibody reaction, so they are not always satisfactory in practical use. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] Patent No. 2109086 [Patent Document 2] Japanese Unexamined Patent Publication No. 54-119292 [Patent Document 3] Japanese Patent Application Publication No. 04-221762 [Patent Document 4] Patent No. 2561134 [Non-patent literature]

[0013] [Non-Patent Document 1] Klann et al., Molecular Cell, 80(1), 2020, pp.164-174 [Non-Patent Document 2] Clin. Chem. 34 / 1,261-264(1988) [Non-Patent Document 3] Clin. Chem. 35 / 1,146-151(1989) [Overview of the project] [Problems that the invention aims to solve]

[0014] The present invention aims to provide an immunochromatographic test strip and a kit containing the immunochromatographic test strip that can detect the SARS-CoV-2 N protein with high sensitivity and suppression of false positives. [Means for solving the problem]

[0015] The inventors of the present invention conducted intensive research to solve the above problems and found that by using a mixture of two antibodies of different origins as the capture antibody and detection antibody, the N protein of SARS-CoV-2 can be detected with high sensitivity and with reduced false positives. Furthermore, they found that by using cellulose-based colored microparticles as detection particles, the N protein of SARS-CoV-2 can be detected with even higher sensitivity, thus completing the present invention.

[0016] In other words, a typical example of the present invention is as follows: 1. (1) Sample pad and, (2) A conjugation pad carrying a complex of antibody composition A, which specifically binds to the nucleocapsid protein (N protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the measurement sample, and cellulose-based colored microparticles, (3) An antibody composition B that specifically binds to the N protein of SARS-CoV-2 in a measurement sample, and an antibody composition C that specifically binds to the antibody composition A are linearly fixed at different positions on a membrane; (4) It consists of an absorption pad; The antibody composition A is a mixture of different antibodies A1 and A2; The immunochromatographic test strip is characterized in that the antibody composition B is a mixture of different antibodies B1 and B2. 2. The immunochromatographic test strip according to 1., wherein any one of the antibodies A1 and A2 is an antibody derived from a mouse. 3. The immunochromatographic test strip according to 1. or 2., wherein the antibodies A1 and A2 are carried on the conjugation pad at a mixing ratio (mass ratio) of 10:1 to 1:10. 4. The immunochromatographic test strip according to any one of 1. to 3., wherein any one of the antibodies B1 and B2 is an antibody derived from a mouse. 5. The immunochromatographic test strip according to any one of 1. to 4., wherein the antibodies B1 and B2 are linearly fixed on the membrane at a mixing ratio (mass ratio) of 10:1 to 1:10. 6. The immunochromatographic test strip according to any one of 1. to 3., wherein the antibody composition C is a mixture of an antibody C1 that binds to the antibody A1 and an antibody C2 that binds to the antibody A2. 7. The immunochromatographic test strip according to 6., wherein the antibodies C1 and C2 are linearly fixed on the membrane at a mixing ratio (mass ratio) of 10:1 to 1:10. 8. An immunochromatographic kit characterized by comprising the immunochromatographic test strip according to any one of 1. to 7., a measurement sample sampler, a filter, and a measurement sample diluent.

Advantages of the Invention

[0017] Since the immunochromatographic test strip of the present invention carries a specific antibody and detection particles in a specific arrangement, it can detect the N protein of SARS-CoV-2 with high sensitivity and low false positives.

Brief Description of the Drawings

[0018] [Figure 1] It is a diagram (top view) showing an example of the immunochromatographic test strip of the present invention. [Figure 2] It is a diagram (side view) showing an example of the immunochromatographic test strip of the present invention.

Embodiments for Carrying Out the Invention

[0019] In the present invention, the immunochromatographic test strip is a test strip for detecting the nucleocapsid protein (N protein) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a measurement sample. The measurement sample used in the present invention includes not only the sample as it is collected, but also the sample subjected to pretreatment such as removal of impurities.

[0020] The measurement sample is not particularly limited, and examples include blood, serum, plasma, bone marrow fluid, lymph fluid, tears, nasal discharge, nasal lavage fluid, nasal swab, saliva, gargle, sputum, throat swab, sweat, tracheal aspirate, bronchial lavage fluid, pleural effusion, ascites, amniotic fluid, intestinal lavage fluid, urine, feces, cell extract, tissue extract, organ extract, etc.

[0021] In the present invention, the N protein of SARS-CoV-2 has the amino acid sequence disclosed in GenBank accession number (MN908947). The N protein of SARS-CoV has the amino acid sequence disclosed in GenBank accession number (AY278741).

[0022] In the present invention, the structure of the immunochromatographic test specimen is such that the addition section (dropping section) for the measurement sample solution of the immunochromatographic test specimen is on the upstream side, and the sample pad having the addition section, the conjugation pad, the membrane, and the absorbent pad are arranged in that order in sequence. Next, an example of the immunochromatographic test specimen of the present invention will be described with reference to the drawings. In Figures 1 and 2, 1 is the sample pad, 2 is the conjugation pad, 3 is the membrane, 4 is the absorbent pad, 5 is the backing sheet, 6 is the test line, 7 is the control line, and 8 is the adhesive sheet.

[0023] In the examples shown in Figures 1 and 2, the immunochromatographic test strips are in the form of elongated strips with a width of 3 to 5 mm (preferably about 4 mm) and a length of 40 to 100 mm (preferably about 60 mm). The conjugation pad 2 of the immunochromatographic test strip supports a complex of antibody composition A, which specifically captures the N protein of SARS-CoV-2 in the sample, and cellulose-based colored microparticles. A test line 6 is formed approximately 15 mm from the upstream end of the membrane 3 of the immunochromatographic test strip, with antibody composition B fixed in a linear fashion to specifically capture the N protein of SARS-CoV-2 in the sample. A control line 7 is formed approximately 20 mm from the aforementioned end, with antibody composition C, which specifically binds to antibody composition A, fixed in a linear fashion.

[0024] In the present invention, the sample pad 1 is not particularly limited as long as it is made of a material that can quickly absorb the sample to be measured and then spread to the downstream conjugation pad, membrane, and absorption pad. Examples include cellulose filter paper or nonwoven fabric, glass filter paper or nonwoven fabric, polyester filter paper or nonwoven fabric, and polyethylene filter paper or nonwoven fabric. Among these, cellulose filter paper is preferred. The thickness of the sample pad 1 is preferably 0.1 to 2 mm, and more preferably 0.2 to 1 mm. If the thickness is too small, the flow of the sample to be measured downstream may become uneven, which may reduce the measurement accuracy. On the other hand, if the thickness is too large, the spread downstream may be slower, which may increase the measurement time. Also, the amount of sample to be measured required for downstream spread will increase.

[0025] In the present invention, the conjugation pad 2 is not particularly limited as long as it is made of a material that can hold a complex of antibody composition A, which specifically binds to the N protein of SARS-CoV-2 in the measurement sample, and cellulose-based colored fine particles in a dry state, and can rapidly release the complex as the measurement sample is deployed downstream. Examples include cellulose filter paper or nonwoven fabric, glass filter paper or nonwoven fabric, polyester filter paper or nonwoven fabric, and polyethylene filter paper or nonwoven fabric. Among these, glass filter paper is preferred. The thickness of the conjugation pad 2 is preferably 0.1 to 2 mm, and more preferably 0.2 to 1 mm. If the thickness is too small, it may not be possible to hold the desired amount of the complex in a dry state. On the other hand, if the thickness is too large, the deployment downstream may be slower, resulting in a longer measurement time. Also, the amount of measurement sample required for downstream deployment will be larger.

[0026] In the present invention, the membrane 3 is not particularly limited as long as it can accurately and uniformly spread the sample being measured, but examples include membranes made of cellulose, cellulose derivatives, nitrocellulose, cellulose acetate, polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, or nylon. Among these, a nitrocellulose membrane is preferred.

[0027] In the present invention, the absorbent pad 4 is not particularly limited as long as it is made of a material that can quickly absorb the measurement sample that has been deployed from upstream and hold it in place so that it does not flow back. Examples include cellulose filter paper or nonwoven fabric, glass filter paper or nonwoven fabric, polyester filter paper or nonwoven fabric, and polyethylene filter paper or nonwoven fabric. Among these, cellulose filter paper is preferred. The thickness of the absorbent pad 4 is preferably 0.2 to 5 mm, and more preferably 0.5 to 2 mm. If the thickness is too small, depending on the amount of measurement sample dropped, the measurement sample that has been absorbed into the absorbent pad may flow back to the membrane side. On the other hand, if the thickness is too large, the size of the immunochromatographic test piece and the housing case covering the immunochromatographic test piece will also be large, which is undesirable from the viewpoint of POCT.

[0028] The antibody used in this invention may be either a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferred. The antibody can be any isotype, such as IgG, IgA, IgD, IgE, IgM, etc., but IgG is preferred. The antibody may be a commercially available product or may be manufactured by other known methods.

[0029] The antibody composition A used as the detection antibody in this invention must be a mixture of antibodies A1 and A2 of different origins that specifically bind to the N protein of SARS-CoV-2. Antibodies of different origins have different three-dimensional structures because they bind to different sugar chains. Therefore, they have different reactivity to the antigen, resulting in a higher degree of antibody binding to the antigen and higher sensitivity compared to antibodies used alone. In particular, the way the sugar chains are attached to mouse-derived antibodies is presumed to result in a structure that binds to the antigen more highly than others. Therefore, it is preferable that either antibody A1 or antibody A2 is derived from a mouse. If antibody composition A is not a mixture of antibodies A1 and A2 of different origins, the detection sensitivity may decrease. In addition, false positives may occur.

[0030] The mixing ratio (mass ratio) of the antibodies A1 and A2 from different origins is preferably 10:1 to 1:10, more preferably 8:1 to 1:8, and even more preferably 5:1 to 1:5. If the mixing ratio (mass ratio) exceeds this range, the detection sensitivity may decrease, and false positives may occur.

[0031] The reflectance absorbance of the test line is preferably 40 mAbs or higher, more preferably 60 mAbs or higher, and even more preferably 80 mAbs or higher, because it provides good visibility upon completion of the measurement and allows the line to be seen early from the start of the measurement.

[0032] Cellulose-based colored fine particles have a large number of hydroxyl groups, allowing them to covalently retain many reactive dyes and maintain stable dispersibility in water and other liquids even after intense dyeing. As the cellulose-based colored fine particles, regenerated cellulose, purified cellulose, natural cellulose, etc., can be used, or partially derivatized cellulose may be used. Preferably, 20 to 90% by mass of the cellulose-based colored fine particles are derived from cellulose, more preferably 20 to 80% by mass, and even more preferably 20 to 70% by mass.

[0033] The average particle size of the cellulose-based colored microparticles is not particularly limited, but is preferably 100 nm to 1000 nm, and more preferably 200 nm to 800 nm. If the average particle size is large, downstream spread will be slower, which may increase the measurement time. Also, the particles will be more easily captured on the membrane, and the background itself may become colored, making the color development on the test line and control line unclear. On the other hand, if the average particle size is small, the amount of antibody that can be physically adsorbed or chemically bound will decrease, which may reduce the measurement sensitivity.

[0034] The color of the cellulose-based colored microparticles is not particularly limited, but examples include red, blue, yellow, green, black, white, and fluorescent colors. Among these, red, blue, and black are preferred due to their good visibility. An example of such cellulose-based colored microparticles is colored cellulose nanobeads (NanoAct®) manufactured by Asahi Kasei Corporation.

[0035] The amount of antibody composition A bound to the cellulose-based colored microparticles can be controlled by adjusting the charging mass ratio of the cellulose-based colored microparticles and antibody composition A, and is not particularly limited, but the charging mass ratio of the cellulose-based colored microparticles and antibody composition A is preferably 1:0.01 to 1:1, more preferably 1:0.02 to 1:0.5, and even more preferably 1:0.02 to 1:0.2. If the mass ratio falls outside the above range, the amount of antibody composition A bound to the cellulose-based colored microparticles may be insufficient, or the amount of antibody composition A bound to the cellulose-based colored microparticles may increase too much, resulting in an increase in antibody composition A that does not contribute to the antigen-antibody reaction, which may reduce the measurement sensitivity.

[0036] The method of binding antibody composition A to cellulose-based colored microparticles is not particularly limited, but sensitization by physical adsorption via hydrophobic bonds or chemical bonding via covalent bonds is preferred, with physical adsorption being more preferred because it is simple to operate and inexpensive. In order to improve sensitization efficiency, reactive active groups may be introduced into the cellulose-based colored microparticles. The reactive active groups are not particularly limited, but examples include carboxyl groups, amino groups, aldehyde groups, thiol groups, epoxy groups, and hydroxyl groups. Among these, carboxyl groups and amino groups are preferred. In the case of carboxyl groups, a carbodiimide can be used to form a covalent bond with the amino group of the ligand.

[0037] The method for loading the complex of antibody composition A, which specifically binds to the N protein of SARS-CoV-2 in the measurement sample, and cellulose-based colored microparticles onto the conjugation pad 2 is not particularly limited, but for example, it can be prepared by uniformly coating, spraying, or impregnating the conjugation pad with a solution of the complex, and then drying it in a constant temperature bath at a suitable temperature for a certain period of time. The amount of the solution of the complex to be coated is not particularly limited, but 5 μL to 50 μL per 1 cm of line length is preferred. The concentration of cellulose-based colored microparticles in the solution of the complex is not particularly limited, but 0.01 to 0.5% by mass is preferred, 0.02 to 0.2% by mass is more preferred, and 0.02 to 0.1% by mass is even more preferred. If the concentration is too low, the N protein of SARS-CoV-2 may not be sufficiently captured and detected, and the measurement sensitivity may decrease. On the other hand, even if the concentration is too high, no improvement in measurement sensitivity is observed, and only the cost increases. Next, the coated conjugation pad is dried. The drying temperature is not particularly limited, but is preferably between 20°C and 80°C, and more preferably between 20°C and 60°C. The drying time varies depending on the drying temperature, but is usually between 5 and 120 minutes.

[0038] In this invention, antibody composition B used as the capture antibody forming test line 6 must be a mixture of antibodies B1 and B2 of different origins that specifically bind to the N protein of SARS-CoV-2. Antibodies of different origins have different three-dimensional structures because they bind to different sugar chains. Therefore, they have different reactivity to the antigen, increasing the degree of antibody binding to the antigen and improving sensitivity. In particular, the way the sugar chains are attached to mouse-derived antibodies is presumed to result in a structure that binds to the antigen more highly than others. Therefore, it is preferable that either antibody B1 or antibody B2 is a mouse-derived antibody. If antibody composition B is not a mixture of antibodies B1 and B2 of different origins, the detection sensitivity may decrease. Also, false positives may occur.

[0039] The mixing ratio (mass ratio) of the antibodies B1 and B2 of different origins is preferably 10:1 to 1:10, more preferably 8:1 to 1:8, and even more preferably 5:1 to 1:5. If the mixing ratio (mass ratio) exceeds this range, the detection sensitivity may decrease, and false positives may occur.

[0040] In the present invention, antibody composition C used as the capture antibody that forms the control line 7 must be an antibody that specifically binds to antibody composition A. Furthermore, it is preferable that antibody composition C is a mixture of antibody C1 that binds to antibody A1 and antibody C2 that binds to antibody A2. If antibody composition C is not a mixture of antibody C1 and antibody C2, the control line may become fainter, which may lead to the conclusion that a remeasurement is necessary.

[0041] The mixing ratio (mass ratio) of antibody C1 and antibody C2 is preferably 10:1 to 1:10, more preferably 8:1 to 1:8, and even more preferably 5:1 to 1:5. If the mixing ratio (mass ratio) exceeds this range, the control line may become fainter, which may lead to the need for remeasurement.

[0042] The method for linearly immobilizing the capture antibody forming the test line 6 and the capture antibody forming the control line 7 on the membrane 3 is not particularly limited, but for example, it can be prepared by coating a certain amount of the capture antibody forming the test line and the capture antibody forming the control line at different positions along the line, and then drying them in a constant temperature bath at a suitable temperature for a certain period of time. The amount of each capture antibody coated is not particularly limited, but 0.1 μL to 2 μL per 1 cm of line length is preferred. The coating concentration of each capture antibody is not particularly limited, but 0.1 mg / mL to 10 mg / mL is preferred, 0.2 mg / mL to 8 mg / mL is more preferred, and 0.5 mg / mL to 5 mg / mL is even more preferred. If the concentration is too low, the N protein of SARS-CoV-2 may not be sufficiently captured and detected, and the measurement sensitivity may decrease. On the other hand, even if the concentration is too high, no improvement in measurement sensitivity is observed, and only the cost increases. Next, the membrane 3 after coating is dried. The drying temperature is not particularly limited, but 20°C to 80°C is preferred, and 20°C to 60°C is more preferred. Drying time varies depending on the drying temperature, but is usually between 5 and 120 minutes.

[0043] The immunochromatographic test specimen of the present invention can be prepared by attaching the prepared membrane 3 to the center of an adhesive sheet 8, then attaching the conjugation pad 2 partially overlapping one end of the membrane 3, then attaching the sample pad 1 partially overlapping the end of the conjugation pad 2 opposite to the overlap with the membrane 3, and then attaching the absorption pad 4 partially overlapping the other end of the membrane 3, and finally cutting it into strips of a certain width. The test line 6 and control line 7 may be prepared after the test specimen is prepared or before the test specimen is prepared.

[0044] The immunochromatographic test specimen may be housed in a suitable plastic housing case having at least a first opening for dropping the sample onto the sample pad 1, and a second opening for visually confirming the test line 6 and control line 7 on the membrane 3.

[0045] In the present invention, the immunochromatographic measurement kit preferably includes, in addition to the immunochromatographic test piece, a sample collection device for collecting the measurement sample, a sample diluent for pretreatment and / or dilution of the measurement sample, and a filter for filtering the measurement sample.

[0046] The sample diluent preferably contains a nonionic surfactant that improves the spreadability of the sample and does not affect the immunoassay. Examples of nonionic surfactants are not particularly limited, but include polyoxyethylene alkylphenyl ethers (Triton® surfactants, etc.), polyoxyethylene alkyl ethers (Brij® surfactants, etc.), polyoxyethylene sorbitan fatty acid esters (Tween® surfactants, etc.), polyoxyethylene fatty acid esters, sorbitan fatty acid esters, alkyl glucosides, sucrose fatty acid esters, and the like. Furthermore, the surfactant may be used alone or in combination of two or more. The concentration of the nonionic surfactant is preferably 0.01% to 5.0% by mass, more preferably 0.05% to 4.0% by mass, and even more preferably 0.1% to 3.0% by mass. If the concentration is too low, downstream spread may become difficult. Also, uneven spread may occur, reducing measurement accuracy. On the other hand, at high concentrations, the physically adsorbed detection particles and the antibody, and / or the membrane and the antibody, may separate, making it impossible to obtain measurement results.

[0047] The sample diluent may contain inorganic salts or buffers used for pH adjustment. Any type of buffer may be used as long as it has sufficient buffering capacity in the target pH range. Examples include Tris, phosphoric acid, phthalic acid, citric acid, maleic acid, succinic acid, oxalic acid, boric acid, tartaric acid, acetic acid, carbonic acid, and Good's Buffers (MES, ADA, PIPES, ACES, coramine hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycinamide, bicine). Among these, Tris, phosphoric acid, MES, PIPES, TES, and HEPES are preferred, and Tris, phosphoric acid, and PIPES are more preferred, for reasons such as having sufficient buffering capacity around 7.0, which is the optimal pH range for the antibody used in the present invention. [Examples]

[0048] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0049] (Example 1) (1) Preparation of complexes of antibody A1, antibody A2 and cellulose-based colored microparticles 100 μL of 1.0% by mass of cellulose-based colored microparticles (NanoAct®, BL2: Dark Navy, average particle size 365 nm, manufactured by Asahi Kasei Corporation), 900 μL of 10 mM Tris buffer (204-07885, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (pH 8.0), 50 μL of mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-101, manufactured by Toyobo Co., Ltd.) at a concentration of 1.0 mg / mL as antibody A1, and rabbit-derived anti-SARS-CoV-2 N protein monoclonal antibody (SARS-CoV-2 (COVID-19) nucleocapsid antibody, [HL5511], Cat) at a concentration of 1.0 mg / mL as antibody A2. 50 μL of No. GTX635689 (manufactured by GeneTex) was added to a 15 mL centrifuge tube and mixed by vortex. Then, it was allowed to stand at 37°C for 120 minutes. Next, 12 mL of a blocking solution (pH 8.0) consisting of 1.0% by mass casein (030-01505, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 100 mM borate buffer (021-02195, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and it was allowed to stand at 37°C for another 60 minutes. Then, using a centrifuge (MX-307, manufactured by Tommy Seiko Co., Ltd.), centrifugation was performed at 13,000 × g for 15 minutes at 25°C to settle the antibody-sensitized cellulose-based colored microparticles, after which the supernatant was removed. Next, 12 mL of a washing solution (pH 10.0) consisting of 50 mM borate buffer (021-02195, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was treated with an ultrasonic disperser (UH-50, manufactured by SMT Corporation) for 10 seconds. Then, a centrifuge (MX-307, manufactured by Tommy Seiko Co., Ltd.) was used to centrifuge at 13,000 × g at 25°C for 15 minutes, after which the antibody-sensitized cellulose-based colored microparticles were settled, and the supernatant was removed. Next, 2.0 mL of a coating solution (pH 9.2) consisting of 15% by mass sucrose (196-00015, Fujifilm Wako Pure Chemical Industries, Ltd.), 0.2% by mass casein (030-01505, Fujifilm Wako Pure Chemical Industries, Ltd.), and 62 mM borate buffer (021-02195, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the mixture was treated with an ultrasonic disperser (UH-50, SMT Corporation) for 10 seconds to obtain complexes of antibodies A1 and A2 with cellulose-based colored microparticles.

[0050] (2) Preparation of a membrane card for detecting the N protein of SARS-CoV-2 A 2.0 mg / mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-100, manufactured by Toyobo Co., Ltd.) was used as antibody B1, and a 2.0 mg / mL rabbit-derived anti-SARS-CoV-2 N protein monoclonal antibody (SARS-CoV-2 (COVID-19) nucleocapsid antibody, [HL146], Cat No. GTX635680, manufactured by GeneTex) was used as antibody B2, and these were mixed in a 1:1 mass ratio. Next, 1.0 mg / mL of rabbit-derived anti-mouse IgG polyclonal antibody (Mouse IgG-heavy and light chain antibody, A90-117A, manufactured by BETHYL) was used as antibody C1, and 1.0 mg / mL of goat-derived anti-rabbit IgG polyclonal antibody (Rabbit IgG-heavy and light chain antibody, A120-101A, manufactured by BETHYL) was used as antibody C2, and these were mixed in a 1:1 mass ratio. Next, a 60mm x 300mm membrane card (Hi-Flow Plus 120 Membrane Cards, HF120, Millipore) consisting of a 20mm x 300mm adhesive tape section on the upstream side, a 25mm x 300mm membrane section in the center, and a 15mm x 300mm adhesive tape section on the downstream side was used. Using a dispensing platform (XYZ3060, BIODOT) and a Bio Jet nozzle (BHQHR-XYZ, BIODOT), a mixture of antibody B1 and antibody B2 was applied at a rate of 1.0 μL / cm². The mixture was then dried for 30 minutes in a dryer (WFO-510, Tokyo Rikakikai Co., Ltd.) adjusted to 45°C to form a test line with a line width of approximately 1 mm.Furthermore, a membrane card for detecting the SARS-CoV-2 N protein was obtained by applying a mixture of antibodies C1 and C2 at a rate of 1.0 μL / cm using a dispensing platform (XYZ3060, BIODOT) and a Bio Jet nozzle (BHQHR-XYZ, BIODOT) 15 mm upstream from the membrane portion of the membrane card on which the test line had been formed. The mixture was then dried for 30 minutes in a dryer (WFO-510, Tokyo Rikakikai Co., Ltd.) adjusted to 45°C to form a control line with a line width of approximately 1 mm.

[0051] (3) Preparation of a conjugation pad for detecting the N protein of SARS-CoV-2 A 10mm x 300mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, Millipore) was uniformly coated with a complex of antibody A1, antibody A2, and cellulose-based colored microparticles at a rate of 15 μL / cm using a dispensing platform (XYZ3060, BIODOT) and an Air Jet nozzle (AJQHR-XYZ, BIODOT). The pad was then dried for 30 minutes in a dryer (WFO-510, Tokyo Rikakikai Co., Ltd.) set to 45°C to obtain a conjugation pad for detecting the SARS-CoV-2 N protein.

[0052] (4) Preparation of immunochromatographic test strips and devices for detecting SARS-CoV-2 N protein A SARS-CoV-2 N protein detection conjugation pad was attached to the 20mm x 300mm adhesive tape on the upstream side of the SARS-CoV-2 N protein detection membrane card, overlapping it by 2mm with the membrane. Next, a 15mm x 300mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, Millipore) was attached further upstream, overlapping it by 3mm with the conjugation pad. Then, a 20mm x 300mm absorbent pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, Millipore) was attached to the 15mm x 300mm adhesive tape on the downstream side of the SARS-CoV-2 N protein detection membrane card, overlapping it by 5mm with the membrane. Next, using a guillotine-type cutting module (CM5000, BIODOT), the material was cut into strips 4 mm wide and 60 mm long to obtain an immunochromatographic test piece for detecting the SARS-CoV-2 N protein. The obtained immunochromatographic test piece for detecting the SARS-CoV-2 N protein was then placed in a housing case (K007, Shengfeng Plastic) to obtain the SARS-CoV-2 N protein detection immunochromatographic device 1.

[0053] (5) Preparation of sample diluent A sample dilution solution was prepared by adding 1 L of 100 mM Tris buffer (204-07885, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (pH 8.5), 8.77 g of sodium chloride (191-01665, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 2 g of polyoxyethylene (20) sorbitan monolaurate: Tween® 20 (166-21213, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 9 g of polyoxyethylene (10) octylphenyl ether: TritonX®-100 (160-24751, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to a glass bottle and dissolving the mixture.

[0054] (6) Preparation of positive samples Positive samples were obtained by diluting Recombinant SARS-CoV-2 Nucleocapsid protein (230-30164, RayBiotech), a commercially available SARS-CoV-2 N protein, to 1 ng / mL with the aforementioned sample diluent.

[0055] (7) Preparation of negative samples A negative sample was obtained by diluting a commercially available HAMA (3PH490, manufactured by Scantibodies Laboratory) to 100 ng / mL with the aforementioned sample diluent.

[0056] (8) Evaluation of immunochromatographic specimens for SARS-CoV-2 N protein detection: Evaluation of non-specific adsorption The immunochromatographic device for detecting the SARS-CoV-2 N protein was placed on a horizontal stand. Next, 100 μL of the sample diluent was dispensed using a micropipette and gently dropped onto the sample pad, where it was allowed to stand at 25°C for 15 minutes. Then, the reflectance absorbance (mAbs) of the test line on the membrane was measured using an immunochromatograph reader (C10060-10, measurement mode: Latex, Line, Hamamatsu Photonics). The evaluation results are shown in Table 1. Non-specific adsorption of the SARS-CoV-2 N protein in the immunochromatographic device for detection in Example 1 was confirmed to be acceptable, as the reflectance absorbance of the test line was <10 mAbs.

[0057] (9) Evaluation of immunochromatographic test strips for SARS-CoV-2 N protein detection: Sensitivity evaluation The immunochromatographic device for detecting the SARS-CoV-2 N protein was placed on a horizontal platform. Next, 100 μL of the positive sample was dispensed using a micropipette and gently dropped onto a sample pad, where it was allowed to stand at 25°C for 15 minutes. Subsequently, the reflectance absorbance (mAbs) of the control line and test line on the membrane was measured using an immunochromatograph reader (C10060-10, measurement mode: Latex, Line, Hamamatsu Photonics). The evaluation results are shown in Table 2. The immunochromatographic device for detecting the SARS-CoV-2 N protein in Example 1 demonstrated high-sensitivity measurement capabilities.

[0058] (10) Evaluation of immunochromatographic test strips for SARS-CoV-2 N protein detection: Evaluation of false positives The immunochromatographic device for detecting the SARS-CoV-2 N protein was placed on a horizontal platform. Next, 100 μL of the negative sample was dispensed using a micropipette and gently dropped onto a sample pad, where it was allowed to stand at 25°C for 15 minutes. Subsequently, the reflectance absorbance (mAbs) of the test line on the membrane was measured using an immunochromatograph reader (C10060-10, measurement mode: Latex, Line, Hamamatsu Photonics). The evaluation results are shown in Table 3. The immunochromatographic device for detecting the SARS-CoV-2 N protein in Example 1 suppressed false positives and enabled highly specific measurements.

[0059] (Example 2) An immunochromatographic device 2 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that a rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1137, EastCoast Bio) was used as antibody A2, and a goat-derived anti-rat IgG polyclonal antibody (Rat IgG-heavy and light chain antibody, A110-105A, BETHYL) was used as antibody C2. The results of the evaluation of (8) non-specific adsorption of the obtained device are shown in Table 1, the results of the evaluation of (9) sensitivity are shown in Table 2, and the results of the evaluation of (10) false positives are shown in Table 3.

[0060] (Example 3) An immunochromatographic device 3 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that a rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1138, EastCoast Bio) was used as antibody B2. The results of the evaluation of (8) non-specific adsorption of the obtained device are shown in Table 1, the results of the evaluation of (9) sensitivity are shown in Table 2, and the results of the evaluation of (10) false positives are shown in Table 3.

[0061] (Example 4) An immunochromatographic device 4 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that a rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1137, EastCoast Bio) was used as antibody A2, a rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1138, EastCoast Bio) was used as antibody B2, and a goat-derived anti-rat IgG polyclonal antibody (Rat IgG-heavy and light chain antibody, A110-105A, BETHYL) was used as antibody C2. The results of the evaluation of (8) non-specific adsorption of the obtained device are shown in Table 1, the results of the evaluation of (9) sensitivity are shown in Table 2, and the results of the evaluation of (10) false positives are shown in Table 3.

[0062] (Example 5) An immunochromatographic device 5 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that a rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1137, EastCoast Bio) was used as antibody A1, and a rabbit-derived anti-rat IgG polyclonal antibody (Rat IgG-heavy and light chain antibody, A110-122A, BETHYL) was used as antibody C1. The results of the evaluation of (8) non-specific adsorption of the obtained device are shown in Table 1, the results of the evaluation of (9) sensitivity are shown in Table 2, and the results of the evaluation of (10) false positives are shown in Table 3.

[0063] (Example 6) An immunochromatographic device 6 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that a rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1138, EastCoast Bio) was used as antibody B1. The results of the evaluation of (8) non-specific adsorption of the obtained device are shown in Table 1, the results of the evaluation of (9) sensitivity are shown in Table 2, and the results of the evaluation of (10) false positives are shown in Table 3.

[0064] (Example 7) An immunochromatographic device 7 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that a rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1137, EastCoast Bio) was used as antibody A1, a rat-derived anti-SARS-CoV-2 N protein monoclonal antibody (Coronavirus (COVID-19 NP) Antibody, ECB-HM1138, EastCoast Bio) was used as antibody B1, and a rabbit-derived anti-rat IgG polyclonal antibody (Rat IgG-heavy and light chain antibody, A110-122A, BETHYL) was used as antibody C1. The results of the evaluation of (8) non-specific adsorption of the obtained device are shown in Table 1, the results of the evaluation of (9) sensitivity are shown in Table 2, and the results of the evaluation of (10) false positives are shown in Table 3.

[0065] [Table 1]

[0066] [Table 2]

[0067] [Table 3]

[0068] As shown in Table 1, the non-specific adsorption of the SARS-CoV-2 N protein detection immunochromatographic devices in Examples 2-7 was confirmed to be acceptable, with test line reflectance absorbance <10 mAbs. Furthermore, as shown in Table 2, the sensitivity of the SARS-CoV-2 N protein detection immunochromatographic devices in Examples 2-4 was confirmed to be acceptable, with test line reflectance absorbance >40 mAbs. In addition, as shown in Table 3, the false positives of the SARS-CoV-2 N protein detection immunochromatographic devices in Examples 2-4 were confirmed to be acceptable, with test line reflectance absorbance <10 mAbs.

[0069] (Example 8) An immunochromatographic device 8 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that the concentration of antibody A1 was set to 0.222 mg / mL and the concentration of antibody A2 was set to 1.778 mg / mL. The results of the evaluation of (8) non-specific adsorption of the obtained device are shown in Table 4, the results of the evaluation of (9) sensitivity are shown in Table 5, and the results of the evaluation of (10) false positives are shown in Table 6.

[0070] (Example 9) An immunochromatographic device 9 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that the concentration of antibody A1 was set to 1.818 mg / mL and the concentration of antibody A2 was set to 0.182 mg / mL. The results of the evaluation of (8) non-specific adsorption of the obtained device are shown in Table 4, the results of the evaluation of (9) sensitivity are shown in Table 5, and the results of the evaluation of (10) false positives are shown in Table 6.

[0071] (Example 10) An immunochromatographic device 10 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that the concentration of antibody A1 was set to 1.905 mg / mL and the concentration of antibody A2 was set to 0.095 mg / mL. The results of the evaluation of (8) non-specific adsorption of the obtained device are shown in Table 4, the results of the evaluation of (9) sensitivity are shown in Table 5, and the results of the evaluation of (10) false positives are shown in Table 6.

[0072] (Example 11) An immunochromatographic device 11 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody B1 and antibody B2 were mixed in a mass ratio of 1:8. The results of the evaluation of (8) non-specific adsorption of the obtained device are shown in Table 4, the results of the evaluation of (9) sensitivity are shown in Table 5, and the results of the evaluation of (10) false positives are shown in Table 6.

[0073] (Example 12) An immunochromatographic device 12 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody B1 and antibody B2 were mixed in a mass ratio of 10:1. The results of the evaluation of (8) nonspecific adsorption of the obtained device are shown in Table 4, the results of the evaluation of (9) sensitivity are shown in Table 5, and the results of the evaluation of (10) false positives are shown in Table 6.

[0074] (Example 13) An immunochromatographic device 13 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody B1 and antibody B2 were mixed in a mass ratio of 20:1. The results of the evaluation of (8) nonspecific adsorption of the obtained device are shown in Table 4, the results of the evaluation of (9) sensitivity are shown in Table 5, and the results of the evaluation of (10) false positives are shown in Table 6.

[0075] (Example 14) An immunochromatographic device 14 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody C1 and antibody C2 were mixed in a mass ratio of 1:0. The results of the evaluation of (8) nonspecific adsorption of the obtained device are shown in Table 4, the results of the evaluation of (9) sensitivity are shown in Table 5, and the results of the evaluation of (10) false positives are shown in Table 6.

[0076] (Example 15) An immunochromatographic device 15 for detecting the N protein of S ARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody C1 and antibody C2 were mixed in a mass ratio of 1:8. The results of the evaluation of (8) nonspecific adsorption of the obtained device are shown in Table 4, the results of the evaluation of (9) sensitivity are shown in Table 5, and the results of the evaluation of (10) false positives are shown in Table 6.

[0077] (Example 16) An immunochromatographic device 16 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody C1 and antibody C2 were mixed in a mass ratio of 10:1. The results of the evaluation of (8) nonspecific adsorption of the obtained device are shown in Table 4, the results of the evaluation of (9) sensitivity are shown in Table 5, and the results of the evaluation of (10) false positives are shown in Table 6.

[0078] (Example 17) An immunochromatographic device 17 for detecting the N protein of SARS-CoV-2 was obtained in the same manner as in Example 1, except that antibody C1 and antibody C2 were mixed in a mass ratio of 20:1. The results of the evaluation of (8) non-specific adsorption of the obtained device are shown in Table 4, the results of the evaluation of (9) sensitivity are shown in Table 5, and the results of the evaluation of (10) false positives are shown in Table 6.

[0079] [Table 4]

[0080] [Table 5]

[0081] [Table 6]

[0082] As shown in Table 4, the non-specific adsorption of the SARS-CoV-2 N protein detection immunochromatographic devices in Examples 5-17 was confirmed to be acceptable, with the test line's reflectance absorbance being <10 mAbs. Furthermore, the control line showed higher reflectance absorbance as the C1 and C2 antibody concentrations were closer. Additionally, as shown in Table 5, the sensitivity of the SARS-CoV-2 N protein detection immunochromatographic devices in Examples 5-14 was confirmed to be acceptable, with the test line's reflectance absorbance being >40 mAbs. Finally, as shown in Table 6, the false positives of the SARS-CoV-2 N protein detection immunochromatographic devices in Examples 5-14 were confirmed to be acceptable, with the test line's reflectance absorbance being <10 mAbs.

[0083] (Comparative Example 1) An immunochromatographic device A for detecting the SARS-CoV-2 N protein was obtained in the same manner as in Example 1, except that 100 μL of a 0.5 mg / mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-101, Toyobo Co., Ltd.) was used as antibody A1, and antibody A2 was not used; 1.0 mg / mL of a mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-100, Toyobo Co., Ltd.) was used as antibody B1, and antibody B2 was not used; and 0.5 mg / mL of a rabbit-derived anti-mouse IgG polyclonal antibody (Mouse IgG-heavy and light chain antibody, A90-117A, BETHYL Inc.) was used as antibody C1, and antibody C2 was not used. The results of the evaluation of (8) nonspecific adsorption of the obtained devices are shown in Table 7, the results of the evaluation of (9) sensitivity are shown in Table 8, and the results of the evaluation of (10) false positives are shown in Table 9.

[0084] (Comparative Example 2) An immunochromatographic device B for detecting the SARS-CoV-2 N protein was obtained in the same manner as in Example 1, except that 100 μL of a 0.5 mg / mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody (SCV-101, Toyobo Co., Ltd.) was used as antibody A1, antibody A2 was not used, and a 0.5 mg / mL rabbit-derived anti-mouse IgG polyclonal antibody (Mouse IgG-heavy and light chain antibody, A90-117A, Bethyl) was used as antibody C1, and antibody C2 was not used. The results of the evaluation of (8) non-specific adsorption of the obtained device are shown in Table 7, the results of the evaluation of (9) sensitivity are shown in Table 8, and the results of the evaluation of (10) false positives are shown in Table 9.

[0085] (Comparative Example 3) An immunochromatographic device C for detecting the SARS-CoV-2 N protein was obtained in the same manner as in Example 1, except that a 1.0 mg / mL mouse-derived anti-SARS-CoV-2 N protein monoclonal antibody (Anti-SARS-CoV-2-NP Monoclonal antibody, SCV-100, Toyobo Co., Ltd.) was used as antibody B1, and antibody B2 was not used. The results of the evaluation of (8) non-specific adsorption of the obtained device are shown in Table 7, the results of the evaluation of (9) sensitivity are shown in Table 8, and the results of the evaluation of (10) false positives are shown in Table 9.

[0086] [Table 7]

[0087] [Table 8]

[0088] [Table 9]

[0089] As shown in Table 7, the non-specific adsorption of the SARS-CoV-2 N protein detection immunochromatographic devices in Comparative Examples 1-3 was not problematic, as the reflectance absorbance of the test line was <10 mAbs. Furthermore, as shown in Table 8, the sensitivity of the SARS-CoV-2 N protein detection immunochromatographic devices in Comparative Examples 1-3 was clearly lower than that of Example 1. In addition, as shown in Table 9, false positives occurred in the SARS-CoV-2 N protein detection immunochromatographic devices in Comparative Examples 1-3, confirming their low specificity. [Industrial applicability]

[0090] The present invention provides an immunochromatographic test strip capable of detecting the N protein of SARS-CoV-2 with high sensitivity and suppression of false positives. [Explanation of symbols]

[0091] 1: Sample Pad 2: Conjugation Pad 3: Membrane 4: Absorbent pad 5: Backing sheet 6: Test line 7: Control Line 8: Adhesive sheet

Claims

1. (1) Sample pad and (2) A conjugation pad carrying a complex of antibody composition A, which specifically binds to the nucleocapsid protein (N protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the measurement sample, and cellulose-based colored microparticles, (3) A membrane in which antibody composition B, which specifically binds to the N protein of SARS-CoV-2 in the measurement sample, and antibody composition C, which specifically binds to antibody composition A, are linearly fixed at different positions, (4) Composed of an absorbent pad, The antibody composition A is a mixture of antibodies A1 and A2 of different origins. The immunochromatographic test piece is characterized in that the antibody composition B is a mixture of antibodies B1 and B2 of different origins.

2. The immunochromatographic test piece according to claim 1, characterized in that either antibody A1 or antibody A2 is an antibody derived from a mouse.

3. The immunochromatographic test piece according to claim 1 or 2, characterized in that the antibody A1 and antibody A2 are supported on the conjugation pad in a mixing ratio (mass ratio) of 10:1 to 1:

10.

4. The immunochromatographic test piece according to any one of claims 1 to 3, characterized in that either antibody B1 or antibody B2 is an antibody derived from a mouse.

5. The immunochromatographic test piece according to any one of claims 1 to 4, characterized in that the antibody B1 and antibody B2 are linearly fixed to the membrane in a mixing ratio (mass ratio) of 10:1 to 1:

10.

6. The immunochromatographic test piece according to any one of claims 1 to 3, characterized in that the antibody composition C is a mixture of antibody C1 which binds to antibody A1 and antibody C2 which binds to antibody A2.

7. The immunochromatographic test piece according to claim 6, characterized in that the antibody C1 and antibody C2 are linearly fixed to the membrane in a mixing ratio (mass ratio) of 10:1 to 1:

10.

8. An immunochromatography kit characterized by comprising an immunochromatographic test piece, a sample collection tool, a filter, and a sample diluent according to any one of claims 1 to 7.

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