JP7728100B2 - Immunological detection method and reagent for SARS-CoV-2 - Google Patents

Description

Article 30, Paragraph 2 of the Patent Act applies. Published on the website on May 13, 2020: https://ssl4.eir-parts.net/doc/4544/tdnet/1827081/00.pdf https://www2.tse.or.jp/disc/45440/140120200512411290.pdf Published on the website on June 19, 2020: https://ssl4.eir-parts.net/doc/4544/tdnet/1850470/00.pdf https://www2.tse.or.jp/disc/45440/140120200512411290.pdf jp/disc/45440/140120200619447766.pdf Published on the website on October 30, 2020 https://ssl4.eir-parts.net/doc/4544/tdnet/1895524/00.pdf https://www2.tse.or.jp/disc/45440/140120201030413055. pdf Sold to testing and research entities after May 13, 2020 Sold to testing and research entities after June 19, 2020 Sold to testing and research entities after October 30, 2020 Published on website on May 13, 2020 https://ssl4.eir-parts.net/doc/4544/tdnet/1827081/00.pdf Published on website on June 19, 2020 https://ssl4.eir-parts.net/doc/4544/tdnet/1850470/00.pdf Published on website on October 30, 2020 https://www.fujirebio.co.jp jp/information/pdf/20201030_news. pdf

The present invention relates to a method and reagent for immunological detection of SARS-CoV-2.

SARS coronavirus-2 (SARS-CoV-2) is a virus classified in the Coronaviridae family and the genus Betacoronavirus, and is the causative agent of COVID-19, a novel coronavirus infection. SARS-CoV-2 is closely related to but distinct from the SARS coronavirus (SARS-CoV) that caused the severe acute respiratory syndrome (SARS) that was prevalent in China in the 2000s.

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 sites. The N protein binds to the M protein, which also makes up the envelope, to form the enveloped virus particle.

There have been no reports on the molecular properties of the N protein in the specimens, but it is generally believed that there are two types of N protein: (i) N protein contained in the nucleocapsid of the virus particle, and (ii) N protein released when virus-infected cells are ruptured by viral proliferation or the immune response of the infected host.

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.

Patent Document 1 describes various monoclonal antibodies against SARS-CoV N protein and immunoassay reagents that use 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.

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 N protein, in which the first or second antibody recognizes an epitope present in the region from 283 to 422 amino acids from the N-terminus of the N protein's amino acid sequence (region C). Patent Document 2 describes how SARS-CoV N protein can be measured with higher sensitivity than conventional methods by dividing the SARS-CoV N protein (amino acids 1-422) into three regions (region A: regions 1-141, region B: 142-282, region C: 283-422) and 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.

International Publication No. 2005/042579 International Publication No. 2007/043582

The object of the present invention is to provide a technology that can immunologically detect SARS-CoV-2.

After extensive research, the inventors have discovered 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 inventors have also discovered that these antibodies are easy to use in combination with other antibodies, and that when used in combination with other antibodies, they are a useful antibody combination that can achieve even more sensitive detection of SARS-CoV-2.

Patent Documents 1 and 2 disclose technologies that enable the immunological detection of SARS-CoV by using antibodies against the SARS-CoV N protein (reported to have a full-length of 422 amino acids), but 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 an epitope 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 directed against two epitopes in three different regions in the immunological detection of SARS-CoV, as described in (a) and (b) above, but does not teach or suggest that a combination of two specific antibodies directed against two epitopes in the C-terminal region of the SARS-CoV-2 N protein is superior for detecting SARS-CoV-2.

Based on the above findings, the inventors have successfully developed a method and reagent for immunological detection of SARS-CoV-2, leading to the completion of the present invention.

That is, the present invention is as follows.
[1] A method for immunologically detecting 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):
(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] The method of [1], further comprising using a third antibody against a third epitope in the region of amino acids 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] The method of any one of [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] The method of any one of [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 method of [6] or [7], wherein the fourth antibody is used as a solid-phase antibody and the fifth antibody is used as a labeled antibody, or the fourth antibody is used as a labeled antibody and the fifth antibody is used as a solid-phase antibody.
[9] The method according to any one of [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 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.
[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 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.
[12] The reagent according to [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.

The present invention enables highly sensitive detection of SARS-CoV-2.

FIG. 1 is a schematic diagram showing an example of an immunochromatography cartridge. 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).

The present invention provides an immunological detection method for SARS-CoV-2, which comprises 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 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.

The subject from which the specimen 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.

Any biological sample that may contain SARS-CoV-2 can be used as the specimen. Examples of such specimens include saliva, sputum, nasal discharge, swabs (e.g., nasal swabs, pharyngeal swabs, and other swabs from mucosal sites), 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). From the perspective of obtaining 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 components such as surfactants.

The SARS-CoV-2 N protein detected by the present invention is the N protein (i.e., a native or mutant N protein) of any strain of SARS-CoV-2. Examples of such strains include the major L- 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. Genomic sequence information for SARS-CoV-2 has been disclosed (for example, see GenBank Accession No. 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. For the native N protein of SARS-CoV-2, you may refer to the amino acid sequence of SEQ ID NO: 4.

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 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.

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

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

In one embodiment, the method of the present invention may further include using a third antibody against a third epitope in the region of amino acids 120 to 147 in the SARS-CoV-2 N protein. By further 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 in the SARS-CoV-2 N protein.

In another embodiment, the method of the present invention may further include using a fourth antibody against a fourth epitope in the region of amino acids 44 to 78 in the SARS-CoV-2 N protein. By further 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 in the SARS-CoV-2 N protein.

In yet another embodiment, the method of the present invention may further include using a fifth antibody against a fifth epitope in the region of amino acids 243 to 259 in 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 in the SARS-CoV-2 N protein.

In yet another embodiment, the method of the present invention may further comprise using a sixth antibody against a sixth epitope in the region of amino acids 306 to 339 in 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 in the SARS-CoV-2 N protein.

The present invention also provides a method for immunological detection of SARS-CoV-2, comprising detecting SARS-CoV-2 N protein in a specimen 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 a fourth antibody and a 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.

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, a constant region-deleted antibody (e.g., F(ab') ₂ , Fab', Fab, Fv). The antibody may also be a modified antibody such as a single-chain antibody.

Antibodies can be produced using conventional methods. For example, antibodies can be produced efficiently by using the above-mentioned epitopes as antigens.

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, plastic), 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.

In another embodiment, the antibody may be a labeled antibody. A labeled antibody refers to an antibody labeled with a labeling substance. Examples of labeling substances include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., one of streptavidin and biotin, or one of the 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., ^3H , ^14C , ^32P , ^35S , ^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. Such methods include, for example, the methods described above for immobilizing antibodies on a solid phase.

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.

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.

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.

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

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 utilizes antigen-antibody binding as the principle of specific detection is applicable. The method of the present invention may be a qualitative or quantitative method.

Label detection can be performed using a technique appropriately selected depending on the type of label. For example, if the label is an enzyme, the label can be detected by detecting enzymatic activity using a signal-generating substrate (e.g., a fluorescent substrate, a luminescent substrate, or a chromogenic substrate). If 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. If the label is a fluorescent substance, luminescent substance, or radioactive substance, the label can be detected by detecting the signal generated from the label. If a chromogenic substance is used as the labeling substance, the label can be detected visually.

In certain embodiments, the methods of the present invention may be performed using a sandwich assay. The sandwich assay has excellent sensitivity and specificity.

In the sandwich method, detection of N protein may be carried out, for example, by treating the sample with a solid-phase antibody to bind the N protein in the sample to the solid-phase antibody (e.g., by contacting the sample 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., a 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 sample with a labeled antibody to bind the N protein in the sample to the labeled antibody (e.g., by contacting the sample 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 an antibody that has been previously immobilized on a solid phase. Alternatively, an antibody immobilized on a solid phase may be used when detecting N protein. For example, this may involve treating the 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.

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 devices 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 a device. Below, as a suitable example of immunochromatography, an overview of a lateral flow immunochromatography method and device will be described with reference to Figure 1.

In Figure 1, the immunochromatography cartridge 1 comprises a matrix 2 made of a porous material such as a nitrocellulose membrane, a detection zone 3 (a zone containing a solid-phase antibody) in which a capture antibody is immobilized in a line, and 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), which carries a detection antibody. The matrix 2 is typically formed in a strip shape. The labeled reagent zone 4 is composed of a porous pad onto which the labeled antibody is applied. A developer solution tank 5 containing a developer solution is provided at the upstream end of the matrix. Further downstream from the detection zone, there is a development confirmation section 6 for confirming the flow of the developer solution, and further downstream there is a developer solution absorption zone 7 equipped 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 carrying a substrate for the enzyme is provided upstream of the labeled reagent zone. By providing a member (not shown) near the developer tank 5 that can be pressed to break the developer tank, such as a push-in member with a protrusion, the developer tank can be easily broken and the developer supplied to the matrix. 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.

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. Next, 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 labeled antibody bind through an antigen-antibody reaction. When they reach the detection zone, the solid-phase antibody and N protein bind through an antigen-antibody reaction in the detection zone. As a result, the labeled antibody is immobilized in the detection zone via the N protein. 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. If an anti-labeled antibody has been 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 an absorbent pad further downstream.

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 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.

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 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.

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 a fourth antibody and a 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.

Details of the terms used for the antibodies and other components of the reagents of the present invention (e.g., definitions, examples, and preferred examples) are the same as those described in the method of the present invention.

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 during detection.

Reagents of the present invention may include a solid-phase antibody and/or a labeled antibody. Alternatively, if such reagents do not include a solid-phase antibody and/or a labeled antibody, they may include 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 reagents of the present invention may include 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).

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 method. Therefore, the reagent of the present invention may include a solid phase used in a sandwich method (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 include a device containing a solid phase used in immunochromatography (i.e., an immunochromatography cartridge as described above), or a solid-phase antibody immobilized on such a solid phase. The reagent of the present invention may include an N protein preparation.

The present invention will now be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Note that, with regard to amounts described in the following examples, % means % by weight (wt%).

Example 1: Production of antibodies Antibodies were produced according to the method described in WO 2005/042579. Specifically, the procedure is as follows.

(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 prepare the plasmid pWS-N. This plasmid was used to transform Escherichia coli, and ampicillin-resistant transformants were obtained. The nucleotide sequence and amino acid sequence of the N protein are shown in SEQ ID NOs: 1 and 2, respectively.

The resulting transformant was cultured at 37°C in 2 ml of LB medium containing 50 μg/ml ampicillin. This transformant was grown in a preliminary culture until the density reached an OD at 600 nm of approximately 0.7, after which 0.4 mM IPTG was added to induce expression. After 18 hours of culture, the culture was centrifuged to recover the E. coli. 20 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM PMSF was added to the recovered E. coli, and the cells were 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, 20 mM phosphate buffer (pH 6.9), and purified by elution with 0.2 M NaCl, 8 M urea, 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), yielding a purified recombinant N protein (CoV) solution. The purity of the recombinant N protein was confirmed by SDS-PAGE and Western blotting, revealing a single band.

(2) Preparation of Monoclonal Antibodies. Anti-N protein monoclonal antibodies were prepared 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 initially immunized with 50-100 μg/mouse of recombinant N protein emulsified in Freund's complete adjuvant. 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 from the mice and splenocytes were prepared. Mouse myeloma cells (P3U1) previously cultured in RPMI-1640 medium were mixed with splenocytes at a ratio of 1:2 to 1:5, and cell fusion was carried out using PEG. The fused cells were suspended in HAT medium, dispensed into 96-well culture plates, and cultured in a _CO2 incubator at 37°C.

Antibody screening was performed using the solid-phase ELISA described above. Specifically, recombinant N protein was dispensed into a 96-well ELISA plate at a concentration of 1 μg/mL, at 50 μL/well, and allowed to adsorb by standing overnight at 4°C. After blocking the wells with 1% skim milk, the plates were washed three times with washing buffer (PBS containing 0.05% Tween), and 50 μL of culture supernatant from the cell fusion plate was added and allowed to react at 37°C for 1 hour. Similarly, after washing three times with washing buffer, POD-labeled anti-mouse immunoglobulin antibody (manufactured by DACO) was added and allowed to react for another 1 hour at 37°C. After washing four times with washing 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 cultured in a _CO2 incubator at 37°C. Single clones were obtained by limiting dilution, and each monoclonal antibody was obtained.

In addition, instead of 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.

(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 incubation, the cells were centrifuged at 2,000 rpm for 15 minutes to prepare a virus culture supernatant (TCID50: 7.95 x ¹⁰⁶ /mL). This 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, followed by pipetting 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 a sample for analysis. 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 by shaking at room temperature for 1 hour, followed by washing three times with wash buffer (5 minutes of shaking wash). Next, a POD-labeled anti-mouse immunoglobulin antibody was added and allowed to react for an additional hour at room temperature. After washing four times with wash buffer (5 minutes of shaking wash), 4-chloronaphthol solution was added as a substrate, and bands were confirmed. Monoclonal antibodies that showed a band at the position corresponding to N protein, which has a molecular weight of just under 50 kD, were selected.

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

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

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

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 multiple antibodies reactive with both the NTD and CTD. Antibodies (anti-N protein antibodies C12 and C13) that recognize a region contained in the amino acid sequence at positions 365 to 419 were also obtained, which react with the full-length antigen and C-terminal peptide but not with the NTD or CTD antigen. Furthermore, multiple anti-N protein monoclonal antibodies (P1 and P2) that recognize the amino acid 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.

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

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 to carry out pepsin digestion. After terminating the reaction, gel filtration purification was carried out to obtain an antibody from which the Fc region had been removed. Next, 2-mercaptoethylamine hydrochloride (2-MEA) was added to carry out thiolation. This was then desalted to obtain a Fab' fragment.

N-(4-maleimidobutyryloxy)-succinimide (GMBS)-treated alkaline phosphatase was mixed with Fab' fragments prepared from each anti-N protein monoclonal antibody at a molar ratio of 1-3:1 to perform coupling. 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 1-3:1 were pooled by gel filtration to obtain labeled antibodies (ALP-labeled anti-N protein monoclonal antibodies).

Example 5: Sandwich immunoassay using particles 100 μL of a solution containing recombinant N protein or a 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.

After the reaction, the plate was separated into B/F using a magnet and washed with Lumipulse® washing solution (Fujirebio Inc.). 50 μL of labeled body fluid (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 above was added, and the reaction was allowed to proceed at 37°C for 8 minutes. After B/F separation using a magnet and washing with Lumipulse washing solution, 200 μL of Lumipulse® substrate solution (Fujirebio Inc.) containing 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD) was added, and the enzyme reaction was allowed to proceed at 37°C for 4 minutes, and the luminescence at a wavelength of 463 nm was measured.

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

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

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. 30 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.

Example 7: Identification of antibody reactive region (part 2)
Recombinant N protein (SARS-CoV-2 or SARS-CoV) was measured and grouped according to the reactive region of each N protein monoclonal antibody according to the sandwich immunoassay method described in Example 5. Specifically, 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.

As a result, when recombinant N protein (0 and 10 ng/mL) was measured using N1 and N2 antibodies 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 and 1 ng/mL) was measured using N4, N5, and N6 antibodies (N6 antibody group) as the solid-phase and labeled antibodies, respectively, the difference in luminescence intensity (1 ng/mL minus the luminescence intensity at 0 ng/mL) was very small, confirming that these antibodies recognize overlapping regions (Table 2). On the other hand, when N1 and N3 antibodies 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 exhibited 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.

Similarly, when the C1 to C6 antibodies were examined, it was confirmed that the C1 and C2 antibodies recognize an overlapping region, the C3 and C4 antibodies recognize an overlapping region, and the C5 and C6 antibodies recognize 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) have low reactivity in sandwich immunoassay measurements.

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 for each measurement.

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.

As a result, it was confirmed that the following combinations of solid-phase antibodies and labeled antibodies in sandwich immunoassays are preferable, as they all show a high S/N ratio: 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.

Example 9: Study of antibody combinations (part 2)
Combinations of solid-phase antibodies and labeled antibodies were investigated for the antibodies grouped in Example 7, as well as the P1 and C12 antibodies. Using the immunochromatography method of Example 6, 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 developing solution 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 higher 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.

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.

Furthermore, when the C1 antibody and P1 antibody were evaluated in combination with other antibodies, the C1 antibody showed relatively strong luminescence intensity when combined with the C3 antibody. On the other hand, the P1 antibody showed strong luminescence intensity when combined with the C3 antibody, and relatively strong luminescence intensity when combined with the N1 antibody and N4 antibody.

Example 10: Study of antibody combinations (part 3)
In Examples 8 and 9 above, a combination of a single solid-phase antibody and a single labeled antibody was investigated. Although 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 above as the labeled antibody and multiple other antibodies as solid-phase antibodies, and a sample containing 10 ng/mL of recombinant N protein (SARS-CoV) was evaluated using the same method as in Example 8 above. The solid-phase antibody was prepared by simultaneously chemically binding multiple antibodies to particles according to the manufacturing method of Example 3 above. The results are shown in Table 9.

As a result, when a mixture of C3 and C12 antibodies was used, significant improvements in luminescence (counts at 1000 pg/ml) and S/N ratio were observed. In particular, when N1 or N4 antibodies were used as the labeled antibody, using a mixture of C3 and C12 antibodies as the solid-phase antibody increased luminescence and the S/N ratio compared to using C3 and C12 antibodies alone as the solid-phase antibody. On the other hand, when C3 or C12 antibodies were used as the labeled antibody, using a mixture of N1 and N4 antibodies as the solid-phase antibody did not increase both the count value and the S/N ratio. This confirms that using 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.

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).

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, as well as recombinant antigens consisting of amino acid regions 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 amino acid region 44 to 78 of the N protein. Furthermore, the N6 antibody group recognized the amino acid region 120 to 147 of the N protein.

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

In summary, the epitopes of the analyzed antibodies were as follows:

Example 12: Study of antibody combinations (part 4)
In Example 10 above, it was shown that when the N1 or N4 antibody was used, it was 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.

Specifically, 100 μL of a solution containing recombinant N protein and 20 μL of a treatment solution (Tris buffer, 150 mM NaCl, 0.1% NaN ₃ , 2.5% C16APS) were added to 50 μL of a particle suspension dilution containing a solid-phase antibody, and the mixture was allowed to react at 37°C for 8 minutes. After the reaction, the mixture was subjected to B/F separation with a magnet, washed with Lumipulse washing solution, and then 50 μL of a labeled body solution containing an ALP-labeled antibody was added, and the mixture was allowed to react at 37°C for 8 minutes. After B/F separation with a magnet and washed with Lumipulse washing solution, 200 μL of Lumipulse substrate solution containing AMPPD was added, and an enzymatic 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. The solid-phase antibody was prepared according to Example 3 above by chemically bonding each antibody to particles and then mixing them at a 1:1 ratio. The labeled antibody was prepared according to Example 4 above by simultaneously mixing GMBS-treated ALP and 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.

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.

Example 13: Study of antibody combinations (part 5)
In Examples 10 and 12 above, it was shown that when an N1 antibody or an N4 antibody is used, it is useful to use a mixture of a C3 antibody and a C12 antibody as a solid-phase antibody. As described above, because mutations and the like may occur in SARS-CoV-2, it is more preferable to use a combination of antibodies that recognize different regions. Therefore, the combination of the P1/P2 antibody, which is an antibody from a group with a different reactivity from the N1/N2 antibody, N4/N6 antibody, C3/C4 antibody, and C12/C13 antibody groups and which is highly reactive with the N1 antibody, N4 antibody, and C3 antibody in Example 10 above, was evaluated. The combination with the C1/C2 antibody was also evaluated.

Specifically, the combinations of solid-phase antibodies and labeled antibodies listed in Tables 11-1 to 11-3 were used and evaluated using the same method 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 by simultaneously mixing alkaline phosphatase (ALP) treated with GMBS and 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 ALP-labeled antibodies as in Example 4 above.

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, and 100 μL of the sample was added to 170 μL of sample treatment solution (Tris buffer, 150 mM NaCl, 1% BSA, 0.1% _NaN , 0.25% C16APS).

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), as well as 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.

As a result, it was confirmed that 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 measured with extremely high sensitivity. In other words, it was confirmed that when antibodies recognizing the amino acid region from 260 to 305 and antibodies recognizing the amino acid region from 365 to 419 were used as solid-phase or labeled antibodies, and an antibody recognizing the amino acid region from 120 to 147 was used as the other, SARS-CoV-2 in samples could be measured with extremely high sensitivity.

Furthermore, by combining the N1 antibody and the P1 antibody, SARS-CoV-2 in samples could be measured with higher sensitivity. In other words, it was confirmed that by adding an antibody that recognizes the amino acid region 44 to 78 and an antibody that recognizes the amino acid region 243 to 259 to the above antibody combination, SARS-CoV-2 in samples could be measured with higher sensitivity.

Furthermore, even when the C1 antibody was added to this antibody combination, SARS-CoV-2 in the sample could still be detected with sufficiently high sensitivity. In other words, it was confirmed that combining antibodies that recognize different CTD regions from these antibodies can also be effective.

Example 14: Investigation of surfactants used in reaction solution 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 a surfactant shown in Table 12 (Tris buffer, 150 mM NaCl, 1% BSA, 0.1% _NaN , 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 a solid-phase antibody, and the reaction was allowed to proceed at 37°C for 8 minutes. After the reaction, the sample was subjected to 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 antibody prepared in Example 4 was added, and the reaction was allowed to proceed 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 an enzyme reaction was carried out at 37°C for 4 minutes. The amount of luminescence at a wavelength of 463 nm was measured to determine the N protein in each sample. Note that N6 antibody and P1 antibody were used as solid-phase antibodies, and N1 antibody, C3 antibody, and C12 antibody were used as labeled antibodies.

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 addition condition to the luminescence value under no surfactant addition condition.

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, enabling highly sensitive detection of N protein. These findings demonstrate that zwitterionic surfactants containing hydrocarbon chains in the hydrophobic moiety are effective for measuring SARS-CoV-2 N protein contained in patient samples.

Example 15: Investigation of surfactants used in sample treatment solutions (part 1)
In Example 14, it was found that mixing a sample with a zwitterionic surfactant containing a hydrocarbon chain as a hydrophobic moiety is useful in 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.

The samples used were purchased nasopharyngeal samples (negative and positive samples, Boca Biolistics, LLC). Tris buffer (150 mM NaCl, 1% BSA, 0.1% _NaN3 ) containing each surfactant was used as the sample treatment solution. Furthermore, P1 and N6 antibodies were used as solid-phase antibodies, and N1, C3, and C12 antibodies were used as labeled antibodies. The color development state of 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 a result, as shown in Table 13, it was confirmed that N protein could be detected with sufficient sensitivity.

Example 16: Investigation of surfactants in sample treatment solutions (part 2)
In the above Examples 14 and 15, it was found that treating the sample with a zwitterionic surfactant containing a hydrocarbon chain as the hydrophobic moiety is useful in measuring the SARS-CoV-2 N protein.

Next, we examined the effects of zwitterionic surfactants containing hydrocarbon chains as hydrophobic moieties and their combinations with other surfactants using a particle-based sandwich immunoassay method.

The specimen used was 3 mL of virus transport solution SGVTM-3R (Sugiyamaken) to which 6 μL of a SARS-CoV-2 positive nasopharyngeal specimen (Boca Biolistics, LLC) was added. Of the purchased SARS-CoV-2 positive specimens (Boca Biolistics, LLC), specimens that were found to have different viral loads using RT-PCR were selected for use as low- and medium-level specimens.

20 μL of sample treatment solution (Tris buffer, 150 mM NaCl, 1% BSA, 0.1% NaN ₃ ) 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. Then, 50 μL of particle suspension dilution containing solid-phase antibody was added to the treated sample and allowed to react at 37 ° C for 8 minutes. After the reaction, B/F separation was performed with a magnet, and after washing with Lumipulse washing solution (Fujirebio Inc.), 50 μL of labeled body solution containing the ALP-labeled antibody prepared in Example 4 above was added and allowed to react for 8 minutes at 37 ° C. After B/F separation with a magnet and washing with Lumipulse washing solution, 200 μL of Lumipulse substrate solution containing AMPPD was added, and an enzyme reaction was performed at 37 ° C for 4 minutes, and the N protein of each sample was measured by measuring the luminescence at a wavelength of 463 nm. Standard solutions containing known concentrations of recombinant N protein (0, 100, 5,000, and 10,000 pg/mL) were also measured in the same manner, and a standard curve was created. The N protein concentration of each sample was determined from the luminescence level of each sample and the standard curve. The results are shown in Table 14.

As a result, the addition of deoxycholic acid or cholic acid to the treatment solution increased the amount of luminescence in both low-level samples (LS) and medium-level samples (MS). In particular, the addition of deoxycholic acid significantly increased the amount of luminescence at all concentrations added.

Addition of components such as deoxycholic acid causes the solution to gel. While sample processing is possible even with 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 to make it easier to handle prevents the solution from gelling and produces a high level of luminescence.

Example 17: Capsid destruction by specimen treatment The efficiency of capsid destruction by specimen treatment was investigated.

The method used was to quantitatively quantify the extracted RNA by semi-quantifying the genomic RNA extracted from the virus particles using the RT-PCR method. By adding RNase beforehand 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 will be degraded before the RNA extraction process is carried out. By comparing this with the amount of RNA before the RNase treatment, the amount of RNA extracted from the particles by the treatment solution can be estimated. This allows the efficiency of capsid destruction to be estimated.

Specifically, RNase A (final concentration 200 μg/mL) was added to each of the following samples: sample diluent (Tris buffer, 150 mM NaCl, 1% BSA), sample treatment solution containing 0.25% C16APS (Tris buffer, 150 mM NaCl, 1% BSA, 0.1% _NaN ), and sample treatment solution containing surfactants at the concentrations listed in Table 16 and 0.25% C16APS. Each sample was prepared by adding purchased nasopharyngeal swab (Boca Biolistics, LLC) to the sample diluent, mixing, and allowing 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 attached manual. The resulting RNA was measured using an RT-PCR kit (SARS-CoV-2 Direct Detection RT-qPCR Kit (TaKaRa)), and the RT-PCR Ct value was calculated. A higher Ct value indicates more RNA degradation and higher capsid disruption efficiency. The results are shown in Tables 15 and 16.

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 approximately 1/30, indicating that the RNA extraction efficiency increased 30-fold.

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. In other words, the amount of RNA decreased by approximately 1/14 and 1/30, respectively, and the RNA extraction efficiency increased by 14 and 30 times, respectively.

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, thereby increasing the RNA extraction efficiency.

These results demonstrate that C16APS is effective for extracting capsids, and that adding 1% or more deoxycholic acid and a non-ionic surfactant enhances the RNA extraction effect, i.e., the efficiency of capsid destruction.

REFERENCE SIGNS LIST 1 immunochromatography cartridge 2 matrix 3 detection zone 4 labeled reagent zone 5 developer tank 6 development confirmation section 7 developer absorption zone 8 substrate zone 9 developer pad

Claims (12)

A method for immunological detection of SARS-CoV-2, comprising detecting SARS-CoV-2 nucleocapsid protein (N protein) in a specimen 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. The method of claim 1, further comprising using a third antibody directed against a third epitope in the region of amino acids 120 to 147 of the SARS-CoV-2 N protein. The method of claim 1 or 2, further comprising using a fourth antibody against a fourth epitope in the region of amino acids 44 to 78 of the SARS-CoV-2 N protein. 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. The method of any one of claims 1 to 4, further comprising using a sixth antibody directed against a sixth epitope in the region of amino acids 306 to 339 of the SARS-CoV-2 N protein. 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. The method of claim 4, wherein the fourth antibody is used as a solid-phase antibody and the fifth antibody is used as a labeled antibody, or the fourth antibody is used as a labeled antibody and the fifth antibody is used as a solid-phase antibody. The method according to any one of claims 1 to 7 , wherein the detection is carried out by a sandwich method. 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. 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. The reagent according to claim 9 or 10 , which is used in a sandwich method. The reagent of any one of claims 9 to 11 , wherein the reagent comprises a solid phase.