Immunological detection methods and reagents for SARS-CoV-2

Abstract

To provide a technique for immunologically detecting SARS-CoV-2.SOLUTION: An immunological method of detecting SARS-CoV-2 is provided, comprising detecting a SARS-CoV-2 N protein in a sample collected from a subject using the following antibodies (1) and (2): (1) a first antibody against a first epitope in the 260-305 amino acid regions in the SARS-CoV-2 N protein; and (2) a second antibody against a second epitope in the 365-419 amino acid regions in the SARS-CoV-2 N protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods and reagents for the immunological detection of SARS-CoV-2. [Background technology]

[0002] SARS-CoV-2, a member of the Betacoronavirus genus in the Coronaviridae family, is the causative agent of COVID-19, a novel coronavirus disease. SARS-CoV-2 is closely related to but distinct from the SARS-CoV (Severe Acute Respiratory Syndrome) that caused the severe acute respiratory syndrome (SARS) epidemic in China in the 2000s.

[0003] Coronaviruses are enveloped viruses whose viral genome is a single-stranded, positive-sense RNA. The nucleocapsid protein (N protein) in the virus particle forms dimers via the C-terminal domain (CTD), which then form tetramers. It is thought that the genomic RNA binds to the N-terminal domain (NTD) of the nucleocapsid protein (N protein) to form the nucleocapsid. The nucleocapsid is surrounded by an envelope made of a lipid bilayer membrane. The S protein, E protein, and M protein are bound to the envelope via their transmembrane regions. The N protein binds to the M protein, which also constitutes the envelope, to form the enveloped virus particle.

[0004] Although there have been no reports on the molecular properties of the N protein in the specimens, it is generally believed that (i) it is contained in the nucleocapsid of the virus particle, and (ii) it is released when virus-infected cells are disrupted by viral proliferation or the immune response of the infected host.

[0005] A method using antibodies against the SARS-CoV N protein (full length: 422 amino acids) has been reported as an immunological method for detecting SARS coronavirus.

[0006] Patent Document 1 describes various monoclonal antibodies against SARS-CoV N protein and immunoassay reagents using them. The immunoassay reagent disclosed is a sandwich immunoassay reagent that uses two or more types of antibodies that recognize different epitopes as solid-phase antibodies and labeled antibodies. Patent Document 1 also describes that the solid-phase antibodies and labeled antibodies contained in such immunoassay reagents can each be selected from two or more types of monoclonal antibodies and used in combination.

[0007] Patent Document 2 describes a method for measuring SARS-CoV N protein using a first antibody and a second antibody that specifically bind to the SARS-CoV N protein, wherein the first antibody or the second antibody recognizes an epitope present in the region from amino acids 283 to 422 from the N-terminus of the amino acid sequence of the N protein (region C). Patent Document 2 describes that SARS-CoV N protein (amino acids 1-422) is divided into three regions (region A: region 1-141, region B: 142-282, region C: 283-422), and that SARS-CoV N protein can be measured with higher sensitivity than conventional methods by using a combination of (a) an antibody against the region 283-422 and (b) an antibody against the region 1-141 or the region 142-282. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2005 / 042579 [Patent Document 2] International Publication No. 2007 / 043582 Summary of the Invention [Problem to be solved by the invention]

[0009] The object of the present invention is to provide a technique capable of immunologically detecting SARS-CoV-2. [Means for solving the problem]

[0010] After extensive research, the present inventors have found that SARS-CoV-2 can be detected with high sensitivity by using a combination of two specific antibodies against two epitopes in the C-terminal region of the SARS-CoV-2 N protein: (1) a first antibody against a first epitope in the region of amino acids 260 to 305 in the SARS-CoV-2 N protein; and (2) a second antibody against a second epitope in the region of amino acids 365 to 419 in the SARS-CoV-2 N protein. The present inventors have also found that these antibodies are easy to use in combination with other antibodies and are a useful antibody combination that can achieve more sensitive detection of SARS-CoV-2 when used in combination with other antibodies.

[0011] Although Patent Documents 1 and 2 disclose technologies that enable the immunological detection of SARS-CoV using antibodies against the SARS-CoV N protein (reported to have a full-length of 422 amino acids), they do not disclose the immunological detection of SARS-CoV-2 using antibodies against the SARS-CoV-2 N protein (reported to have a full-length of 419 amino acids). Furthermore, Patent Document 1 does not even describe the epitopes in the N protein that should be targeted in the immunological detection of SARS-CoV, which is a different virus from SARS-CoV-2. Patent Document 2 describes the use of a combination of two specific antibodies against two epitopes in three distinct regions, as described in (a) and (b) above, in the immunological detection of SARS-CoV, but does not teach or suggest that a combination of two specific antibodies against two epitopes in the C-terminal region of the SARS-CoV-2 N protein is superior for detecting SARS-CoV-2.

[0012] Based on the above findings, the inventors have succeeded in developing an immunological detection method and reagent for SARS-CoV-2, and have completed the present invention.

[0013] That is, the present invention is as follows. [1] A method for immunological detection of SARS-CoV-2, comprising detecting SARS-CoV-2 nucleocapsid protein (N protein) in a sample collected from a subject using the antibodies (1) and (2) below: (1) a first antibody directed against a first epitope in the region of amino acids 260 to 305 of the SARS-CoV-2 N protein; and (2) A second antibody directed against a second epitope in the region of amino acids 365 to 419 in the SARS-CoV-2 N protein. [2] The method of [1], further comprising using a third antibody against a third epitope in the amino acid region of positions 120 to 147 in the SARS-CoV-2 N protein. [3] The method of [1] or [2], further comprising using a fourth antibody against a fourth epitope in the region of amino acids 44 to 78 in the SARS-CoV-2 N protein. [4] Any of the methods described in [1] to [3], further comprising using a fifth antibody against a fifth epitope in the region of amino acids 243 to 259 in the SARS-CoV-2 N protein. [5] Any of the methods described in [1] to [4], further comprising using a sixth antibody against a sixth epitope in the region of amino acids 306 to 339 in the SARS-CoV-2 N protein. [6] The method according to any one of [1] to [5], wherein both the first antibody and the second antibody are used as solid-phase antibodies or labeled antibodies. [7] When both the first antibody and the second antibody are used as solid-phase antibodies, the third antibody is used as a labeled antibody; The method according to [6], wherein when both the first antibody and the second antibody are used as labeled antibodies, the third antibody is used as a solid-phase antibody. [8] The fourth antibody is used as a solid-phase antibody, and the fifth antibody is used as a labeled antibody; or The method according to [6] or [7], wherein the fourth antibody is used as a labeled antibody and the fifth antibody is used as a solid-phase antibody. [9] Any of the methods [1] to [8], wherein the detection is carried out by a sandwich method.

[10] An immunological detection reagent for SARS-CoV-2, comprising the following antibodies (1) and (2): (1) a first antibody directed against a first epitope in the region of amino acids 260 to 305 of the SARS-CoV-2 N protein; and (2) A second antibody directed against a second epitope in the region of amino acids 365 to 419 in the SARS-CoV-2 N protein.

[11] The reagent according to

[10] , further comprising one or more antibodies selected from the group consisting of the following (3) to (6): (3) A third antibody against a third epitope in the region of amino acids 120 to 147 of the SARS-CoV-2 N protein: (4) a fourth antibody against a fourth epitope in the region of amino acids 44 to 78 in the SARS-CoV-2 N protein; (5) a fifth antibody directed against a fifth epitope in the region of amino acids 243 to 259 in the SARS-CoV-2 N protein; and (6) A sixth antibody against a sixth epitope in the region of amino acids 306 to 339 in the SARS-CoV-2 N protein.

[12] The reagent of

[10] or

[11] , wherein both the first antibody and the second antibody are solid-phase antibodies or labeled antibodies.

[13] The reagent according to any one of

[10] to

[12] , wherein the reagent is a reagent used in a sandwich method.

[14] The reagent according to any one of

[11] to

[13] , wherein the reagent comprises a solid phase. [Effects of the Invention]

[0014] According to the present invention, SARS-CoV-2 can be detected with high sensitivity. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram showing an example of an immunochromatography cartridge. [Figure 2] FIG. 2 shows (A) the amino acid sequence of the SARS-CoV N protein (SEQ ID NO: 1), and (B) the amino acid sequence of the SARS-CoV-2 N protein (SEQ ID NO: 4). DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides an immunological detection method for SARS-CoV-2, comprising detecting SARS-CoV-2 N protein in a sample collected from a subject using the following antibodies (1) and (2): (1) a first antibody directed against a first epitope in the region of amino acids 260 to 305 of the SARS-CoV-2 N protein; and (2) A second antibody directed against a second epitope in the region of amino acids 365 to 419 in the SARS-CoV-2 N protein.

[0017] The subject from which the sample is obtained can be any subject that can be infected with SARS-CoV-2. Examples of such subjects include mammals (e.g., primates such as humans and monkeys; rodents such as mice, rats, and rabbits; ungulates such as cows, pigs, goats, horses, and sheep; and carnivores such as dogs and cats) and birds (e.g., chickens). Preferably, the subject is a mammal such as a human. From the perspective of clinical application, the subject is preferably a human.

[0018] The specimen may be any biological sample that may contain SARS-CoV-2. Examples of such specimens include saliva, sputum, nasal discharge, swabs (e.g., nasal swabs, pharyngeal swabs, and other mucosal swabs), lavage fluids (e.g., nasal washes, oral washes, bronchial washes, and lung washes), blood (e.g., whole blood, plasma, and serum), feces, and other specimens (e.g., specimens containing infected cells). To obtain specimens that may contain large amounts of SARS-CoV-2 in a minimally invasive, rapid, and simple manner, the specimen is preferably saliva, sputum, nasal discharge, or swab. The specimen may be pre-treated. Examples of such treatments include centrifugation, extraction, dilution, filtration, precipitation, heating, freezing, refrigeration, and agitation, as well as treatment with a surfactant or other component.

[0019] The SARS-CoV-2 N protein detected in the present invention is the N protein (i.e., native or mutant N protein) of any strain of SARS-CoV-2. Examples of such strains include the major L-type and S-type strains and their substrains. Numerous strains of SARS-CoV-2 have been reported. For example, the influenza virus gene database GISAID (Global Initiative on Sharing All Influenza Data) can be referenced for such numerous strains. The genome sequence information of SARS-CoV-2 has been disclosed (for example, see GenBank accession number MN908947 for the SARS-CoV-2 Wuhan-Hu-1 strain). Therefore, the N protein encoded by such a genome sequence can be referred to as the SARS-CoV-2 N protein. Furthermore, the amino acid sequence of SEQ ID NO: 4 can be referenced for the native N protein of SARS-CoV-2.

[0020] In the present invention, SARS-CoV-2 N protein is detected using the following antibodies (1) and (2): (1) a first antibody directed against a first epitope in the region of amino acids 260 to 305 of the SARS-CoV-2 N protein; and (2) A second antibody directed against a second epitope in the region of amino acids 365 to 419 in the SARS-CoV-2 N protein.

[0021] The first epitope is located in the region of amino acids 260 to 305 in the SARS-CoV-2 N protein.

[0022] The second epitope is located in the region of amino acids 365 to 419 in the SARS-CoV-2 N protein.

[0023] In one embodiment, the method of the present invention may further include the use of a third antibody directed against a third epitope in the region of amino acids 120 to 147 of the SARS-CoV-2 N protein. By using such a third antibody in combination with the antibodies (1) and (2) above, SARS-CoV-2 N protein can be detected with high sensitivity. The third epitope is located in the region of amino acids 120 to 147 of the SARS-CoV-2 N protein.

[0024] In another embodiment, the method of the present invention may further include the use of a fourth antibody directed against a fourth epitope in the region of amino acids 44 to 78 of the SARS-CoV-2 N protein. By using such a fourth antibody in combination with the antibodies (1) and (2) above, SARS-CoV-2 N protein can be detected with high sensitivity. The fourth epitope is located in the region of amino acids 44 to 78 of the SARS-CoV-2 N protein.

[0025] In yet another embodiment, the method of the present invention may further include the use of a fifth antibody directed against a fifth epitope in the region of amino acids 243 to 259 of the SARS-CoV-2 N protein. By further using such a fifth antibody in combination with the antibodies (1) and (2) above, SARS-CoV-2 N protein can be detected with high sensitivity. The fifth epitope is located in the region of amino acids 243 to 259 of the SARS-CoV-2 N protein.

[0026] In yet another embodiment, the method of the present invention may further include the use of a sixth antibody directed against a sixth epitope in the region of amino acids 306 to 339 of the SARS-CoV-2 N protein. By further using such a sixth antibody in combination with the antibodies (1) and (2) above, SARS-CoV-2 N protein can be detected with high sensitivity. The sixth epitope is located in the region of amino acids 306 to 339 of the SARS-CoV-2 N protein.

[0027] The present invention also provides a method for immunological detection of SARS-CoV-2, comprising detecting SARS-CoV-2 N protein in a sample collected from a subject using the following antibody combinations: (1) A combination of a third antibody and a first antibody; (2) a combination of a third antibody and a second antibody; (3) a combination of the fourth antibody and the first antibody; (4) a combination of a fourth antibody and a second antibody; (5) a combination of the sixth antibody and the first antibody; (6) a combination of the fifth antibody and the first antibody; or (7) A combination of the fifth antibody and the third antibody.

[0028] The antibody may be either a polyclonal antibody or a monoclonal antibody. The antibody may be of any immunoglobulin isotype (e.g., IgG, IgM, IgA, IgD, IgE, IgY). The antibody may also be a full-length antibody. A full-length antibody refers to an antibody comprising a heavy chain and a light chain, each of which comprises a variable region and a constant region (e.g., an antibody comprising two Fab portions and an Fc portion). The antibody may also be an antibody fragment derived from such a full-length antibody. An antibody fragment is a portion of a full-length antibody, and includes, for example, constant region-deleted antibodies (e.g., F(ab')2, Fab', Fab, Fv). The antibody may also be a modified antibody such as a single-chain antibody.

[0029] The antibody can be produced using a conventionally known method. For example, the antibody can be efficiently produced by using the above-mentioned epitope as an antigen.

[0030] In one embodiment, the antibody may be a solid-phase antibody. A solid-phase antibody refers to an antibody immobilized on a solid phase. Examples of solid phases include solid phases that can be suspended or dispersed in a liquid phase (e.g., solid-phase carriers such as particles and beads) and solid phases that can accommodate or carry a liquid phase (e.g., supports such as plates, membranes, and test tubes, and containers such as well plates, microchannels, glass capillaries, nanopillars, and monolith columns). Examples of solid phase materials include glass, silica, polymers (e.g., polystyrene, plastics), metals, and carbon. Nonmagnetic or magnetic materials can also be used as solid phase materials. Immobilization of an antibody on a solid phase can be performed by any method. Examples of such methods include covalent bonding, methods using affinity substances (e.g., biotin, streptavidin), ionic bonding, and physical adsorption. Examples of covalent bonding methods include periodic acid, glutaraldehyde, maleimide, and N-hydroxysuccinimide.

[0031] In another embodiment, the antibody may be a labeled antibody. A labeled antibody refers to an antibody labeled with a labeling substance. Examples of the labeling substance include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., one of streptavidin and biotin, one of complementary sense and antisense strand nucleic acids), fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridyl)ruthenium, luminol), radioactive substances (e.g., 3 H, 14 C. 32 P, 35 S, 125I), metal colloids (e.g., gold colloid, silver colloid, platinum colloid, iron oxide colloid, aluminum hydroxide colloid), and coloring substances (e.g., latex particles colored with dyes, dyes, pigments, etc.). Labeling of antibodies with labeling substances can be carried out by any method. Examples of such methods include the methods described above for immobilizing antibodies on solid phases.

[0032] In certain embodiments, both the first antibody and the second antibody may be used as solid-phase antibodies or labeled antibodies. Using both the first antibody and the second antibody together as solid-phase antibodies or labeled antibodies has the advantage of enabling more sensitive detection of SARS-CoV-2 N protein and making it easier to avoid binding competition that is often observed between solid-phase antibodies and labeled antibodies. More preferably, both the first antibody and the second antibody may be used as solid-phase antibodies.

[0033] In another specific embodiment, the third antibody may be used as a labeled antibody or a solid-phase antibody. More specifically, when both the first antibody and the second antibody are used as solid-phase antibodies, the third antibody may be used as a labeled antibody. Also, when both the first antibody and the second antibody are used as labeled antibodies, the third antibody may be used as a solid-phase antibody. When the third antibody is used in a form different from that of the first antibody or the second antibody, it can detect SARS-CoV-2 N protein with higher sensitivity.

[0034] In another specific embodiment, the fourth antibody and the fifth antibody may be used in different formats. More specifically, when the fourth antibody is used as a solid-phase antibody, the fifth antibody may be used as a labeled antibody. Also, when the fourth antibody is used as a labeled antibody, the fifth antibody may be used as a solid-phase antibody. When the fourth antibody and the fifth antibody are used in different formats, SARS-CoV-2 N protein can be detected with higher sensitivity.

[0035] In another specific embodiment, the sixth antibody may be used as a labeled antibody or a solid phase antibody.

[0036] The method of the present invention can detect SARS-CoV-2 N protein by an immunological method using the above-mentioned antibody. Examples of such immunological methods include direct competitive assays, indirect competitive assays, sandwich assays, Western blotting, and immunohistochemical staining. Such immunoassays may preferably be sandwich assays. Examples of such immunoassays include chemiluminescent immunoassays (CLIA) (e.g., chemiluminescent enzyme immunoassays (CLEIA)), turbidimetric immunoassays (TIA), enzyme immunoassays (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioimmunoassays (RIA), latex agglutination assays, fluorescent immunoassays (FIA), and immunochromatography. Any measurement system that uses antigen-antibody binding as the principle of specific detection is applicable. The method of the present invention may be a qualitative or quantitative method.

[0037] The detection of the label can be carried out by a method appropriately selected depending on the type of label. For example, when the label is an enzyme, the label can be detected by detecting the enzymatic activity using a signal-generating substrate (e.g., a fluorescent substrate, a luminescent substrate, or a chromogenic substrate). When the label is an affinity substance, the label can be detected by using an enzyme or signal-generating substance capable of binding to the affinity substance and detecting the enzyme or signal-generating substance bound to the affinity substance. Such an enzyme or signal-generating substance capable of binding to an affinity substance may be an enzyme or signal-generating substance bound to a substance capable of binding to the affinity substance. When the label is a fluorescent substance, a luminescent substance, or a radioactive substance, the label can be detected by detecting the signal generated from the label. When a chromogenic substance is used as the labeling substance, the label can be detected visually.

[0038] In a particular embodiment, the method of the present invention may be carried out by a sandwich method, which has excellent sensitivity and specificity.

[0039] In the sandwich method, detection of N protein may be carried out, for example, by treating the specimen with a solid-phase antibody to bind the N protein in the specimen to the solid-phase antibody (e.g., by contacting the specimen with the solid-phase antibody), and detecting the N protein bound to the solid-phase antibody with a labeled antibody. Detection of N protein may further include a step of removing N protein that is not bound to the solid-phase antibody (e.g., (B / F separation or washing step). In addition, in the sandwich method, detection of N protein may be carried out, for example, by treating the specimen with a labeled antibody to bind the N protein in the specimen to the labeled antibody (e.g., by contacting the specimen with the labeled antibody), further treating the N protein with the solid-phase antibody to bind the N protein to the solid-phase antibody, and detecting the N protein bound to the solid-phase antibody and the labeled antibody with the labeled antibody. In addition, the solid-phase antibody may be a antibody that has been previously immobilized on a solid phase. For example, an antibody that can be immobilized on a solid phase may be used when detecting N protein. For example, the method may include the steps of treating a sample with an antibody that can be immobilized on a solid phase to bind the N protein in the sample to the antibody (e.g., contacting the sample with a solid-phase antibody), binding the N protein bound to the antibody that can be immobilized on a solid phase to a labeled antibody, immobilizing the antibody that can be immobilized on a solid phase to the solid phase, and detecting the N protein bound to the antibody immobilized on the solid phase (solid-phase antibody) and the labeled antibody with the labeled antibody. The affinity substances described above can be used to bind the antibody immobilized on the solid phase to the solid phase.

[0040] In another specific embodiment, the method of the present invention may be carried out by immunochromatography. Immunochromatography is preferred as a rapid and simple qualitative method. Immunochromatography and the instruments used therein are well known. For example, an immunochromatography cartridge (which may further include a reservoir for a developing solution) having a zone containing a solid-phase antibody and a zone containing a labeled antibody can be used as such an instrument. Below, as a preferred example of immunochromatography, an outline of a lateral flow immunochromatography method and instrument will be described with reference to FIG. 1.

[0041] In FIG. 1, the immunochromatography cartridge 1 comprises a matrix 2 made of a porous material such as a nitrocellulose membrane. The matrix 2 comprises a detection zone 3 (a zone containing a solid-phase antibody) in which a capture antibody is immobilized in a line. The matrix 2 is also provided with a labeled reagent zone 4 (a zone containing a labeled antibody) upstream of the detection zone 3 (upstream in the direction of the flow of the developer solution described below). The labeled reagent zone 4 is typically formed in a strip shape. The labeled antibody is deposited on a porous pad. The upstream end of the matrix is ​​provided with a developer solution tank 5 containing a developer solution. Further downstream from the detection zone, a development confirmation section 6 is provided to confirm the flow of the developer solution. Further downstream, a developer solution absorption zone 7 is provided with a porous absorbent pad for absorbing the developer solution. In the development confirmation section, a probe (e.g., an antibody) that has affinity for a substance other than the detection substance (N protein), such as an anti-labeled antibody, that flows with the developer solution is immobilized in a line. Furthermore, if the label is an enzyme, a substrate zone 8 containing a substrate for the enzyme is provided upstream of the labeled reagent zone. The developer tank can be easily broken and the developer supplied to the matrix by providing a member (not shown), such as a push-in part with a protrusion, that can break the developer tank by pushing it, near the developer tank 5. Furthermore, a developer pad 9 may be provided to cover the developer tank and the upper end of the matrix so that the developer in the broken developer tank can be easily supplied to the upper end of the matrix.

[0042] During use, the sample is added to the labeled reagent zone 4, and the developer tank 5 is broken, allowing the developer to contact the upper end of the matrix and supply it. The developer supplied to the matrix flows downstream due to the matrix's capillary action. As the developer passes through the substrate zone, the substrate is eluted into the developer, and the developer containing the substrate flows. Subsequently, as the developer passes through the labeled reagent zone, the labeled antibody and sample are eluted into the developer, and the developer containing the substrate, labeled antibody, and sample flows. If the sample contains N protein, the N protein and the labeled antibody bind via an antigen-antibody reaction. When they reach the detection zone, the solid-phase antibody and N protein bind via an antigen-antibody reaction in the detection zone. As a result, the labeled antibody is immobilized in the detection zone via the N protein. The N protein is detected by detecting the label immobilized in the detection zone. If the sample does not contain N protein, nothing binds to the solid-phase antibody, and the labeled antibody is not immobilized in the detection zone. The developer continues downstream and reaches the developer confirmation section. For example, if an anti-labeled antibody is immobilized in the developer confirmation section, the labeled antibody that did not bind to the N protein will bind to the anti-labeled antibody through an antigen-antibody reaction, resulting in the labeled antibody being immobilized in the developer confirmation section. If a label is detected in the developer confirmation section, it can be confirmed that the developer has been properly developed up to the development confirmation section. The developer is then absorbed by the absorbent pad further downstream.

[0043] The present invention also provides an immunological detection reagent for SARS-CoV-2, comprising the following antibodies (1) and (2): (1) a first antibody directed against a first epitope in the region of amino acids 260 to 305 of the SARS-CoV-2 N protein; and (2) A second antibody directed against a second epitope in the region of amino acids 365 to 419 in the SARS-CoV-2 N protein.

[0044] The reagent of the present invention may further comprise one or more antibodies selected from the group consisting of the following (3) to (6): (3) A third antibody against a third epitope in the region of amino acids 120 to 147 of the SARS-CoV-2 N protein: (4) a fourth antibody against a fourth epitope in the region of amino acids 44 to 78 in the SARS-CoV-2 N protein; (5) a fifth antibody directed against a fifth epitope in the region of amino acids 243 to 259 in the SARS-CoV-2 N protein; and (6) A sixth antibody against a sixth epitope in the region of amino acids 306 to 339 in the SARS-CoV-2 N protein.

[0045] The present invention also provides an immunological detection reagent for SARS-CoV-2, comprising the following antibody combination: (1) A combination of a third antibody and a first antibody; (2) a combination of a third antibody and a second antibody; (3) a combination of the fourth antibody and the first antibody; (4) a combination of a fourth antibody and a second antibody; (5) a combination of the sixth antibody and the first antibody; (6) a combination of the fifth antibody and the first antibody; or (7) A combination of the fifth antibody and the third antibody.

[0046] Details of the terms such as the above-mentioned antibodies in the reagents of the present invention (eg, definitions, examples, and preferred examples) are the same as those described in the method of the present invention.

[0047] The reagent of the present invention can contain the first and second antibodies in the form of a mixture (e.g., contained in the same container, or in the form of solid-phase antibodies immobilized on the same solid phase), or in a form separated from each other (e.g., contained in different containers). The reagent of the present invention can also contain, in addition to the first and second antibodies, one or more antibodies selected from the group consisting of (3) to (6) below, in the form of a mixture or separated from each other. The reagent of the present invention can be provided in the form of a kit. The reagent of the present invention can also be provided in the form of a device. Specifically, all of the antibodies can be contained in the device. Alternatively, some of the antibodies can be contained in the device, and the remaining antibodies can be not contained in the device (e.g., contained in different containers). In this case, the antibodies not contained in the device can be injected into the device at the time of detection.

[0048] The reagent of the present invention may contain a solid-phase antibody and / or a labeled antibody. Alternatively, if such a reagent does not contain a solid-phase antibody and / or a labeled antibody, it may contain a solid phase and / or a labeled substance. The solid phase and labeled substance are the same as those described above. When the labeled substance is an enzyme, the reagent of the present invention may contain a substrate for the enzyme (e.g., a substrate that generates a detectable signal, or a substrate that is converted by the enzyme into a product that generates a detectable signal, or a substrate in a reaction that can be coupled to another enzymatic reaction that utilizes a substrate that generates a detectable signal, or a substrate that is converted by the enzyme into a product that generates a detectable signal).

[0049] The reagent of the present invention is not particularly limited as long as it can be used in immunoassays, and can have a configuration appropriate for the type of immunoassay. Preferably, the reagent of the present invention may be a reagent used in a sandwich assay. Therefore, the reagent of the present invention may contain a solid phase used in a sandwich assay (e.g., particles such as magnetic particles, a well plate, a membrane), or a solid-phase antibody immobilized on such a solid phase. The reagent of the present invention may also contain a device containing a solid phase used in immunochromatography (i.e., the immunochromatography cartridge as described above), or a solid-phase antibody immobilized on such a solid phase. The reagent of the present invention may also contain an N protein preparation. [Example]

[0050] The present invention will now be described in more detail with reference to examples, but is not limited to the following examples. Note that, insofar as the amounts described in the following examples are concerned, % means % by weight (wt%).

[0051] Example 1: Antibody production The antibody was produced according to the method described in WO 2005 / 042579. Specifically, the procedure is as follows.

[0052] (1) Production and purification of recombinant N protein The nucleocapsid protein (N protein) gene of SARS coronavirus (SARS-CoV) was inserted into an expression plasmid to construct the plasmid pWS-N. This plasmid was used to transform Escherichia coli, resulting in ampicillin-resistant transformants. The nucleotide and amino acid sequences of the N protein are shown in SEQ ID NOs: 1 and 2, respectively.

[0053] The resulting transformants were cultured at 37°C in 2 ml of LB medium containing 50 μg / ml ampicillin. The transformants were grown in a preliminary culture until the density reached approximately 0.7 (OD at 600 nm), and then 0.4 mM IPTG was added to induce expression. After 18 hours of culture, the cells were centrifuged to recover E. coli. The recovered E. coli was added to 20 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM PMSF and sonicated under ice cooling. After centrifugation, ammonium sulfate was added to the soluble fraction, and a 20-40% ammonium sulfate fraction was recovered. This ammonium sulfate fraction was applied to SP Sepharose Fast Flow (Amersham) equilibrated with 0.1 M NaCl, 8 M urea, and 20 mM phosphate buffer (pH 6.9) and purified by elution with 0.2 M NaCl, 8 M urea, and 20 mM phosphate buffer (pH 6.9). The eluted fraction was dialyzed against 0.2 M NaCl, 20 mM Tris-HCl buffer (pH 8.0) to obtain a purified recombinant CoV N protein solution. The purity of the recombinant N protein was confirmed by SDS-PAGE and Western blotting, revealing a single band.

[0054] (2) Production of monoclonal antibodies Anti-N protein monoclonal antibodies were generated by immunizing mice with the recombinant N protein (CoV) prepared above and fusing the splenic lymphocytes of the mice with myeloma cells. Specifically, BALB / C mice were immunized with 50–100 μg / mouse of recombinant N protein emulsified in Freund's complete adjuvant as a primary immunization. Two to three weeks later, they were boosted with 50–100 μg / mouse of the same antigen emulsified in Freund's incomplete adjuvant. Antibody titers were assessed by solid-phase ELISA using a 96-well ELISA plate coated with recombinant N protein. Mice showing elevated antibody titers were intravenously administered 25–100 μg of free recombinant N protein. Three to four days later, the spleens were removed and splenocytes were prepared. Mouse myeloma cells (P3U1) cultured in RPMI-1640 medium were mixed with splenocytes at a ratio of 1:2 to 1:5, and cell fusion was performed using PEG. The fused cells were suspended in HAT medium, then dispensed into 96-well culture plates and cultured in a CO2 incubator at 37°C.

[0055] Antibody screening was performed using the solid-phase ELISA described above. Specifically, recombinant N protein was dispensed into 96-well ELISA plates at a concentration of 1 μg / mL, at 50 μL / well, and allowed to adsorb overnight at 4°C. After blocking with 1% skim milk, the wells were washed three times with wash buffer (PBS containing 0.05% Tween). 50 μL of culture supernatant from the cell fusion plate was added and incubated at 37°C for 1 hour. After washing three times with wash buffer, a POD-labeled anti-mouse immunoglobulin antibody (DACO) was added and incubated for another hour at 37°C. After washing four times with wash buffer, the substrate ABTS was added, and wells showing color development were selected. Next, the cells from the selected wells were transferred to a 24-well culture plate and incubated in a CO2 incubator at 37°C. Single clones were isolated by limiting dilution, and each monoclonal antibody was obtained.

[0056] In addition, instead of the recombinant N protein (CoV), mice were immunized with a KLH conjugate of a synthetic peptide (N5 peptide: GQTVTKKSAAEASKKPRC: SEQ ID NO: 3) as an immunogen, and anti-N protein monoclonal antibodies were similarly obtained.

[0057] (3) Confirmation of monoclonal antibody reactivity The reactivity of each established monoclonal antibody against the native antigen (N protein derived from SARS-CoV) was confirmed by Western blot (WB) using concentrated virus suspension as a sample. VeroE6 cells were infected with the SARS virus Hanoi strain and cultured in a CO2 incubator for 48 hours. After centrifugation at 2,000 rpm for 15 minutes, the virus culture supernatant (TCID50 of 7.95 × 10 6 A 100-μL (100 μg / mL) solution was prepared. The culture supernatant was inactivated at 56°C for 90 minutes, and 31.5 mL of the supernatant was centrifuged at 30,000 rpm for 3 hours in a Hitachi ultracentrifuge (40T rotor). 0.3 mL of Tris-NaCl-EDTA buffer was added to the resulting precipitate, and the mixture was pipetted to prepare a concentrated virus suspension. An equal volume of sample treatment solution for electrophoresis was added to this suspension, followed by heating to prepare the analytical sample. After SDS-PAGE using a 12.5% ​​gel, the sample was transferred to a nitrocellulose membrane to prepare a WB transfer membrane (antigen transfer WB membrane). After blocking the transfer membrane with skim milk, each monoclonal antibody was reacted with the antigen transfer WB membrane at room temperature for 1 hour with shaking, followed by washing three times with wash buffer (5 minutes with shaking). Next, a POD-labeled anti-mouse immunoglobulin antibody was added and reacted for another 1 hour at room temperature. After washing four times with washing buffer (5 minutes of shaking wash), 4-chloronaphthol solution was added as a substrate, and bands were confirmed. Monoclonal antibodies that showed bands at positions corresponding to N protein with a molecular weight of just under 50 kD were selected.

[0058] The selected monoclonal antibodies were further subjected to WB using the recombinant SARS-CoV-2 N protein and the common coronavirus N protein prepared in the same manner as in (1) above, and those that bound to the recombinant SARS-CoV-2 N protein but not to the N protein of the common coronavirus were designated as anti-N protein monoclonal antibodies. In the following examples, unless otherwise specified, the N protein used was the SARS-CoV-2 N protein.

[0059] Example 2: Identification of antibody reactive regions (part 1) The epitopes of the multiple anti-N protein monoclonal antibodies obtained in Example 1 above were identified by the WB method using a recombinant antigen containing a partial sequence of the N protein.

[0060] First, an equal amount of electrophoresis sample treatment solution was added to a solution containing recombinant antigens of the full-length SARS-CoV-2 N protein (419 amino acids), NTD (amino acids 44 to 180), CTD (amino acids 247 to 364), and the C-terminal peptide of the N protein (amino acids 330 to 419), prepared in the same manner as in Example 1 above. The solution was then heated to prepare a sample for analysis. SDS-PAGE and WB were performed in the same manner as in Example 1 (3) above to confirm the reactivity of each N protein monoclonal antibody.

[0061] As a result, six antibodies (anti-N protein antibodies N1 to N6) were confirmed to react with the full-length antigen and NTD antigen, and six antibodies (anti-N protein antibodies C1 to C6) were confirmed to react with the full-length antigen and CTD antigen, confirming the isolation of several antibodies reactive with both the NTD and CTD. Furthermore, antibodies (anti-N protein antibodies C12 and C13) were also isolated that recognized a region contained in the amino acid sequence at positions 365 to 419 of the N protein, which reacted with the full-length antigen and C-terminal peptide but not with the NTD or CTD antigen. Furthermore, several anti-N protein monoclonal antibodies (P1 and P2) that recognized the region at positions 243 to 259 of the N protein were obtained by immunizing mice with a KLH conjugate of a synthetic peptide (N5 peptide: GQTVTKKSAAEASKKPRC; SEQ ID NO: 3) as an immunogen.

[0062] Example 3: Preparation of solid-phase antibody (antibody-immobilized magnetic particles) The anti-N protein monoclonal antibody obtained above was chemically bound to magnetic particles (Fujirebio) using a carboxyl-amine crosslinker (carbodiimide, Thermo-Fisher Scientific) according to the product manual to obtain a solid-phase antibody (antibody-immobilized magnetic particles).

[0063] Example 4: Preparation of labeled antibody (alkaline phosphatase-labeled anti-N protein monoclonal antibody) The anti-N protein monoclonal antibody obtained above was mixed with pepsin in 0.1 M citrate buffer (pH 3.5) and allowed to stand at 37°C for 1 hour for pepsin digestion. After terminating the reaction, the antibody was purified by gel filtration to obtain an antibody from which the Fc region had been removed. 2-Mercaptoethylamine hydrochloride (2-MEA) was then added to thiolate the antibody. The resulting antibody was then desalted to obtain the Fab' fragment.

[0064] N-(4-maleimidobutyryloxy)-succinimide (GMBS)-treated alkaline phosphatase and Fab' fragments prepared from each anti-N protein monoclonal antibody were mixed at a molar ratio of 1 to 3:1, and coupling was performed. 2-Mercaptoethylamine hydrochloride and iodoacetamide were added to the coupling solution to terminate the reaction. Further, peaks with molecular weights where the Fab' to ALP ratio was 1 to 3:1 were pooled by gel filtration to obtain labeled antibodies (ALP-labeled anti-N protein monoclonal antibodies).

[0065] Example 5: Particle-based sandwich immunoassay 100 μL of the solution containing the recombinant N protein or the sample was added to 50 μL of a diluted particle suspension (50 mM Tris, 1% BSA, 150 mM NaCl, 1 mM EDTA, 0.1% NaN3) containing 0.03% antibody-immobilized magnetic particles prepared in Example 3 above, and the mixture was allowed to react at 37°C for 8 minutes.

[0066] After the reaction, the plate was separated into B / F using a magnet and washed with Lumipulse® washing solution (Fujirebio). 50 μL of labeled solution (50 mM MES, 150 mM NaCl, 1% BSA, 3 mM MgCl2, 0.3 mM ZnCl2) containing 1 μg / mL of the ALP-labeled antibody prepared in Example 4 was added and incubated for 8 minutes at 37°C. After B / F separation using a magnet and washing with Lumipulse® washing solution, 200 μL of Lumipulse® substrate solution (Fujirebio) containing 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD) was added. The enzyme reaction was carried out at 37°C for 4 minutes, and the luminescence at 463 nm was measured.

[0067] Example 6: Immunochromatography As shown in Figure 1, a 0.5 μL solution containing an anti-N protein monoclonal antibody was spotted on a 3.7 mm wide, 50 mm long nitrocellulose membrane matrix 15 mm from the end of the developer absorption zone, followed by drying, to create a detection zone. An anti-alkaline phosphatase (ALP) antibody solution (sodium carbonate buffer containing 0.15 M NaCl) was then spotted 12 mm from the end of the developer absorption zone, followed by drying, to create a development confirmation zone. A 0.9 μL line of 5-bromo-4-chloro-3-indolyl phosphate disodium salt (BCIP) solution was then spotted as a substrate, creating a substrate zone. Next, 3 μL of a solution containing an ALP-labeled anti-N protein monoclonal antibody (labeled antibody) was spotted on the matrix and dried, creating a labeled reagent zone consisting of an enzyme-labeled reagent pad.

[0068] The matrix, developer pad, enzyme-labeled reagent pad, and absorbent pad (highly water-retentive filter paper) were stacked and secured with adhesive tape as shown in Figure 1, and then secured to a plastic case having a developer tank 11 to produce an immunochromatography cartridge for detecting SARS-N protein.

[0069] 20 μL of a solution containing recombinant N protein or a sample was mixed with 100 μL of treatment solution and allowed to stand at room temperature for approximately 5 minutes. The N protein in the resulting mixture was detected using the manufactured immunochromatography cartridge. Specifically, 20 μL of the mixture was dropped into the labeled antibody zone of the immunochromatography cartridge, the developer tank was broken, and the developer was supplied to the developer pad and the top of the matrix to begin measurement. Thirty minutes after the start of measurement, the development of the developer was confirmed by the color development in the development confirmation section 10, and the color development in the detection zone was then visually measured.

[0070] Example 7: Identification of antibody reactive region (part 2) Recombinant N protein (SARS-CoV-2 or SARS-CoV) was measured using the sandwich immunoassay method described in Example 5, and the samples were grouped according to the reactive region of each N protein monoclonal antibody. Specifically, the recombinant N protein was measured using N1 to N6 antibodies, C1 to C6 antibodies, and C12 antibody as antibody-immobilized magnetic particles (solid-phase antibodies) and ALP-labeled antibodies, respectively.

[0071] As a result, when recombinant N protein (0, 10 ng / mL) was measured using N1 antibody and N2 antibody as the solid-phase and labeled antibodies, respectively, the ratio of the luminescence intensity at 10 ng / mL to the luminescence intensity at 0 ng / mL (background value) was very low, confirming that the two antibodies react to overlapping regions (Table 1). Similarly, when recombinant N protein (0, 1 ng / mL) was measured using N4, N5, and N6 antibodies (N6 antibody group), respectively, as the solid-phase and labeled antibodies, the difference in luminescence intensity (1 ng / mL minus the luminescence intensity at 0 ng / mL) was very low, confirming that these antibodies recognize overlapping regions (Table 2). On the other hand, when N1 antibody and N3 antibody were used as the solid-phase and labeled antibodies, the difference in luminescence intensity was smaller than with other antibody combinations, confirming partial competition. Since the N1 antibody showed higher reactivity with other NTD antibodies than the N3 antibody in sandwich immunoassays, the N1 antibody group (N1 and N2 antibodies) was considered to be more useful than the N3 antibody in sandwich immunoassays using multiple antibodies.

[0072] [Table 1]

[0073] [Table 2]

[0074] Similarly, when the C1 to C6 antibodies were examined, it was confirmed that the C1 and C2 antibodies recognized an overlapping region, the C3 and C4 antibodies recognized an overlapping region, and the C5 and C6 antibodies recognized an overlapping region (Tables 3 to 5). However, the luminescence intensity was low in all combinations of the C5 and C6 antibodies, confirming that antibodies against the region recognized by the C5 antibody group (C5 and C6 antibodies) had low reactivity in measurements by sandwich immunoassay.

[0075] [Table 3]

[0076] [Table 4]

[0077] [Table 5]

[0078] Example 8: Examination of antibody combinations (part 1) Combinations of solid-phase antibodies and labeled antibodies were examined for the antibodies grouped in Example 7 above and the C12 antibody. Recombinant N protein was measured using each of the antibody combinations shown in Table 6, using the same sandwich immunoassay method as in Example 5 above. N=2 was measured for each.

[0079] The results are shown in Table 6. Table 6 shows the ratio (S / N ratio) of the average luminescence intensity from 1 ng / mL samples to the average luminescence intensity from 0 ng / mL samples.

[0080] [Table 6]

[0081] As a result, it was confirmed that the following combinations of solid-phase antibodies and labeled antibodies for sandwich immunoassays were preferable, as they all showed high S / N ratios: N1 antibody and C3 antibody, N1 antibody and C12 antibody, N4 antibody and C3 antibody, N4 antibody and C12 antibody, and C3 antibody and C12 antibody.

[0082] Example 9: Examination of antibody combinations (part 2) Combinations of solid-phase antibodies and labeled antibodies were investigated for the antibodies grouped in Example 7 above, as well as the P1 and C12 antibodies. Using the immunochromatography method of Example 6 above, samples containing 10 ng / mL of recombinant N protein were measured using the antibody combinations shown in Tables 7 and 8. Furthermore, using the antibody combinations shown in Tables 7 and 8, the development of the developer was confirmed by the color development in the development confirmation section 10 30 minutes (Table 7) or 60 minutes (Table 8) after the start of measurement, and the color development in the detection zone was then visually measured. The results are shown in Tables 7 and 8. Tables 7 and 8 show the color development intensity of the detection zone under each condition. The larger the number, the stronger the color development, and "w" indicates weaker color development. In other words, the color development intensity decreases in the following order: 4.5 > 4 > 3 > 2 > 1 > w. Blank cells indicate that no experiment was performed.

[0083] [Table 7]

[0084] [Table 8]

[0085] As a result, similar to the sandwich immunoassay using particles, strong luminescence intensities were confirmed for the combinations of N1 antibody, N2 antibody and C3 antibody, N1 antibody and C12 antibody, N4 antibody and C3 antibody, N4 antibody and C12 antibody, and C3 antibody and C12 antibody. Furthermore, N1 antibody, N2 antibody and N4 antibody also showed relatively high luminescence intensities.

[0086] Furthermore, when the combination of the C1 antibody and the P1 antibody with other antibodies was also evaluated, the C1 antibody showed relatively strong luminescence intensity in combination with the C3 antibody, whereas the P1 antibody showed strong luminescence intensity in combination with the C3 antibody, and in combination with the N1 antibody and the N4 antibody.

[0087] Example 10: Examination of antibody combinations (part 3) In Examples 8 and 9, a combination of a single solid-phase antibody and a single labeled antibody was investigated. While a sufficiently sensitive assay system could be constructed using such a combination, it is more preferable to use a combination of solid-phase antibodies and / or labeled antibodies that recognize different regions. Therefore, an immunoassay system was constructed using one of the antibodies shown to be effective alone in Examples 8 and 9 as the labeled antibody and multiple other antibodies as solid-phase antibodies. A sample containing 10 ng / mL of recombinant N protein (SARS-CoV) was evaluated using the same method as in Example 8. The solid-phase antibody was prepared by simultaneously chemically binding multiple antibodies to particles according to the manufacturing method in Example 3. The results are shown in Table 9.

[0088] The results showed that the use of a mixture of C3 and C12 antibodies significantly improved the luminescence intensity (counts at 1000 pg / ml) and S / N ratio. In particular, when N1 or N4 antibodies were used as the labeled antibody, the use of a mixture of C3 and C12 antibodies as the solid-phase antibody increased the luminescence intensity and S / N ratio compared to the use of C3 and C12 antibodies alone. On the other hand, when C3 or C12 antibodies were used as the labeled antibody, the use of a mixture of N1 and N4 antibodies as the solid-phase antibody did not increase either the count value or the S / N ratio. This demonstrates that the use of a combination of C3 and C12 antibodies is effective in improving sensitivity in sandwich immunoassays, not simply because multiple antibodies are used as the solid-phase antibody.

[0089] [Table 9]

[0090] Example 11: Identification of antibody reactive region (part 3) The antibodies confirmed to be effective in Examples 8 to 10 above were analyzed for epitopes of the N protein (SARS-CoV-2).

[0091] For the N1 antibody group (N1 and N2 antibodies) and the N6 antibody group (N4 to N6 antibodies), which recognize the NTD, the reactive regions were analyzed by WB using recombinant antigens consisting of the N-terminal region (amino acids 44 to 112), the central region (amino acids 79 to 147), and the C-terminal region (amino acids 113 to 180) of the NTD, which were prepared in the same manner as in Example 1 above, and recombinant antigens consisting of amino acids 1 to 119, 110 to 229, 220 to 339, and 330 to 419 of the N protein (trNP1, trNP2, trNP3, and trNP4 antigens). The results showed that the N1 antibody group recognized the region of amino acids 44 to 78 of the N protein. Furthermore, the N6 antibody group was shown to recognize the region of amino acids 120 to 147 of the N protein.

[0092] Similarly, the C3 and C1 antibody groups, which recognize the CTD, were analyzed for their reactive regions by WB and sandwich assays using recombinant antigens consisting of the N-terminal region (amino acids 247-305), the central region (amino acids 276-334), and the C-terminal region (amino acids 306-364) of the CTD, as well as trNP1, trNP2, trNP4, and trNP4 antigens, prepared as in Example 1 above, and by their reactivity with synthetic peptides and other antibodies. The results showed that the C3 antibody group recognized the region of amino acids 260-305 of the N protein. Furthermore, the C1 antibody group was shown to recognize the region of amino acids 306-339 of the N protein.

[0093] In summary, the epitopes of the analyzed antibodies were as follows: [Table A]

[0094] Example 12: Examination of antibody combinations (part 4) In Example 10 above, it was shown that when using the N1 or N4 antibody, it is useful to use a mixture of the C3 and C12 antibodies as the solid-phase antibody. To confirm that these antibody combinations are also useful for SARS-CoV-2, a solution containing 100 pg / mL of recombinant N protein was measured using the combinations of solid-phase and labeled antibodies listed in Table 10 in the same manner as in Example 5 above.

[0095] Specifically, 100 μL of a solution containing recombinant N protein and 20 μL of a treatment solution (Tris buffer, 150 mM NaCl, 0.1% NaN3, 2.5% C16APS) were added to 50 μL of a diluted particle suspension containing solid-phase antibody, and the mixture was allowed to react for 8 minutes at 37°C. After the reaction, the mixture was separated into B / F groups using a magnet, washed with Lumipulse washing solution, and then 50 μL of a labeled body solution containing ALP-labeled antibody was added and allowed to react for 8 minutes at 37°C. After B / F separation using a magnet and washed with Lumipulse washing solution, 200 μL of Lumipulse substrate solution containing AMPPD was added, and the enzyme reaction was carried out for 4 minutes at 37°C. The N protein content of each sample was measured by measuring the amount of luminescence at a wavelength of 463 nm. The solid-phase antibody was prepared by chemically coupling each antibody to particles and then mixing them 1:1 as described in Example 3 above. The labeled antibody was prepared according to Example 4 above by simultaneously mixing GMBS-treated ALP with Fab' fragments prepared from multiple anti-N protein monoclonal antibodies so that the molar ratio of each Fab' fragment to ALP was 1:1, and coupling was performed to prepare the ALP-labeled antibody.

[0096] The results are shown in Table 10. As a result, when a mixture of C3 and C12 antibodies was used, a significant improvement in luminescence (counts at 100 pg / ml) and S / N ratio was observed. It was confirmed that using a combination of C3 and C12 antibodies is also effective in improving the sensitivity of sandwich immunoassays for SARS-CoV-2.

[0097] [Table 10]

[0098] Example 13: Examination of antibody combinations (part 5) In Examples 10 and 12 above, it was demonstrated that when using the N1 or N4 antibody, it is useful to use a mixture of the C3 and C12 antibodies as the solid-phase antibody. As mentioned above, because mutations and other factors may occur in SARS-CoV-2, it is preferable to use a combination of antibodies that recognize different regions. Therefore, we evaluated the combination of the P1 / P2 antibody, which is an antibody from a group with a different reactivity from the N1 / N2, N4 / N6, C3 / C4, and C12 / C13 antibody groups and which is highly reactive with the N1, N4, and C3 antibodies in Example 10 above. Combinations with the C1 / C2 antibody were also evaluated.

[0099] Specifically, the combinations of solid-phase antibodies and labeled antibodies listed in Tables 11-1 to 11-3 were used and evaluated in the same manner as in Example 8 above. The solid-phase antibodies were prepared by simultaneously chemically binding multiple antibodies to particles as in Example 3 above. The labeled antibodies were prepared as in Example 4 above by simultaneously mixing and coupling alkaline phosphatase (ALP) treated with GMBS with Fab' fragments prepared from multiple anti-N protein monoclonal antibodies so that the molar ratio of each Fab' fragment to ALP was 1:1, to prepare ALP-labeled antibodies.

[0100] The samples used were SARS-CoV-2-negative saliva samples to which recombinant N protein or nasal swabs 1 to 5 from SARS-CoV-2-positive patients (purchased samples: Boca Biolistics, LLC) had been added. 100 μL of sample was added to 170 μL of sample treatment solution (Tris buffer, 150 mM NaCl, 1% BSA, 0.1% NaN3, 0.25% C16APS).

[0101] The results are shown in Tables 11-1 to 11-3. Tables 11-1 to 11-3 show the ratio of the luminescence intensity when measuring 10 ng / mL of recombinant N protein to the luminescence intensity when measuring 0 ng / mL of recombinant N protein (S / N ratio), and the ratio of the luminescence intensity when measuring each SARS-CoV-2 positive sample to the luminescence intensity when measuring 0 ng / mL of recombinant N protein.

[0102] [Table 11-1]

[0103] [Table 11-2]

[0104] [Table 11-3]

[0105] As a result, when C3 and C12 antibodies were used as solid-phase or labeled antibodies and N6 antibody was used as the other, SARS-CoV-2 in samples could be detected with very high sensitivity.In other words, it was confirmed that SARS-CoV-2 in samples could be detected with very high sensitivity when antibodies recognizing the amino acid region of 260-305 and antibodies recognizing the amino acid region of 365-419 were used as solid-phase or labeled antibodies and an antibody recognizing the amino acid region of 120-147 was used as the other.

[0106] Furthermore, combining the N1 and P1 antibodies enabled the detection of SARS-CoV-2 in samples with higher sensitivity. In other words, it was confirmed that combining an antibody that recognizes the region of amino acids 44-78 and an antibody that recognizes the region of amino acids 243-259 in addition to the above antibody combination enables the detection of SARS-CoV-2 in samples with higher sensitivity.

[0107] Furthermore, even when the C1 antibody was added to the combination of these antibodies, SARS-CoV-2 in the samples could be detected with sufficiently high sensitivity, confirming that combining antibodies that recognize different CTD regions from these antibodies is also effective.

[0108] Example 14: Examination of surfactants used in reaction solutions A sample was prepared by adding a nasal swab (Boca Biolistics, LLC) from a SARS-CoV-2-positive patient to a SARS-CoV-2-negative saliva sample. 100 μL of this sample and 20 μL of a treatment solution containing the surfactants listed in Table 12 (Tris buffer, 150 mM NaCl, 1% BSA, 0.1% NaN3, 2.5% concentration of each surfactant in the treatment solution, 0.19% concentration after mixing of the sample and particle suspension) were added to 150 μL of a diluted particle suspension containing solid-phase antibodies and reacted at 37°C for 8 minutes. After the reaction, the samples were separated by B / F separation using a magnet and washed with Lumipulse washing solution (Fujirebio Inc.). Then, 150 μL of the labeled body solution containing the ALP (alkaline phosphatase)-labeled antibodies prepared in Example 4 was added and reacted at 37°C for 8 minutes. After B / F separation using a magnet and washing with Lumipulse wash solution, 200 μL of Lumipulse substrate solution containing AMPPD (3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane disodium salt) was added and the enzyme reaction was carried out at 37°C for 4 minutes. The N protein in each sample was measured by measuring the luminescence at a wavelength of 463 nm. N6 and P1 antibodies were used as solid-phase antibodies, and N1, C3, and C12 antibodies were used as labeled antibodies.

[0109] The results are shown in Table 12. In addition to the measured luminescence values, Table 12 also shows the ratio (%) of the luminescence value under each surfactant-added condition to the luminescence value under no surfactant-added condition.

[0110] [Table 12]

[0111] As a result, when zwitterionic surfactants containing hydrocarbon chains in the hydrophobic moiety (C12APS and C14APS) were used, very high luminescence values ​​were obtained in positive samples, and the N protein could be detected with high sensitivity. These results demonstrate that zwitterionic surfactants containing hydrocarbon chains in the hydrophobic moiety are effective for measuring SARS-CoV-2 N protein in patient samples.

[0112] Example 15: Examination of surfactants used in sample treatment solutions (part 1) In Example 14, it was found that mixing a zwitterionic surfactant containing a hydrocarbon chain as a hydrophobic moiety with a sample is useful for measuring SARS-CoV-2 N protein. Therefore, N protein was detected by the immunochromatography method described in Example 6 using a sample treated with a zwitterionic surfactant containing a hydrocarbon chain as a hydrophobic moiety.

[0113] The samples used were purchased nasopharyngeal samples (negative and positive samples, Boca Biolistics, LLC). The sample treatment solution was a Tris buffer solution (150 mM NaCl, 1% BSA, 0.1% NaN3) containing each surfactant. The solid-phase antibodies used were P1 and N6, and the labeled antibodies used were N1, C3, and C12. The color development in the detection zone under each condition is shown in Table 13. The larger the number, the stronger the color development, and "w" indicates weaker color development. In other words, the color development intensity decreases in the order 1 > 1w > 1ww. As shown in Table 13, the results confirmed that N protein could be detected with sufficient sensitivity.

[0114] [Table 13]

[0115] Example 16: Examination of surfactants in sample treatment solutions (part 2) In Examples 14 and 15 above, it was found that treating samples with zwitterionic surfactants containing hydrocarbon chains as hydrophobic moieties is useful in measuring SARS-CoV-2 N protein.

[0116] Next, we investigated the effects of amphoteric surfactants containing hydrocarbon chains as hydrophobic moieties and their combinations with other surfactants in a particle-based sandwich immunoassay.

[0117] The sample used was 3 mL of virus transport solution SGVTM-3R (Sugiyamaken Co., Ltd.) plus 6 μL of a SARS-CoV-2-positive nasopharyngeal specimen (Boca Biolistics, LLC). Among the purchased SARS-CoV-2-positive specimens (Boca Biolistics, LLC), samples with different viral loads determined by RT-PCR were selected as low- and medium-level samples.

[0118] 20 μL of sample treatment solution (Tris buffer, 150 mM NaCl, 1% BSA, 0.1% NaN3) containing the surfactants shown in Table 14 and 0.25% C16APS was added to 100 μL of sample and incubated at 37 °C for 6.5 minutes. 50 μL of a particle suspension dilution containing a solid-phase antibody was then added to the treated sample and allowed to react at 37 °C for 8 minutes. After the reaction, B / F separation was performed using a magnet, and the sample was washed with Lumipulse washing solution (Fujirebio Inc.). 50 μL of the labeled body solution containing the ALP-labeled antibody prepared in Example 4 above was added, and the reaction was allowed to continue for 8 minutes at 37 °C. After B / F separation using a magnet and washing with Lumipulse washing solution, 200 μL of Lumipulse substrate solution containing AMPPD was added, and the enzyme reaction was carried out at 37 °C for 4 minutes. The N protein of each sample was measured by measuring the amount of luminescence at a wavelength of 463 nm. In addition, standard solutions containing known concentrations of recombinant N protein (0, 100, 5000, and 10000 pg / mL) were measured in the same manner, and a standard curve was created. The N protein concentration of each sample was determined from the luminescence intensity of each sample and the standard curve. The results are shown in Table 14.

[0119] As a result, the addition of deoxycholic acid or cholic acid to the treatment solution increased the luminescence intensity in both the low value sample (LS) and the medium value sample (MS). In particular, the addition of deoxycholic acid significantly increased the luminescence intensity at all concentrations.

[0120] Furthermore, the addition of components such as deoxycholic acid causes the solution to gel. Although sample processing is possible even in a gelled solution, it has been found that adding a nonionic surfactant such as Tween 80, Tween 20, or NP-40 to the processing solution for easier handling prevents the solution from gelling and results in a high luminescence output.

[0121] [Table 14]

[0122] Example 17: Capsid disruption by specimen treatment The efficiency of capsid destruction by sample treatment was examined.

[0123] The method used was to quantitatively quantify the extracted RNA by semi-quantitating the genomic RNA extracted from the virus particles using RT-PCR. By adding RNase to the sample treatment solution or the sample dilution solution used as a control, if the virus capsid is destroyed by the sample treatment solution, the RNA is degraded before the RNA extraction process. By comparing the amount of RNA before RNase treatment with that before RNase treatment, the amount of RNA extracted from the particles by the treatment solution can be estimated. This allows us to estimate the efficiency of capsid destruction.

[0124] Specifically, each of the following samples was prepared: sample diluent (Tris buffer, 150 mM NaCl, 1% BSA), sample treatment solution (Tris buffer, 150 mM NaCl, 1% BSA, 0.1% NaN3) containing RNase A (final concentration 200 μg / mL), sample treatment solution containing 0.25% C16APS (Tris buffer, 150 mM NaCl, 1% BSA, 0.1% NaN3), and sample treatment solution containing surfactants at the concentrations listed in Table 16 and 0.25% C16APS. Each sample was added to a nasopharyngeal swab (Boca Biolistics, LLC) in equal amounts, mixed, and left to stand at room temperature for 5 minutes. RNA was then extracted using an RNA extraction kit (GeneJET Viral DNA / RNA Purification kit (Thermo Fisher)) according to the accompanying manual. The resulting RNA was measured using an RT-PCR kit (SARS-CoV-2 Direct Detection RT-qPCR Kit (TaKaRa)), and the Ct values ​​of the RT-PCR were calculated. The higher the Ct value, the more RNA is degraded and the more efficient the capsid disruption. The results are shown in Tables 15 and 16.

[0125] [Table 15]

[0126] [Table 16]

[0127] As a result, the Ct value increased by 4.9 when the sample dilution solution was treated with the sample treatment solution (no additions). In other words, the amount of RNA was reduced to about 1 / 30, indicating that the efficiency of RNA extraction was increased 30-fold.

[0128] Furthermore, when 0.5% deoxycholic acid was added to the sample treatment solution, there was no significant change in the Ct value, but as the concentration increased to 1% and 2%, the Ct value increased by 3.8 and 4.9, respectively. In other words, the RNA amount decreased by approximately 1 / 14 and 1 / 30, respectively, and the RNA extraction efficiency increased by 14 and 30 times, respectively.

[0129] Furthermore, when a nonionic surfactant (Tween 20) was added to the sample treatment solution, the Ct value increased by 1. In other words, the amount of RNA decreased by half, and therefore the efficiency of RNA extraction increased.

[0130] These results indicate that C16APS is effective for extracting capsids, and that the addition of 1% or more deoxycholic acid and a non-ionic surfactant enhances the RNA extraction effect, i.e., the efficiency of capsid destruction. [Explanation of symbols]

[0131] 1 Immunochromatography Cartridge 2. Matrix 3 Detection Zone 4 Labeling Reagent Zone 5. Development fluid tank 6 Deployment confirmation section 7. Developer absorption zone 8. Substrate Zone 9 Spreading liquid pad

Claims

1. A method for immunological detection of SARS-CoV-2, comprising detecting SARS-CoV-2 nucleocapsid protein (N protein) in a sample collected from a subject using the following antibodies (1) and (2), wherein both of the following antibodies (1) and (2) are used as solid-phase antibodies or labeled antibodies: (1) a first antibody directed against a first epitope in the region of amino acids 260 to 305 in the SARS-CoV-2 N protein; and (2) a second antibody directed against a second epitope in the region of amino acids 365 to 419 in the SARS-CoV-2 N protein;

2. 10. The method of claim 1, further comprising using a third antibody directed against a third epitope in the region of amino acids 120-147 in the SARS-CoV-2 N protein.

3. 3. The method of claim 1 or 2, further comprising using a fourth antibody directed against a fourth epitope in the region of amino acids 44 to 78 in the SARS-CoV-2 N protein.

4. 4. The method of claim 3, further comprising using a fifth antibody directed against a fifth epitope in the region of amino acids 243-259 in the SARS-CoV-2 N protein.

5. 5. The method of claim 1, further comprising using a sixth antibody directed against a sixth epitope in the region of amino acids 306 to 339 in the SARS-CoV-2 N protein.

6. When both the first antibody and the second antibody are used as solid-phase antibodies, the third antibody is used as a labeled antibody; The method according to claim 2, wherein when both the first antibody and the second antibody are used as labeled antibodies, the third antibody is used as a solid-phase antibody.

7. the fourth antibody is used as a solid phase antibody and the fifth antibody is used as a labeled antibody; or The method of claim 4, wherein the fourth antibody is used as a labeled antibody and the fifth antibody is used as a solid-phase antibody.

8. The method according to any one of claims 1 to 7, wherein the detection is carried out by a sandwich method.

9. An immunological detection reagent for SARS-CoV-2, comprising the following antibodies (1) and (2), wherein both of the following antibodies (1) and (2) are solid-phase antibodies or labeled antibodies: (1) a first antibody directed against a first epitope in the region of amino acids 260 to 305 in the SARS-CoV-2 N protein; and (2) a second antibody directed against a second epitope in the region of amino acids 365 to 419 in the SARS-CoV-2 N protein;

10. The reagent according to claim 9, further comprising one or more antibodies selected from the group consisting of the following (3) to (6): (3) A third antibody directed against a third epitope in the region of amino acids 120 to 147 in the SARS-CoV-2 N protein: (4) a fourth antibody directed against a fourth epitope in the region of amino acids 44 to 78 in the SARS-CoV-2 N protein; (5) a fifth antibody directed against a fifth epitope in the region of amino acids 243 to 259 in the SARS-CoV-2 N protein; and (6) A sixth antibody directed against a sixth epitope in the region of amino acids 306 to 339 in the SARS-CoV-2 N protein.

11. The reagent according to claim 9 or 10, which is used in a sandwich method.

12. The reagent of any one of claims 9 to 11, wherein the reagent comprises a solid phase.

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