SARS-CoV-2 detection method and kit for same

Abstract

Disclosed are a high performance anti-SARS-CoV-2 antibody, and a test reagent using the same. The method for detecting the SARS-CoV-2 comprises the step of detecting the SARS-CoV-2 in a test sample by an immunoassay method, wherein the immunoassay method uses a monoclonal antibody or an antigen binding fragment thereof which specifically reacts with an RNA binding domain of an amino acid sequence of an N protein of the SARS-CoV-2. The SARS-CoV-2 detection kit comprises a monoclonal antibody or an antigen binding fragment of the monoclonal antibody, wherein the monoclonal antibody specifically reacts with an RNA binding domain of an N protein of the SARS-CoV-2.

Description

Technical Field

[0001] The present invention relates to a method for detecting SARS-CoV-2 and a kit therefor. Background Art

[0002] SARS-CoV-2 is a virus belonging to the Coronaviridae family, with a single-stranded, positive-sense RNA genome. In March 2020, the infectious disease (COVID-19) caused by SARS-CoV-2 reached pandemic proportions, with a large number of infections reported worldwide. As of 2022, countries are developing vaccines and promoting vaccination globally, but the emergence of variants suggests that the pandemic shows no sign of ending.

[0003] COVID-19 is a respiratory disease, and 80% of patients recover with mild symptoms. However, severe cases can lead to breathing difficulties, requiring oxygen inhalation and ECMO treatment. According to data from the Ministry of Health, Labor, and Welfare's Novel Coronavirus Infection Countermeasures Advisory Committee published on September 7, 2022, the mortality rate for the sixth wave of COVID-19 in Japan was 2.67% for those in their 80s and 4.05% for those in their 90s. Early medical intervention is crucial for those at high risk.

[0004] In the case of COVID-19, SARS-CoV-2 infection is diagnosed using PCR and antigen testing. An immunochromatographic method using anti-SARS-CoV-2 antibodies has been developed as a rapid and convenient method for detecting SARS-CoV-2. However, with no sign of an end to the pandemic, there is a demand for more accurate testing.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: WO2021 / 181994 Summary of the Invention

[0008] Problems to be solved by the invention

[0009] Currently, various test kits using anti-SARS-CoV-2 antibodies are on the market. However, these existing SARS-CoV-2 test kits have insufficient performance when used to detect SARS-CoV-2.

[0010] In view of the above-mentioned current situation, an object of the present invention is to provide high-performance anti-SARS-CoV-2 antibodies and test reagents using the same.

[0011] Means for solving problems

[0012] The inventors of the present application conducted in-depth research and found that, for SARS-CoV-2 contained in the test sample, the performance of detecting SARS-CoV-2 can be improved by using antibodies against the RNA binding domain of the N protein (nucleoprotein, nucleocapsid protein) of SARS-CoV-2, thereby completing the present invention.

[0013] That is, the present invention provides the following solutions.

[0014] (1) A method for detecting SARS-CoV-2, comprising detecting SARS-CoV-2 in a sample by an immunoassay using a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with the RNA binding domain of the amino acid sequence of the N protein of SARS-CoV-2.

[0015] (2) The method according to (1), wherein the monoclonal antibody specifically reacts with a region contained within the 114aa to 170aa region of the amino acid sequence of the N protein of SARS-CoV-2.

[0016] (3) The method according to (2), wherein the monoclonal antibody specifically reacts with a region contained within the 126aa to 165aa region of the amino acid sequence of the N protein of SARS-CoV-2.

[0017] (4) The method according to (1), wherein the immunoassay is an immunochromatographic method.

[0018] (5) A SARS-CoV-2 detection kit comprising a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with the RNA binding domain of the N protein of SARS-CoV-2.

[0019] (6) The detection kit as described in (5), wherein the aforementioned monoclonal antibody specifically reacts with a region contained in the 114aa to 170aa region of the amino acid sequence of the N protein of SARS-CoV-2.

[0020] (7) The detection kit as described in (6), wherein the aforementioned monoclonal antibody specifically reacts with a region contained in the 126aa to 165aa region of the amino acid sequence of the N protein of SARS-CoV-2.

[0021] (8) The detection kit according to any one of (5) to (7), which is an immunochromatographic test piece.

[0022] Effects of the Invention

[0023] The method of the present invention uses an antibody that reacts with the RNA-binding domain of the SARS-CoV-2 N protein in an immunoassay, resulting in high sensitivity. Furthermore, the present invention provides a detection kit for use in the novel detection method of the present invention. DETAILED DESCRIPTION

[0024] The method of the present invention includes detecting SARS-CoV-2 in a sample by an immunoassay using a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with the RNA binding domain of the amino acid sequence of the N protein of SARS-CoV-2. Here, the "RNA binding domain" refers to the region ranging from 41 to 186 (hereinafter, described as "41aa to 186aa" from the N terminus of the N protein. The same applies to others.) The monoclonal antibody used in the present invention specifically reacts with the region contained in the region of 41aa to 186aa. Even within the range of 41aa to 186aa, a monoclonal antibody that specifically reacts with the region contained in the region of 114aa to 170aa is particularly preferred, and a monoclonal antibody that specifically reacts with the region contained in the region of 126aa to 165aa is further preferred. It should be noted that "specific reaction" refers to an antigen-antibody reaction. Therefore, in a system where the protein is mixed with the antibody, the antibody does not react with proteins other than the target protein component in the antigen at a detectable level; or, even if coexisting substances other than the antibody and antigen undergo some binding reaction or association reaction with the antibody and antigen, only a reaction that is significantly weaker than the antigen-antibody reaction between the antibody and the antigen occurs. It should be noted that the amino acid sequence of the N protein of SARS-CoV-2 is shown in sequence number 1. It should be noted that the amino acid sequence shown in sequence number 1 is the amino acid sequence of the wild strain, but the present invention is not limited to wild strains and can also be applied to other strains derived from wild strains.

[0025] Antigen-binding fragments obtained by isolating only the antigen-binding site of the monoclonal antibody used in the method of the present invention can also be used in the method of the present invention. That is, the use of Fab, Fab', F(ab')2, single-chain antibody (scFv), recombinant antibody or modified antibody (e.g., chimeric antibody, humanized antibody, human antibody, CDR-grafted antibody, primatized antibody, deimmunized antibody, synhumanized antibody, dsFv, diabody, minibody, etc.) produced by known methods and binding to a specific region of the SARS-CoV-2 N protein (antigen-binding fragment) is also included in the scope of the present invention. In addition, the type of monoclonal antibody is not limited to IgG and can also be IgM or IgY.

[0026] The monoclonal antibodies used in the method of the present invention can be obtained by immunizing an immunized animal with a polypeptide consisting of a corresponding epitope (a region contained in the RNA binding domain) or a polypeptide, complex or extract containing the epitope using a known immunological method, and using cells from the immunized animal to prepare a hybridoma using conventional methods. Immunogens can also be obtained from viral culture fluids, or by integrating DNA encoding any antigen into a plasmid vector and introducing it into a host cell for expression. Any antigen or its partial peptide as an immunogen can also be expressed in the form of a fusion protein with the protein exemplified below, and used as an immunogen after purification or in an unpurified state. In the preparation of fusion proteins, those skilled in the art can utilize glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (TRX), Nus tags, S tags, HSV tags, FRAG tags, polyhistidine tags, etc., which are commonly used as "protein expression and purification tags". The fusion protein formed with them is preferably used as an immunogen after using a digestive enzyme to cut off any antigen or its partial peptide portion and the tag portion other than this, and is separated and purified.

[0027] Monoclonal antibodies can be easily prepared from immunized animals by the well-known method of Kohler et al. (Kohler et al., Nature, vol. 256, pp. 495-497 (1975)). Specifically, antibody-producing cells such as spleen cells and lymphocytes are recovered from the immunized animal, and these antibody-producing cells are fused with mouse myeloma cells using conventional methods to produce hybridomas. The resulting hybridomas are cloned using limiting dilution methods, and monoclonal antibodies produced by each cloned hybridoma are selected for those that react with the antigen used for animal immunization.

[0028] The purification of monoclonal antibodies from ascites and culture supernatants can utilize known immunoglobulin purification methods. For example, fractionation based on salting out using ammonium sulfate or sodium sulfate, PEG fractionation, ethanol fractionation, DEAE ion exchange chromatography, gel filtration, etc. can be cited. In addition, depending on the type of immunized animal and the type of monoclonal antibody, purification may also be performed by affinity chromatography using a carrier bound to any one of protein A, protein G, and protein L.

[0029] Furthermore, the monoclonal antibodies used in the present invention can be produced by genetically modifying plants and can be produced using a plant transient expression system.

[0030] In addition, the monoclonal antibody used in the present invention can also be obtained using mammalian cells as the form of the gene recombinant of expression host.As mammalian cells in this case, CHO (Chinese hamster ovary cell, Chinese Hamster Ovary) cell, HEK293 (human embryonic kidney cell, Human Embryonic Kidney cells293) cell etc. can be enumerated, but are not limited to this.In addition, about the method obtained with the form of gene recombinant, the transient (transient) expression system of the plasmid vector lacking autonomous replication ability, viral vector, the semi-stable (semi-stable) expression system of the episomal vector (episomal vector) that uses to give nuclear localization signal and has autonomous replication ability and the stable (stable) expression system of the genome of target gene inserted into expression host etc. can be enumerated, are not particularly limited.

[0031] The N protein (nucleoprotein, nucleocapsid protein; hereinafter, in this specification, also referred to as "N protein") of SARS-CoV-2 consists of a 419 amino acid sequence (see SEQ ID NO: 1). As described above, the monoclonal antibodies used in the present invention specifically react with the RNA binding domain in the N protein of SARS-CoV-2. Whether a monoclonal antibody specifically reacts with the RNA binding domain can be determined by immunoassay to investigate whether an antigen-antibody reaction occurs between a polypeptide consisting of an RNA binding domain and the monoclonal antibody.

[0032] The antibodies of the present invention that specifically react with the RNA-binding domain in the anti-SARS-COV-2 N protein are flexible in reactivity and, for example, may recognize the three-dimensional structure formed by the polypeptide.

[0033] In the immunoassay method of the present invention, the determination is performed by immunoassay of the antigen-antibody reaction of the monoclonal antibody or its antigen-binding fragment (hereinafter, in the description before the examples, except for the case where it is clear that it has other meanings based on the context) prepared in the above manner and the antigen in the sample. As the immunoassay method used therefor, any method known to those skilled in the art such as competitive method, agglutination method, Western blotting method, immunostaining method, sandwich method, etc. can be used. It should be noted that in the present invention, "determination" includes any of quantitative, semi-quantitative, and detection.

[0034] As the immunoassay method of the present invention, the sandwich method is preferred. In the sandwich method, a complex is formed by sandwiching an antigen between two antibodies, and the complex is detected. The sandwich method itself is well known in the field of immunoassays and can be performed, for example, using immunochromatography or ELISA. These sandwich methods are well known in themselves, and the method of the present invention can be performed using the aforementioned monoclonal antibody that recognizes the hydrophobic region of the N protein as an antigen.

[0035] In the sandwich method, one or more antibodies (antibodies immobilized on a solid phase and labeled antibodies) that recognize the antigen are used. When two or more antibodies are used, at least one of the two antibodies is a monoclonal antibody that recognizes the above-mentioned specific region in the N protein of anti-SARS-COV-2 as an antigen. Alternatively, the same two antibodies can be used to sandwich the antigen to form a complex. Preferably, one or two antibodies that recognize the peptide are used.

[0036] In the immunoassay based on the sandwich method as the detection principle, as the solid phase for antibody immobilization, any solid phase that can utilize known technology to immobilize the antibody can be used, for example, any known article such as a porous film (membrane), a particulate material, a test tube, a resin plate with capillary action can be selected. In addition, as the material for labeling the antibody, enzymes, radioisotopes, fluorescent substances, luminescent substances, colored particles, colloidal particles, etc. can be used. In the immunoassay utilizing the aforementioned various materials, particularly from the viewpoint of simplicity and rapidity of clinical examination, it is preferred to use an immunochromatographic method as a lateral flow immunoassay utilizing a membrane.

[0037] The present invention also provides a SARS-CoV-2 detection kit, which contains a monoclonal antibody that specifically reacts with the RNA binding domain of the amino acid sequence of the N protein of SARS-CoV-2. As such a kit, an immunochromatographic test piece that can perform immunoassays in a lateral flow manner is particularly preferred. The immunoassay device provided by the present invention comprises: a support having a detection area on which an antibody (antibody 1) that captures the assay object (antigen) is immobilized; a labeling area having a removable labeled antibody (antibody 2); a sample pad for dripping the sample; an absorption band that absorbs the developed sample solution; and a backing sheet for bonding these components together, wherein at least one of antibody 1 and antibody 2 is a monoclonal antibody that specifically reacts with the above-mentioned specific area in the N protein of anti-SARS-COV-2 of the present invention. The immunoassay device is also referred to as an immunochromatographic test piece. It should be noted that the support having a detection area immobilized with an antibody (antibody 1) that captures the assay object (antigen) and the removable labeled antibody (antibody 2) can use a variety of monoclonal antibodies. In addition, polyclonal antibodies can also be combined. It should be noted that the kit may further include a brochure, a sample collection device, and the like.

[0038] The support is a material with the performance of the antibody immobilization for capturing the detected substance (antigen), and has the performance that does not hinder the passage of liquid in the horizontal direction. It is preferably a porous film with capillary action, which is a material that can transport liquid and the components dispersed therein by absorption. The material forming the support is not particularly limited, and for example, cellulose, nitrocellulose, cellulose acetate, polyvinylidene fluoride (PVDF), glass fiber, nylon, polyketone, etc. can be cited. Wherein, nitrocellulose is more preferably used to make a film. The film immobilized with the antibody is referred to as an antibody immobilized membrane.

[0039] The labeled region is formed from a porous substrate containing the labeled antibody. Commonly used materials for the substrate include glass fiber and nonwoven fabrics. To allow for the impregnation of a large amount of labeled antibody, the substrate is preferably in the form of a pad with a thickness of approximately 0.3 mm to 0.6 mm. A porous substrate impregnated with labeled antibody and dried is also referred to as a dry pad.

[0040] The labeling of labeled antibodies mostly uses enzymes such as alkaline phosphatase and horseradish peroxidase, metal colloids such as gold colloids, silica particles, cellulose particles, colored polystyrene particles and colored latex particles. When using colored particles such as metal colloid particles, colored polystyrene particles and colored latex particles, since these labeling reagents produce coloration due to aggregation, the coloration is measured. The particles immobilized with the antibody are referred to as antibody-immobilized particles. The immobilized amount of the antibody is not particularly limited, and the presence of several ng to tens of μg in the labeled region is sufficient.

[0041] The detection region refers to a portion of the support where antibodies that capture the substance to be detected (antigen) are immobilized. The detection region is provided with at least one region where antibodies that capture the antigen are immobilized. The detection region only needs to be contained within the support, and the antibodies can be immobilized on the support. The amount of immobilized antibody is not particularly limited, and a few ng to tens of μg of antibody can be immobilized within the detection region.

[0042] The sample pad is a porous material used to drip the sample. It is located at the uppermost part of the immunoassay instrument. Typical materials used for this pad include filter paper, fiberglass, and non-woven fabric. To accommodate large quantities of sample for immunoassays, a pad with a thickness of approximately 0.3 mm to 1 mm is preferred. Samples prepared using the sample are also included, such as those obtained by suspending the sample in another solution.

[0043] The absorbent tape is used to absorb components supplied to the support but not involved in the reaction in the detection area. This material can be made of commonly used natural or synthetic polymers, such as filter paper or sponge with high water retention. However, to facilitate sample development, highly absorbent materials are preferred.

[0044] The base plate is a component to which all the aforementioned materials, namely the support, specimen pad, marker area, absorbent tape, etc., are partially overlapped and fixed. As long as these materials are arranged and fixed at optimal intervals, a base plate is not essential, but it is generally preferred for ease of manufacture and use.

[0045] The immunoassay device of the present invention may further include a control display area (component). The control display area is a portion that indicates that the test has been correctly performed. For example, the control display area is located downstream of the detection area, and when the test sample passes through the detection area and reaches the control display area, a signal is emitted by coloring, etc. The control display area may be pre-immobilized with a substance that binds to an antibody bound to a labeled carrier, or may be pre-immobilized with a pH indicator or other reagent that changes color when the test sample arrives. In the case where the antibody bound to the labeled carrier is a mouse monoclonal antibody, an anti-mouse IgG antibody may be used.

[0046] The size of the immunoassay instrument is not limited, and may be, for example, several centimeters to several dozen centimeters in length and several millimeters to several centimeters in width.

[0047] The immunoassay device of the present invention can be stored in a storage container, which can prevent degradation caused by, for example, ultraviolet light or air humidity. Furthermore, when using contaminating or infectious test samples, the storage container can prevent contamination or infection of the test subject. For example, a resin housing of an appropriately sized storage container can be used as the storage container, and the device of the present invention can be housed within the housing. The storage container and the immunochromatographic test strip housed therein are sometimes collectively referred to as an immunoassay device.

[0048] In the method of the present invention using an immunoassay device, capillary action is utilized to cause a complex of an antibody 2 capable of binding to a target substance (labeled reagent) labeled with an appropriate labeling substance, such as colored polystyrene particles or gold colloid, and the target substance to develop and migrate within a solid support immobilized with antibody 1. As a result, a complex of the immobilized substance, target substance, and labeled reagent forms on the solid support. The target substance can be detected by detecting the labeled reagent signal emitted by this complex (in the case of gold colloid, the portion of the solid support immobilized with the target substance-binding substance turns red). This immunoassay method can be performed at 5 to 35°C, preferably at room temperature.

[0049] The number of detection regions and the type of labeled antibodies contained in the marker region are not limited to one. By using a plurality of antibodies corresponding to the analyte, two or more antigens can be detected using the same immunoassay instrument.

[0050] The method of the present invention can detect whether a person is infected with SARS-CoV-2. If N protein is detected in a test sample, it can be determined that the person is infected with SARS-CoV-2.

[0051] When the antibody of the present invention that specifically reacts with the RNA binding domain in the N protein is used, SARS-CoV-2 can be specifically recognized. The antibody that recognizes the RNA binding domain in the N protein does not recognize other viruses, such as adenovirus, coxsackievirus, echovirus, herpes simplex virus, human metapneumovirus, influenza virus, measles virus, mumps virus, parainfluenza virus, RS virus (respiratory syncytial virus infection), etc., and will not cause false detection of these viruses.

[0052] Regarding the sample to be tested, as a sample, for example, there can be mentioned: body fluids such as blood, serum, plasma, urine, semen, cerebrospinal fluid, saliva, sweat, tears, ascites or amniotic fluid of humans or animals; mucus; feces; organs such as blood vessels or liver; tissues; cells or their extracts, etc., which may contain SARS-CoV-2 proteins, preferably cells and secretions from the oral cavity, tonsils, nasal cavity, pharynx, larynx, trachea, bronchi or lungs, etc., which are easy to collect, nasal swabs, throat swabs, mouthwashes, sputum, tracheal aspirates, bronchoalveolar lavage fluid, saliva, etc. The method for collecting these samples is not particularly limited, and known methods can be used. Specifically, a method using a cotton swab can be mentioned.

[0053] The present invention will be described in more detail below based on examples. However, the present invention is not limited to the following examples.

[0054] Example

[0055] Example 1 Preparation of Monoclonal Antibodies Recognizing the N Protein of SARS-CoV-2

[0056] 1. Preparation of SARS-CoV-2 N protein antigen

[0057] The DNA encoding the SARS-CoV-2N protein was expressed in Escherichia coli using an expression vector, and the protein was purified after several days of culture, and the resulting product was used.

[0058] 2. Preparation of monoclonal antibodies against SARS-CoV-2N protein

[0059] BALB / c mice were immunized with the SARS-CoV-2 N protein antigen described in 1. After a period of feeding, the iliac lymph nodes were removed from the mice, and the "mouse iliac lymph node method" (Sado Y et al., Acta Histochem. Cytochem. 39: 89-94 (2006)) was used to obtain multiple hybridoma cell lines that produced anti-SARS-CoV-2 N protein antibodies.

[0060] The obtained cell line was intraperitoneally administered to pristane-treated BALB / c mice. Approximately two weeks later, ascites containing antibodies was collected. IgG was purified from the resulting ascites using affinity chromatography using a Protein A column to obtain several purified anti-SARS-CoV-2 N protein monoclonal antibodies (hereinafter sometimes referred to as "anti-N protein antibodies").

[0061] In the following examples, antibodies selected from a plurality of obtained anti-SARS-CoV-2 N protein monoclonal antibodies were used in consideration of reactivity and specificity.

[0062] Example 2 Analysis of the binding region of anti-N protein antibodies

[0063] Investigation of the binding sites of the anti-N protein antibodies produced in Example 1 revealed monoclonal antibodies that bind to the 419 amino acid sequence of the SARS-CoV-2 N protein and the sequences shown in Table 1 below. The sequences bound by monoclonal antibodies Nos. 1, 8, 14, 18, 20, 22, 23, 24, 25, 27, and 34 were specifically determined as follows. Partial chains consisting of 5, 25, or 50 consecutive amino acids of the SARS-CoV-2 N protein antigen were immobilized on a plate, and their reactivity with the anti-N protein antibodies produced in Example 1 was evaluated by ELISA. Partial chains showing reactivity with the anti-N protein antibodies were designated as the binding sites of the anti-N protein antibodies.

[0064] The sequences bound by monoclonal antibodies Nos. 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15, 16, 17, 19, 21, 26, 28, 29, 30, 31, 32, 33, 35, 36, 37, and 38 were specifically determined as follows. A partial chain consisting of 15 to 25 consecutive amino acids of the SARS-CoV-2 N protein antigen was immobilized on microbeads, and the reactivity with the anti-N protein antibody obtained in Example 1 was evaluated using xMAP (registered trademark) (Luminex (registered trademark)). The partial chain that showed reactivity with the anti-N protein antibody was defined as the binding site of the anti-N protein antibody.

[0065] [Table 1]

[0066] Monoclonal antibody number. sequence 1 1～50 2 3～28 3 6～24 4 22～42 5 22～43 6 23～42 7 60～81 8 76～100 9 88～106 10 88～107 11 88～108 12 90～106 13 90～107 14 101～125 15 114～131 16 114～135 17 114～136 18 126～150 19 155～175 20 156～160 21 156～174 22 161～165 23 166～170 24 171～175 25 176～200 26 185～207 27 226～250 28 266～282 29 301～319 30 301～320 31 326～369 32 337～369 33 344～364 34 351～375 35 366～389 36 369～389 37 372～389 38 376～419

[0067] The RNA-binding domain of the N protein refers to the amino acid sequence ranging from positions 41 to 186. Therefore, it can be seen that numbers 7 to 24 are antibodies that recognize the RNA-binding domain of the N protein.

[0068] Example 3 Immunoassay Device for SARS-CoV-2

[0069] 1. Immobilization of anti-SARS-CoV-2N protein antibodies on nitrocellulose membranes

[0070] A solution of the anti-N protein antibodies classified in Example 2 diluted with a buffer solution and anti-mouse IgG antibodies were prepared. The anti-N protein antibodies were applied in a line pattern to the sample pad side of a nitrocellulose membrane backed with a PET film, and the anti-mouse IgG antibodies were applied in a line pattern to the absorbent side. The nitrocellulose membrane was then thoroughly dried with hot air to obtain an anti-N protein antibody-immobilized membrane.

[0071] 2. Immobilization of Anti-SARS-CoV-2N Protein Antibodies on Colored Polystyrene Particles

[0072] The anti-N protein antibody prepared in Example 1 was bound to colored polystyrene particles, suspended in a buffer solution, and fully dispersed by ultrasonic treatment to obtain anti-N protein antibody-bound colored polystyrene particles. These are referred to herein as anti-N protein antibody-immobilized particles.

[0073] 3. Coating and Drying of Anti-SARS-CoV-2N Protein Antibody-Conjugated Colored Polystyrene Particles

[0074] The anti-N protein antibody-immobilized particles prepared in step 2 were applied in a predetermined amount onto a glass fiber nonwoven fabric and dried thoroughly by thermal evaporation. In this specification, this is referred to as a marker pad.

[0075] 4. Fabrication of SARS-CoV-2 Testing Equipment

[0076] The anti-N protein antibody-immobilized membrane prepared in step 1 and the marker pad prepared in steps 2 and 3 were bonded to other components (base plate, absorption belt, sample pad), and cut into 5 mm widths to prepare a SARS-CoV-2 inspection device.

[0077] 5. Confirmation of the specificity and accuracy of SARS-CoV-2 detection equipment

[0078] 50 μL of a buffer solution containing viruses that cause respiratory infections (10 mM Tris (pH 7.0), 1% (w / v) polyoxyethylene octylphenyl ether, 3% (w / v) arginine, 3% (w / v) BSA) was added to the SARS-CoV-2 inspection device prepared in 4 and allowed to stand for 8 minutes.

[0079] If color development was visually confirmed at the application site for both the anti-mouse IgG antibody and the anti-N protein antibody, the result was considered positive. If color development was visually confirmed only at the application site for the anti-mouse IgG antibody, and no color development was visually confirmed at the application site for the anti-N protein antibody, the result was considered negative. Table 2 shows several items from the evaluation results for each monoclonal antibody combination. Positive results were assigned in order of color development intensity: 3, 2, and 1, and negative results were assigned as 0.

[0080] [Table 2]

[0081]

[0082] As shown in Table 2, when monoclonal antibodies (12, 15, 18, 19, 20, 22, 23, 24) that recognize the RNA binding domain were used on either or both of the anti-N protein antibody-immobilized membrane and the anti-N protein antibody-immobilized particles, the results were judged as strongly positive. In contrast, when monoclonal antibodies other than these that do not recognize the RNA binding domain were used on either or both of the anti-N protein antibody-immobilized membrane and the anti-N protein antibody-immobilized particles, the results were judged as negative or weakly positive.

Claims

1. A method for detecting SARS-CoV-2, comprising detecting SARS-CoV-2 in a sample by an immunoassay using a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with the RNA-binding domain of the amino acid sequence of the SARS-CoV-2 N protein.

2. The method according to claim 1, wherein The monoclonal antibody specifically reacts with a region contained in the 114aa to 170aa region of the amino acid sequence of the N protein of SARS-CoV-2.

3. The method according to claim 2, wherein: The monoclonal antibody specifically reacts with a region contained in the 126aa to 165aa region of the amino acid sequence of the N protein of SARS-CoV-2.

4. The method according to any one of claims 1 to 3, wherein The immunoassay is an immunochromatographic method.

5. A SARS-CoV-2 detection kit comprising a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with the RNA-binding domain of the N protein of SARS-CoV-2.

6. The detection kit according to claim 5, wherein The monoclonal antibody specifically reacts with a region contained in the 114aa to 170aa region of the amino acid sequence of the N protein of SARS-CoV-2.

7. The detection kit according to claim 6, wherein The monoclonal antibody specifically reacts with a region contained in the 126aa to 165aa region of the amino acid sequence of the N protein of SARS-CoV-2. 8 . The detection kit according to claim 5 , which is an immunochromatographic test piece.

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