Antibody against SARS-cov-2, method for detecting SARS-cov-2 using the same, and kit comprising the same

Abstract

To provide a means for detecting a protein constituting SARS-CoV-2-derived nucleocapsid.SOLUTION: Provided are an antibody or a fragment thereof that binds to a protein that constitutes a SARS-CoV-2-derived nucleocapsid, a method for detecting a protein constituting a SARS-CoV-2-derived nucleocapsid using the antibody or a fragment thereof, and a kit for detecting a protein constituting a SARS-CoV-2-derived nucleocapsid, the kit comprising the antibody or a fragment thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an antibody against SARS-CoV-2, a method for detecting SARS-CoV-2 using the antibody, and a kit containing the antibody. [Background technology]

[0002] The 2019 novel coronavirus (hereinafter sometimes referred to as "SARS-CoV-2") is a coronavirus belonging to the SARS-related coronaviruses that was first identified in 2019 near Wuhan, China. It is pathogenic to humans and causes the novel coronavirus disease (COVID-19). Currently, the WHO has recognized that the spread of SARS-CoV-2 infection is equivalent to a pandemic (global epidemic). However, there is no established treatment for patients infected with SARS-CoV-2. Therefore, in order to take appropriate measures to prevent the spread of infection, it is extremely important to detect patients infected with SARS-CoV-2 through rapid testing and to investigate the infection rate and route of infection by examining the antibody possession rate in serum using immunological tests.

[0003] As a method for detecting SARS-CoV-2, genetic tests such as real-time reverse transcription PCR using collected nasal swabs, pharyngeal swabs, sputum, etc. are mainly used (Non-Patent Documents 1 and 2). This makes it possible to determine the presence or absence of SARS-CoV-2 infection. On the other hand, in the genetic tests, there are many cases where SARS-CoV-2 is not detected from the sample despite infection with SARS-CoV-2 (false negatives) due to the influence of the sample collection location, collection time, storage method, etc., which is one of the factors contributing to the spread of SARS-CoV-2 infection. In addition, genetic tests require special equipment that detects fluorescence in real time during PCR and take several hours to obtain results, so there are issues with practicality from the perspective of early detection in places where facilities such as infection sites, quarantine sites, and clinical sites are not sufficient. Therefore, there is a demand for immunological tests that utilize antigen-antibody reactions against SARS-CoV-2-derived proteins as a test that combines accuracy, speed, and simplicity.

[0004] As a protein derived from SARS-CoV-2, spike (hereinafter sometimes referred to as "S") protein is known, which is present on the surface of the SARS-CoV-2 envelope and is involved in infection of host cells (Non-Patent Document 3). However, the S protein derived from SARS-CoV-2 is a glycoprotein with many glycans bound thereto (Non-Patent Document 3), and it is considered extremely difficult to artificially produce a protein that accurately reproduces its structure using chemical synthesis or recombinant microorganisms. In addition, it is known that the S protein derived from SARS coronavirus, which is genetically closely related to SARS-CoV-2, is prone to mutations in its amino acid sequence as SARS coronavirus grows (Non-Patent Document 4), and it is considered that the S protein derived from SARS-CoV-2 is also prone to mutations in its amino acid sequence as SARS-CoV-2 grows. For this reason, antibodies generated against the SARS-CoV-2 S protein may not show an antigen-antibody reaction against mutant versions of the S protein. Therefore, immunological tests using antibodies against the SARS-CoV-2 S protein may produce false negative test results depending on the sample.

[0005] On the other hand, a protein that constitutes the SARS-CoV-2-derived nucleocapsid (hereinafter, sometimes referred to as "NP") is also known as a protein derived from SARS-CoV-2. It is known that NP derived from SARS coronavirus, which is genetically closely related to SARS-CoV-2, is produced in the largest amount by infection among various SARS coronavirus-derived proteins (Non-Patent Document 5), and it is also thought that SARS-CoV-2-derived NP is produced in the largest amount by infection among various SARS-CoV-2-derived proteins. For this reason, it is expected that SARS-CoV-2-derived NP can be relatively easily detected from a sample. In fact, a case has already been reported in which an antibody against a partial peptide of SARS-CoV-2-derived NP was produced (Non-Patent Document 6). However, the antibody produced in that case also showed an antigen-antibody reaction against NP derived from MERS coronavirus and NP derived from SARS coronavirus, which are genetically closely related to SARS-CoV-2, and did not have specificity for SARS-CoV-2-derived NP (Non-Patent Document 6). Therefore, in immunological tests using such antibodies, there is a risk that false positive test results may occur depending on the sample. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Manual for specimen collection and transportation for patients suspected of 2019-nCoV (novel coronavirus) infection (National Institute of Infectious Diseases, April 16, 2020) [Non-Patent Document 2] Pathogen Detection Manual 2019-nCoV Ver.2.9.1 (National Institute of Infectious Diseases, March 19, 2020) [Non-Patent Document 3] Chen X., et al., Cell. Mol. Immunol., (2020) doi: https: / / doi.org / 10.1038 / s41423-020-0426-7 [Non-Patent Document 4] Qin E., et al., Geno., Prot. Bioinfo., 1(2), 101-107 (2003) [Non-Patent Document 5] Wang J., et al., Geno., Prot. Bioinfo., 1(2), 145-154 (2003) [Non-Patent Document 6] Li M., et al., medRxiv, (2020) doi: https: / / doi.org / 10.1101 / 2020.02.20.20025999 Summary of the Invention [Problem to be solved by the invention]

[0007] The objective of the present invention is to provide a means for detecting proteins that constitute the SARS-CoV-2 nucleocapsid. [Means for solving the problem]

[0008] As a result of analyzing SARS-CoV-2-derived NP and other coronavirus-derived NP, the present inventors found a region with low identity between the amino acid sequence of SARS-CoV-2-derived NP and the amino acid sequence of other coronavirus-derived NP. In view of the above problem, the present inventors prepared a fragment of SARS-CoV-2-derived NP corresponding to the region, and used it as an immunogen to prepare an antibody according to a conventional method, thereby completing the present invention.

[0009] That is, the present invention relates to the following. [1] An antibody or fragment thereof that binds to a protein that constitutes the nucleocapsid derived from SARS-CoV-2, wherein the epitope recognized by the antibody or fragment thereof is located at positions 121 to 419 of the amino acid sequence represented by SEQ ID NO:1. [2] The antibody or fragment thereof according to [1], wherein the epitope is located at positions 208 to 222, 332 to 351, 374 to 397, or 398 to 419 of the amino acid sequence represented by SEQ ID NO:1. [3] An antibody or fragment thereof of [1] or [2] that specifically binds to a protein that constitutes the nucleocapsid of SARS-CoV-2. [4] Any of the antibodies or fragments thereof according to [1] to [3], wherein the antibody is a monoclonal antibody. [5] An antibody or a fragment thereof according to any one of [1] to [4], wherein the fragment is a Fab fragment, a Fab / c fragment, an Fv fragment, a Fab' fragment or an F(ab')2 fragment. [6] A method for detecting a protein constituting a SARS-CoV-2 nucleocapsid from a sample, the method comprising a step of contacting the sample with any one of the antibodies or fragments thereof according to [1] to [5]. [7] The method of [6], wherein the sample is a biological sample. [8] The method of [6] or [7], further comprising a step of detecting proteins constituting the SARS-CoV-2 nucleocapsid by at least one immunological test selected from the group consisting of ELISA, immunochromatography and filter antigen assay. A kit for detecting proteins that constitute the SARS-CoV-2 nucleocapsid, comprising any of the antibodies or fragments thereof listed in [9][1] to [5].

[10] The kit of [9] in the form of immunochromatographic strips. Effect of the Invention

[0010] The antibody or fragment thereof that specifically binds to SARS-CoV-2-derived NP of the present invention, a method for detecting SARS-CoV-2-derived NP using the antibody or fragment thereof, and a kit for detecting a protein constituting SARS-CoV-2-derived nucleocapsid containing the antibody or fragment thereof, can accurately, simply, and quickly detect the presence or absence of SARS-CoV-2 infection or its medical history. In other words, the present invention can provide an immunological test that is highly practical for early detection in places where facilities such as infection sites, quarantine sites, and clinical sites are not sufficient. This is because the epitope recognized by the antibody or fragment thereof that specifically binds to SARS-CoV-2-derived NP of the present invention is composed of a region that has low identity between the amino acid sequence of SARS-CoV-2-derived NP and the amino acid sequence of NP derived from other coronaviruses, making it possible to specifically detect SARS-CoV-2-derived NP from a sample. In addition, by using the method of the present invention to detect SARS-CoV-2-derived NP using an antibody or a fragment thereof that specifically binds to SARS-CoV-2-derived NP, it is possible to reduce the occurrence of false positives and false negatives. [Brief description of the drawings]

[0011] [Figure 1] Figure 1 shows the identity of the amino acid sequence between SARS-CoV-2-derived NP and NP derived from other coronaviruses (SARS, MERS, OC43, NL63, HKU1, and 229E). A fragment of SARS-CoV-2-derived NP (region 121 to 419 of the amino acid sequence represented by SEQ ID NO: 1), which has high antigenicity among SARS-CoV-2-derived NP and has a motif common to NP derived from other coronaviruses, is used as an immunogen for antibody production. [Diagram 2] Figure 2 shows the results of preparation of SARS-CoV-2-derived NP fragments. Figure 2A shows the results of confirming the expression of SARS-CoV-2-derived NP fragments. Figure 2B shows the results of purifying SARS-CoV-2-derived NP fragments. [Diagram 3]Figure 3 shows the results of screening hybridoma cells that produce monoclonal antibodies that show high reactivity to fragments of SARS-CoV-2-derived NP. Figure 3A shows the results of screening using the ELISA method in which fragments of SARS-CoV-2-derived NP were immobilized. Figure 3B shows the results of screening using the AlphaScreen method using fragments of SARS-CoV-2-derived NP.

[0012] [Figure 4] Figure 4 shows the results of epitope analysis of each monoclonal antibody prepared in Example 2. Figure 4A shows the results of investigating the epitopes recognized by each monoclonal antibody by preparing three types of recombinant proteins (deletion mutants) d1 to d3 in which each region of the amino acid sequence of the fragment of SARS-CoV-2-derived NP is deleted (schematic diagram in the upper row) and comparing the reactivity with them (Western blot results in the lower row). Figure 4B shows the results of investigating the epitopes recognized by each monoclonal antibody by preparing eight types of recombinant proteins (deletion mutants) d4 to d11 in which each region of the amino acid sequence of the fragment of SARS-CoV-2-derived NP is deleted (schematic diagram in the upper row) and comparing the reactivity with them (Western blot results in the lower row). [Diagram 5] 5 shows the results of specificity analysis of each monoclonal antibody prepared in Example 2. The specificity of each monoclonal antibody was analyzed by Western blot using each NP derived from other human coronaviruses (SARS, MERS, OC43, NL63, HKU1, and 229E). [Figure 6] Figure 6 shows the results of immunostaining of SARS-CoV-2 infected cells. Figure 6A shows the expression of SARS-CoV-2 derived NP in the infected cells. Figure 6B shows the results of immunostaining the infected cells with the monoclonal antibodies (#7, #98) prepared in Example 2. [Figure 7] Figure 7 shows the results of SARS-CoV-2-derived NP detection by ELISA using the monoclonal antibodies (#9, #98) prepared in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications and other publications and information referenced herein are incorporated herein by reference in their entirety.

[0014] [Antibody or fragment thereof that binds to a protein that constitutes the SARS-CoV-2 nucleocapsid] One aspect of the present invention relates to an antibody or a fragment thereof that binds to a protein constituting a SARS-CoV-2-derived nucleocapsid, the antibody or a fragment thereof having an epitope recognized by the antibody or a fragment thereof at positions 121 to 419 of the amino acid sequence represented by SEQ ID NO: 1 (hereinafter, sometimes referred to as "the antibody or a fragment thereof of the present invention"). In one embodiment, the epitope recognized by the antibody or a fragment thereof of the present invention is at positions 208 to 222, 332 to 351, 374 to 397, or 398 to 419 of the amino acid sequence represented by SEQ ID NO: 1. In one embodiment, the antibody or a fragment thereof of the present invention specifically binds to a protein constituting a SARS-CoV-2-derived nucleocapsid.

[0015] In the present invention, "SARS-CoV-2" refers to a coronavirus belonging to the SARS-related coronavirus, whose outbreak was first confirmed in 2019 near Wuhan, China, and is used interchangeably with "2019 novel coronavirus." SARS-CoV-2 is pathogenic to humans and causes acute respiratory disease (COVID-19). Like other coronaviruses, SARS-CoV-2 is composed of spikes, nucleocapsids, integral membrane proteins, envelope proteins, and RNA. Of these, the nucleocapsid binds to RNA to form NP, and the spikes, integral membrane proteins, and envelope proteins bound to lipids surround it to form an envelope. The genome sequence of SARS-CoV-2 is published under GenBank accession number MN908947.3.

[0016] In the present invention, the term "protein constituting the nucleocapsid" refers to a protein constituting a complex consisting of nucleocapsid and RNA, and is used interchangeably with "NP." The amino acid sequence of SARS-CoV-2-derived NP is represented by SEQ ID NO:1.

[0017] In the present invention, the term "fragment of SARS-CoV-2-derived NP" refers to a protein fragment consisting of a region (the region of 121st to 419th positions of the amino acid sequence represented by SEQ ID NO: 1) of SARS-CoV-2-derived NP that has low identity between the amino acid sequence of SARS-CoV-2-derived NP and the amino acid sequence of NP derived from other coronaviruses. The fragment of SARS-CoV-2-derived NP has a sequence identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 2. From the viewpoint of detecting SARS-CoV-2-derived NP accurately, simply, and quickly, the fragment of SARS-CoV-2-derived NP preferably has a sequence identity of 95% or more with the amino acid sequence represented by SEQ ID NO: 2, particularly preferably has a sequence identity of 98% or more, and further preferably has a sequence identity of 100%. In one embodiment, the fragment of SARS-CoV-2-derived NP consists of the amino acid sequence represented by SEQ ID NO: 2. Since a fragment of SARS-CoV-2-derived NP corresponds to a region that has low identity between the amino acid sequence of SARS-CoV-2-derived NP and the amino acid sequence of NP derived from other coronaviruses, the fragment can be prepared and used as an immunogen to obtain the antibody of the present invention or a fragment thereof according to conventional methods.

[0018] The antibody of the present invention may be, for example, a complete immunoglobulin molecule, preferably IgM, IgD, IgE, IgA or IgG, more preferably IgG1, IgG2a, IgG2b, IgG3 or IgG4. The antibody of the present invention may also be a modified and / or altered antibody, such as a chimeric or humanized antibody. The antibody of the present invention may also be a monoclonal or polyclonal antibody, a modified or altered monoclonal or polyclonal antibody, or a recombinantly or synthetically produced or synthesized antibody. The antibody of the present invention is preferably a monoclonal antibody from the viewpoint of detecting SARS-CoV-2-derived NP accurately, easily, and quickly.

[0019] The fragment of the antibody of the present invention may be a portion of such an immunoglobulin molecule, such as a Fab fragment or a VL-, VH- or CDR-region, but the fragment of the antibody of the present invention may specifically recognize and bind to NP derived from SARS-CoV-2 to the same extent as the antibody of the present invention. The fragment of the antibody of the present invention may be an antibody fragment and a portion thereof, such as separated light and heavy chains, Fab, Fab / c, Fv, Fab', F(ab')2, etc. In one embodiment, the fragment of the antibody of the present invention is a Fab fragment, a Fab / c fragment, an Fv fragment, a Fab' fragment or a F(ab')2 fragment. The antibody or fragment thereof of the present invention may also be an antibody derivative, such as a bifunctional antibody, or an antibody construct, such as a single chain Fv (scFv), a bispecific scFv or an antibody fusion protein.

[0020] [Method for detecting proteins that compose the SARS-CoV-2 nucleocapsid from samples] Another aspect of the present invention relates to a method for detecting a protein constituting a SARS-CoV-2-derived nucleocapsid from a sample, the method comprising a step of contacting the sample with an antibody of the present invention or a fragment thereof (hereinafter, sometimes referred to as the "method of the present invention"). In one embodiment, in the method of the present invention, the sample is a biological sample. In one embodiment, the method of the present invention further comprises a step of detecting a protein constituting a SARS-CoV-2-derived nucleocapsid by at least one immunological test selected from the group consisting of an ELISA method, an immunochromatography method, and a filter antigen assay method.

[0021] In one embodiment, the method of the present invention includes a step of performing an antigen-antibody reaction between the antibody or fragment thereof of the present invention and SARS-CoV-2-derived NP. In one embodiment, the method of the present invention includes a step of performing an antigen-antibody reaction between the antibody or fragment thereof of the present invention and SARS-CoV-2-derived NP multiple times. In one embodiment, the antibody or fragment thereof of the present invention has a different epitope for each of the steps. In one embodiment, the antibody or fragment thereof of the present invention is labeled with a labeling substance. For example, the method of the present invention includes a step of performing an antigen-antibody reaction between an unlabeled antibody or fragment thereof of the present invention and SARS-CoV-2-derived NP, and a step of performing an antigen-antibody reaction between an antibody or fragment thereof of the present invention labeled with a labeling substance (having an epitope different from that of the unlabeled antibody or fragment thereof of the present invention) and SARS-CoV-2-derived NP.

[0022] In one embodiment, the method of the present invention further comprises a step of performing an antigen-antibody reaction between the antibody or fragment thereof of the present invention and SARS-CoV-2-derived NP, and / or a step of performing an antigen-antibody reaction between the antibody or fragment thereof of the present invention and an antibody or fragment thereof that recognizes the antibody or fragment thereof. In one embodiment, the antibody or fragment thereof of the present invention, and / or the antibody or fragment thereof that recognizes the antibody or fragment thereof is labeled with a labeling substance.

[0023] In the present invention, the term "sample" refers to any sample that may contain SARS-CoV-2-derived NP, including, but not limited to, a biological sample. In the present invention, the term "sample" is used interchangeably with the term "specimen." In the present invention, the "biological sample" refers to any sample that may contain SARS-CoV-2-derived NP, such as human or animal blood, plasma, serum, urine, semen, cerebrospinal fluid, saliva, oral mucosa, nasal mucus, sweat, tears, ascites, amniotic fluid, feces, blood vessels, or organs such as the liver, tissues, cells, or extracts thereof, and is preferably easy to collect, such as cells and secretions from the oral cavity, tonsils, nasal cavity, pharynx, larynx, trachea, bronchi, and lungs, nasal swabs, pharyngeal swabs, gargles, sputum, tracheal aspirates, and bronchoalveolar lavage fluid. The method of collecting these specimens is not particularly limited, and known methods can be adopted. Specifically, a method using a cotton swab is often used. The animal is not particularly limited as long as it can be infected with SARS-CoV-2, and examples thereof include dogs and cats.

[0024] Biological samples can be collected according to routine methods. For example, as described in Non-Patent Document 1, 1 to 2 ml of serum after separation is placed in a plastic tube that can be sealed, and the tube is then capped and sealed with parafilm. A coagulant may be included, and when a blood collection tube containing a serum separating agent is used, 1 to 2 ml of serum after centrifugation is transferred to a plastic tube (preferably a sterilized tube), capped, and sealed with parafilm. For example, as described in Non-Patent Document 1, whole blood is collected in a blood collection tube containing a blood coagulation inhibitor (EDTA-Na or K), and 1 to 2 ml is dispensed into a plastic tube that can be sealed, and after the tube is capped, it is sealed with parafilm. If possible, blood cells are separated, and peripheral blood mononuclear cells are suspended in a cell preservation solution and frozen and stored. The separation of peripheral blood mononuclear cells is easily performed by using a BD Vacutainer (registered trademark) CPT (trademark) blood collection tube for mononuclear cell separation. In addition, if it is not possible to dispense or separate blood cells after blood collection, blood may be collected using a PAXgene (registered trademark) RNA blood collection tube and then frozen and stored as is.

[0025] For example, as described in Non-Patent Document 1, sputum is collected from the lower respiratory tract if the patient is expectorating sputum. If the patient is under artificial respirator management, tracheal aspirate is collected using a sterilized catheter under aseptic technique. If there are no clinical contraindications, collection of bronchoalveolar lavage fluid may also be considered. The collected sputum or aspirate is placed in a plastic tube with a screw cap, closed, and sealed with parafilm. For example, as described in Non-Patent Document 1, a nasopharyngeal swab is prepared by inserting a sterile cotton swab (such as a flocked swab or a swab made of rayon or polyester; a thin one for use in the nasal cavity) through the nostril, wiping the nasopharynx thoroughly, and then placing the swab in a sterile Spitz tube containing 1-3 ml of virus transport solution (VTM / UTM), closing the tube, and sealing it with parafilm. If no virus transport solution is available, PBS or physiological saline can be used. For example, as described in Non-Patent Document 1, 1 to 2 ml of urine is placed in a test tube (such as a Falcon tube), and the tube is capped and sealed with parafilm. For example, as described in Non-Patent Document 1, about 0.1 g (the size of a red bean) of stool is collected in a plastic tube that can be sealed, and the tube is then capped and sealed with parafilm.

[0026] In the present invention, the "labeling substance" refers to any substance that labels the antibody or fragment thereof of the present invention, and / or the antibody or fragment thereof that recognizes the antibody or fragment thereof, and is not particularly limited as long as it can detect an antigen-antibody reaction. From the viewpoint of detecting SARS-CoV-2-derived NP accurately, simply, and quickly, the labeling substance is preferably one or more selected from enzymes, dyes, metal colloid particles, latex particles, cellulose particles, etc.

[0027] In the present invention, "an antibody or a fragment thereof that recognizes the antibody or a fragment thereof of the present invention" refers to an antibody or a fragment thereof that can specifically bind to the antibody or a fragment thereof of the present invention. From the viewpoint of accurately, simply, and quickly detecting NP derived from SARS-CoV-2, the antibody that recognizes the antibody or a fragment thereof of the present invention is preferably one or more selected from the group consisting of IgG, IgM, IgA, IgD, and IgE, and is particularly preferably IgG and / or IgM, and is even more preferably IgG. In addition, the antibody that recognizes the antibody or a fragment thereof of the present invention may be a modified and / or altered antibody, such as a chimeric or humanized antibody. In addition, the antibody that recognizes the antibody or a fragment thereof of the present invention may be a monoclonal or polyclonal antibody, a modified or altered monoclonal or polyclonal antibody, or a recombinantly or synthetically produced or synthesized antibody.

[0028] The antibody fragment recognizing the antibody of the present invention or a fragment thereof may be a part of such an immunoglobulin molecule, such as a Fab fragment or a VL-, VH- or CDR-region, but the antibody fragment recognizing the antibody of the present invention or a fragment thereof may specifically recognize and bind to the antibody of the present invention or a fragment thereof to the same extent as an antibody recognizing the antibody of the present invention or a fragment thereof. The antibody fragment recognizing the antibody of the present invention or a fragment thereof may be an antibody fragment, as well as a part thereof, such as separated light and heavy chains, Fab, Fab / c, Fv, Fab', F(ab')2, etc. In one embodiment, the antibody fragment recognizing the antibody of the present invention or a fragment thereof is a Fab fragment, a Fab / c fragment, an Fv fragment, a Fab' fragment or an F(ab')2 fragment. Furthermore, an antibody or a fragment thereof that recognizes an antibody or a fragment thereof of the present invention may be an antibody derivative such as a bifunctional antibody, or an antibody construct such as a single chain Fv (scFv), a bispecific scFv or an antibody fusion protein.

[0029] In the present invention, SARS-CoV-2-derived NP is detected by any immunological test that can detect the antigen-antibody reaction between the antibody or fragment thereof of the present invention and SARS-CoV-2-derived NP. Immunological tests include, for example, enzyme immunoassay (ELISA, EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay (LIA), enzyme antibody method, fluorescent antibody method, immunochromatography (immunochromatography), filter antigen assay, immunoturbidimetry, latex turbidimetry, latex agglutination assay, erythrocyte agglutination assay, or particle agglutination assay. From the viewpoint of detecting SARS-CoV-2-derived NP accurately, simply, and quickly, SARS-CoV-2-derived NP is preferably detected by ELISA and / or immunochromatography. In addition, a calibration curve can be created from the antigen-antibody reaction between a predetermined amount of the antibody or fragment thereof of the present invention and SARS-CoV-2-derived NP, and the amount of SARS-CoV-2-derived NP contained in a sample can be quantified by interpolating the measured values ​​onto the calibration curve.

[0030] [A kit for detecting proteins that compose the SARS-CoV-2 nucleocapsid] Another aspect of the present invention relates to a kit for detecting a protein constituting a SARS-CoV-2-derived nucleocapsid, comprising the antibody of the present invention or a fragment thereof (hereinafter, may be referred to as the "kit of the present invention"). From the viewpoint of accurately, simply, and quickly detecting SARS-CoV-2-derived NP, from the viewpoint of detecting SARS-CoV-2-derived NP by medical personnel in places where facilities such as infection sites, quarantine sites, and clinical sites are not sufficient, and from the viewpoint of epidemiological surveys, etc., the kit of the present invention is preferably in the form of an immunochromatographic strip.

[0031] For example, an immunochromatographic strip is one in which the antibody or fragment thereof of the present invention and an anti-mouse immunoglobulin antibody are immobilized on separate sites, i.e., a test line and a control line. The sample pad of the immunochromatographic strip is pre-impregnated with the antibody or fragment thereof of the present invention labeled with gold colloid particles, latex particles, fluorescent particles, magnetic particles, etc. The sample dropped onto the sample pad is developed by capillary action, and when it reaches the test line, only the SARS-CoV-2-derived NP in the sample reacts, and when it reaches the control line, only the antibody or fragment thereof labeled with gold colloids, etc. reacts. Other components in the sample move to the absorbent pad without reacting. By measuring the color intensity, reflection intensity, absorbance, fluorescence intensity, magnetic intensity, etc. of the test line and control line with an immunochromatographic reader, not only can SARS-CoV-2-derived NP in the sample be detected, but also the amount of SARS-CoV-2-derived NP in the sample can be quantified.

[0032] [Composition for detecting proteins that compose the SARS-CoV-2 nucleocapsid] Another aspect of the present invention relates to a composition for detecting a protein constituting a SARS-CoV-2 nucleocapsid, comprising an antibody of the present invention or a fragment thereof (hereinafter sometimes referred to as the "composition of the present invention").

[0033] [Methods for diagnosing SARS-CoV-2 infection] Another aspect of the present invention relates to a method for diagnosing SARS-CoV-2 infection (hereinafter, sometimes referred to as the "diagnostic method of the present invention"), which comprises a step of contacting a sample with the antibody of the present invention or a fragment thereof. In one embodiment, the diagnostic method of the present invention further comprises a step of detecting a protein constituting the SARS-CoV-2-derived nucleocapsid by at least one immunological test selected from the group consisting of ELISA, immunochromatography, and filter antigen assay.

[0034] In one embodiment, the diagnostic method of the present invention includes a step of performing an antigen-antibody reaction between the antibody or fragment thereof of the present invention and SARS-CoV-2-derived NP. In one embodiment, the diagnostic method of the present invention includes a step of performing an antigen-antibody reaction between the antibody or fragment thereof of the present invention and SARS-CoV-2-derived NP multiple times. In one embodiment, the antibody or fragment thereof of the present invention has a different epitope for each of the steps. In one embodiment, the antibody or fragment thereof of the present invention is labeled with a labeling substance. For example, the diagnostic method of the present invention includes a step of performing an antigen-antibody reaction between an unlabeled antibody or fragment thereof of the present invention and SARS-CoV-2-derived NP, and a step of performing an antigen-antibody reaction between an antibody or fragment thereof of the present invention labeled with a labeling substance (having an epitope different from that of the unlabeled antibody or fragment thereof of the present invention) and SARS-CoV-2-derived NP.

[0035] In one embodiment, the diagnostic method of the present invention further comprises a step of performing an antigen-antibody reaction between the antibody or fragment thereof of the present invention and SARS-CoV-2-derived NP, and / or a step of performing an antigen-antibody reaction between the antibody or fragment thereof of the present invention and an antibody or fragment thereof that recognizes the antibody or fragment thereof. In one embodiment, the antibody or fragment thereof of the present invention and / or the antibody or fragment thereof that recognizes the antibody or fragment thereof are labeled with a labeling substance. EXAMPLES

[0036] Example 1 Preparation of fragments of SARS-CoV-2-derived NP [Immunogen design] Based on the sequences published in the database, we analyzed the identity between the amino acid sequence of SARS-CoV-2-derived NP and the amino acid sequences of NP derived from other coronaviruses (SARS, MERS, OC43, NL63, HKU1, 229E) (Figure 1). As a result, we found that the amino acid sequence of SARS-CoV-2-derived NP has approximately 90% amino acid sequence identity to the amino acid sequence of SARS-coronavirus-derived NP, approximately 47% amino acid sequence identity to the amino acid sequence of MERS-coronavirus-derived NP, and approximately 30% or less amino acid sequence identity to the amino acid sequences of other coronavirus-derived NP. Therefore, we decided to use the region of amino acids 121 to 419 in the SARS-CoV-2-derived NP, excluding the motif (FYYLGTGP) that is commonly conserved among coronaviruses, as the immunogen.

[0037] [Cell-free protein synthesis of fragments of SARS-CoV-2-derived NP] A fragment of SARS-CoV-2-derived NP consisting of the amino acid sequence represented by SEQ ID NO: 2 was prepared by the following method. SARS-CoV-2-derived NP consists of the amino acid sequence represented by SEQ ID NO: 1, the RNA sequence corresponding to the amino acid sequence is represented by SEQ ID NO: 3, and the DNA sequence corresponding to the RNA sequence is represented by SEQ ID NO: 4. The DNA sequence was codon-optimized for protein synthesis in eukaryotic cells based on the amino acid sequence.

[0038] First, the DNA sequence represented by SEQ ID NO: 4 was artificially synthesized, and the region of 361 to 1260 of the DNA sequence represented by SEQ ID NO: 4, which corresponds to the region of 121 to 419 of the amino acid sequence represented by SEQ ID NO: 1 (FIG. 1), was amplified by PCR using the DNA sequence as a template. 5 μL of 1 μM forward primer (SEQ ID NO: 5), 5 μL of 1 μM reverse primer (SEQ ID NO: 6), 1 μL of 10 ng / μL DNA vector (pUC57 (GeneWiz)) into which the DNA sequence represented by SEQ ID NO: 4 was inserted, 25 μL of PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio Inc.), and 14 μL of ultrapure water were mixed to prepare a reaction solution of a total volume of 50 μL. The PCR method was performed with the thermal cycler settings shown in Table 1. As a result, a DNA sequence including the region of 361 to 1260 of the DNA sequence represented by SEQ ID NO: 4 was obtained. Then, a DNA vector was amplified in a similar manner using the primers shown in SEQ ID NOs: 7 and 8 by PCR with the pEU-E01-His-TEV-MCS-N2 vector (Cell Free Sciences) as a template.

[0039] [Table 1]

[0040] Next, the DNA sequence and DNA vector obtained by the PCR method were purified by agarose gel electrophoresis, and then the DNA was ligated to prepare a DNA vector using In-Fusion (registered trademark) HD ​​Cloning Kit (Takara Bio Inc.). The obtained DNA vector was transformed into Escherichia coli JM109 strain, and the transformed Escherichia coli was seeded on an ampicillin-containing LB medium. A DNA vector was prepared from the grown Escherichia coli using a plasmid purification kit (Promega). Next, mRNA was transcribed from the obtained DNA vector using SP6 RNA polymerase, and a His-tagged SARS-CoV-2 NP fragment (the region from amino acid 121 to 419 of the amino acid sequence represented by SEQ ID NO: 1 (Figure 1)) was subjected to cell-free protein synthesis using wheat germ extract according to the instructions for use of the WEPRO7240 kit from Cell Free Sciences.

[0041] [Confirmation of expression of SARS-CoV-2-derived NP fragment] The expression of SARS-CoV-2-derived NP fragments was confirmed by Western blotting. First, His-tagged SARS-CoV-2-derived NP fragments were mixed with 2xSDS sample buffer (125mM Tris-HCl, 4% SDS, 20% glycerol, 0.01% bromophenol blue, and 10% 2-mercaptoethanol), heat-treated, and subjected to polyacrylamide gel electrophoresis. Next, the gel after electrophoresis was transferred to a PVDF membrane. After blocking the membrane with 2% skim milk solution, it was reacted with peroxidase-labeled anti-His tag antibody. After that, it was reacted with HRP-labeled secondary antibody, and then peroxidase substrate solution was added to cause light emission, which was detected using a chemiluminescence imaging device. As a result, the molecular weight of the His-tagged SARS-CoV-2-derived NP fragment was 33.7 kDa, and a band was confirmed at the position corresponding to this molecular weight (Figure 2A), confirming the expression of the SARS-CoV-2-derived NP fragment.

[0042] [Purification of a fragment of SARS-CoV-2-derived NP] A fragment of SARS-CoV-2-derived NP with a His tag was purified from the cell-free protein synthesis reaction solution using a Ni column. A series of steps, including transcription, cell-free protein synthesis, and purification, were performed using an automated synthesizer (Cell Free Sciences). The fragment of SARS-CoV-2-derived NP with a His tag was purified three times, and the first to third purification fractions were designated E1 to E3, respectively. E1 to E3 were mixed with 2×SDS sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 0.01% bromophenol blue, and 10% 2-mercaptoethanol), heat-treated, subjected to electrophoresis on a polyacrylamide gel, and stained with Rapid CBB KANTO 3S (Kanto Chemical Co., Ltd.). As a result, the amount of contaminating proteins was reduced in E1 to E3 compared to the solution before purification (S: soluble fraction) (Figure 2B), confirming that a fragment of SARS-CoV-2-derived NP had been purified.

[0043] Example 2: Generation of antibodies against fragments of SARS-CoV-2 NP [Immunization of mice and production of hybridoma cells] 300 μg of purified SARS-CoV-2-derived NP fragment was mixed and emulsified with Freund's adjuvant and injected subcutaneously multiple times into BALB / c mice for immunization. B cells were collected from the immunized mice one month later and fused with SP2 / 0 myeloma cells using a polyethylene glycol solution. The fused hybridoma cells were seeded into 96-well microplates with selection medium.

[0044] [Hybridoma cell screening and cloning] Next, the reactivity of the monoclonal antibodies produced by each hybridoma to fragments of SARS-CoV-2-derived NP was examined using ELISA and AlphaScreen. In the ELISA method, first, a fragment of SARS-CoV-2-derived NP was immobilized on an ELISA plate. After blocking the plate, the hybridoma culture supernatant was added to the plate and reacted, followed by the addition of a peroxidase-labeled goat anti-mouse immunoglobulin antibody. After that, a peroxidase substrate solution was added to develop color, and the absorbance was measured (Figure 3A).

[0045] The AlphaScreen method was performed according to the manufacturer's recommended method. First, a biotin-labeled protein was synthesized from a fragment of SARS-CoV-2-derived NP using a cell-free protein synthesis method similar to that described in Example 1. The synthesized biotin-labeled protein and the hybridoma culture supernatant were then added to the plate, mixed, and incubated at room temperature for 30 minutes. Then, 10 μl of acceptor beads (Protein G coating) and donor beads (streptavidin coating) were added. After further incubation at room temperature for 30 minutes, measurements were performed using EnVision (PerkinElmer) (Figure 3B). By cloning the hybridomas identified in the above screening, we obtained 27 types of hybridoma cells that produce monoclonal antibodies that are highly reactive to fragments of SARS-CoV-2-derived NP.

[0046] Example 3 Analysis of epitopes recognized by each monoclonal antibody Three types of recombinant proteins (deletion mutants) were created by deleting different regions of the amino acid sequence of a fragment of SARS-CoV-2-derived NP, and the epitopes recognized by each monoclonal antibody were investigated by comparing their reactivity with these proteins. To prepare each recombinant protein, first, the wheat cell-free expression vector pEU-E01-His-TEV-SARS-CoV-2-NP (121-419) (SEQ ID NO: 9) encoding the 121st to 419th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 prepared in Example 1 was used as a template, and expression vectors were prepared according to the method of PrimeSTAR (registered trademark) Mutagenesis Basal Kit (Takara Bio Inc.) in which cDNA fragments encoding the 121st to 231st (d1), 232nd to 340th (d2), and 341st to 419th (d3) amino acid sequence regions of the amino acid sequence represented by SEQ ID NO: 1 were deleted. From the prepared DNA vector, mRNA was synthesized using SP6 RNA polymerase, and each recombinant protein was subjected to cell-free protein synthesis in the same procedure as in Example 1 according to the method of using the WEPRO7240 kit of Cell Free Sciences.

[0047] The epitopes of each monoclonal antibody were analyzed by Western blotting using each of the obtained proteins. First, each of the obtained proteins was mixed with 2xSDS sample buffer (125mM Tris-HCl, 4% SDS, 20% glycerol, 0.01% bromophenol blue, and 10% 2-mercaptoethanol), heat-treated, and subjected to polyacrylamide gel electrophoresis. Next, the gel after electrophoresis was transferred to a PVDF membrane. After blocking the membrane with a 2% skim milk solution, it was reacted with each of the monoclonal antibodies prepared in Example 2. After that, it was reacted with an HRP-labeled secondary antibody, and a peroxidase substrate solution was added to cause light emission, which was detected using a chemiluminescence imaging device.

[0048] As a result, #7 did not react with d1 among the three deletion mutants of SARS-CoV-2-derived NP prepared, and therefore it was found to recognize the region from 121 to 231 of the amino acid sequence represented by SEQ ID NO: 1. The 11 clones #2, #4, #9, #13, #40, #45, #55, #97, #104, #111, and #128 did not react with d2 and d3 among the three deletion mutants of SARS-CoV-2-derived NP prepared, and therefore it was found to recognize the region from 232 to 419 of the amino acid sequence represented by SEQ ID NO: 1. Of the three deletion mutants of SARS-CoV-2-derived NP that were prepared, 15 clones, #28, #35, #47, #60, #65, #67, #86, #96, #98, #102, #103, #112, #131, #135, and #144, were the only ones that did not react with d3, indicating that they recognize the region from amino acid 341 to 419 in the amino acid sequence represented by sequence number 1. From the above, the prepared antibodies were classified as antibodies having epitopes in the regions of positions 121 to 231, 232 to 340, or 341 to 419 of the amino acid sequence shown in SEQ ID NO: 1 (FIG. 4A).

[0049] Next, to investigate the recognition region in more detail, expression vectors were prepared from which cDNA fragments encoding the amino acid sequence regions 208 to 222 (d4), 203 to 222 (d5), 198 to 222 (d6), 190 to 222 (d7), 147 to 162 (d8), 398 to 419 (d9), 374 to 419 (d10), and 332 to 351 (d11) of the amino acid sequence represented by SEQ ID NO:1 were deleted, and tested in a manner similar to that described above.

[0050] As a result, #7 did not react with d4, d5, d6, and d7 out of the five deletion mutants (d4, d5, d6, d7, and d8), and was therefore found to recognize the region from 208 to 222 in the amino acid sequence shown in SEQ ID NO: 1. Eleven clones, #2, #4, #9, #13, #40, #45, #55, #97, #104, #111, and #128, did not react with d11 out of the three deletion mutants (d9, d10, and d11), and were therefore found to recognize the region from 332 to 351 in the amino acid sequence shown in SEQ ID NO: 1. Ten clones, #35, #47, #65, #67, #86, #96, #98, #103, #112, and #131, did not react with d10 among the three deletion mutants (d9, d10, and d11), indicating that they recognize the region from 374 to 397 of the amino acid sequence shown in SEQ ID NO: 1. Five clones, #28, #60, #102, #135, and #144, did not react with d9 and d10 among the three deletion mutants (d9, d10, and d11), indicating that they recognize the region from 398 to 419 of the amino acid sequence shown in SEQ ID NO: 1. From the above, the antibodies prepared were classified as antibodies having epitopes in the regions of 208 to 222, 332 to 351, 374 to 397, or 398 to 419 of the amino acid sequence shown in SEQ ID NO: 1 (Figure 4B).

[0051] Example 4 Confirmation of the specificity of each monoclonal antibody We synthesized recombinant proteins of each NP to examine whether each monoclonal antibody specifically recognizes SARS-CoV-2-derived NP and does not react with NPs derived from other human coronaviruses (SARS, MERS, OC43, NL63, HKU1, and 229E). First, DNA sequences encoding each NP derived from other human coronaviruses (SARS, MERS, OC43, NL63, HKU1, and 229E) were synthesized (commissioned to GeneWiz) (represented by SEQ ID NOs: 10, 11, 12, 13, 14, and 15, respectively). Each DNA sequence was codon-optimized for protein synthesis in eukaryotic cells based on the amino acid sequence. Next, NPs derived from other human coronaviruses (SARS, MERS, OC43, NL63, HKU1, and 229E) were subjected to cell-free protein synthesis using a procedure similar to that described in Example 1.

[0052] Using each of the obtained proteins, the specificity of each monoclonal antibody was analyzed in the same manner as the Western blot method described in Example 3. The results are shown in Figure 5. A total of eight antibodies (one antibody recognizing the 208-222 region, three antibodies recognizing the 332-351 region, three antibodies recognizing the 374-397 region, and one antibody recognizing the 398-419 region) were obtained that recognized the NP derived from SARS-CoV-2 and did not react with the NP derived from other human coronaviruses (SARS, MERS, OC43, NL63, HKU1, and 229E). For example, #7, #9, and #98 did not react with the NP derived from other human coronaviruses (SARS, MERS, OC43, NL63, HKU1, and 229E), and specifically recognized only the NP derived from SARS-CoV-2. On the other hand, both #128 and #144 reacted with SARS-CoV-2-derived NP and SARS coronavirus-derived NP and specifically recognized SARS-CoV-2-derived NP and SARS coronavirus-derived NP.

[0053] Example 5. Immunostaining of SARS-CoV-2-infected cells We investigated whether the monoclonal antibodies (#7, #98) prepared in Example 2 exhibited reactivity against SARS-CoV-2-infected cells. First, African green monkey kidney-derived cells VeroE6 / TMPRSS2 (JCRB1819) were infected with SARS-CoV-2 provided by the National Institute of Infectious Diseases for 24 hours at 37°C.

[0054] [Confirmation of expression of SARS-CoV-2-derived NP] The expression of SARS-CoV-2-derived NP was confirmed in the same manner as the Western blot method described in Example 1. First, the lysate of the infected cells was mixed with 2xSDS sample buffer (125mM Tris-HCl, 4% SDS, 20% glycerol, 0.01% bromophenol blue, and 10% 2-mercaptoethanol), heat-treated, and subjected to polyacrylamide gel electrophoresis. Next, the gel after electrophoresis was transferred to a PVDF membrane. The membrane was blocked with a 2% skim milk solution and then reacted with peroxidase-labeled monoclonal antibodies (#7, #98). After that, it was reacted with an HRP-labeled secondary antibody, and then a peroxidase substrate solution was added to cause light emission, which was then detected using a chemiluminescence imaging device. As a result, the molecular weight of SARS-CoV-2-derived NP was 45.6 kDa, and a band was identified at the position corresponding to this molecular weight, confirming the expression of SARS-CoV-2-derived NP (Figure 6A).

[0055] [Immunostaining of SARS-CoV-2 infected cells] The monoclonal antibodies (#7, #98) prepared in Example 2 were added to the culture medium of the infected cells to react, and then fluorescently labeled goat anti-mouse immunoglobulin antibodies were added to immunostain the infected cells. The immunostained infected cells were then imaged using a fluorescent microscope. Non-infected cells were used as a negative control. As a result, a fluorescent reaction resulting from the binding of the monoclonal antibodies (#7, #98) prepared in Example 2 was observed only in the infected cells (FIG. 6B).

[0056] Example 6 Detection of SARS-CoV-2-derived NPs by ELISA Among the monoclonal antibodies obtained in the present invention, ELISA was performed using #9 and #98, which have different epitopes. SARS-CoV-2-derived NP (full length) was synthesized as a cell-free protein based on SEQ ID NO: 1 in the same manner as in Example 1. #9 was diluted to 2.5 μg / mL with PBS, immobilized on an ELISA plate, and blocked with a 2% skim milk solution. After washing the plate with PBS-T, SARS-CoV-2-derived NP serially diluted to 0, 1.25, 5, and 20 ng / mL was dispensed into the wells and reacted. After washing the plate with PBS-T, it was reacted with peroxidase-labeled #98. After washing the plate with PBS-T, a peroxidase substrate solution (KPL) was added to develop color, and the absorbance was measured. As a result, SARS-CoV-2-derived NPs could be detected at each concentration (Figure 7).

[0057] The various features of the present invention described herein can be combined in various ways, and all aspects obtained by such combinations, including combinations not specifically described herein, are within the scope of the present invention. In addition, those skilled in the art will understand that many different modifications are possible without departing from the spirit of the present invention, and equivalents including such modifications are also included in the scope of the present invention. Therefore, it should be understood that the embodiments described herein are merely examples and are not intended to limit the scope of the present invention.

Claims

1. An antibody or a fragment thereof that binds to a protein constituting a SARS-CoV-2-derived nucleocapsid, wherein the epitope recognized by the antibody or the fragment thereof is located at amino acid positions 121 to 419 of the amino acid sequence represented by SEQ ID NO:

1.

2. The antibody or fragment thereof according to claim 1, wherein the epitope is located at positions 208 to 222, 332 to 351, 374 to 397, or 398 to 419 of the amino acid sequence represented by SEQ ID NO:

1.

3. The antibody or fragment thereof according to claim 1 or 2, which specifically binds to a protein constituting the nucleocapsid derived from SARS-CoV-2.

4. The antibody or fragment thereof according to any one of claims 1 to 3, wherein the antibody is a monoclonal antibody.

5. The fragment may be a Fab fragment, a Fab / c fragment, an Fv fragment, a Fab' fragment or an F(ab') fragment. ２ The antibody or fragment thereof according to any one of claims 1 to 4, which is a fragment.

6. A method for detecting a protein constituting a SARS-CoV-2 nucleocapsid from a sample, the method comprising a step of contacting the sample with an antibody or a fragment thereof according to any one of claims 1 to 5.

7. The method of claim 6 , wherein the sample is a biological sample.

8. The method according to claim 6 or 7, further comprising detecting a protein constituting the SARS-CoV-2-derived nucleocapsid by at least one immunological test selected from the group consisting of an ELISA method, an immunochromatography method, and a filter antigen assay method.

9. A kit for detecting a protein constituting a SARS-CoV-2 nucleocapsid, comprising the antibody or fragment thereof according to any one of claims 1 to 5.

10. The kit according to claim 9, which is in the form of an immunochromatographic strip.

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