Immunological Detection Methods and Reagents for SARS-CoV-2

Abstract

To provide a technique useful for immunologically detecting SARS-CoV-2.SOLUTION: An immunological method of detecting SARS-CoV-2 is provided, comprising mixing a SARS-CoV-2-containing sample collected from a subject with a solution containing a zwitterionic surfactant containing a hydrocarbon chain having a chain length of 12-18 carbon atoms.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an immunological detection method and reagent for SARS-CoV-2.

Background Art

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative virus of coronavirus disease 2019 (COVID-19), which is a novel coronavirus infection classified in the genus Betacoronavirus of the family Coronaviridae. SARS-CoV-2 is closely related to, but different from, the severe acute respiratory syndrome coronavirus (SARS-CoV) that caused severe acute respiratory syndrome (SARS) prevalent in China in the 2000s.

[0003] Coronaviruses are enveloped viruses having a positive-sense single-stranded RNA as their viral genome. The nucleocapsid protein (N protein) in the virus particle forms dimers by its C-terminal domain (CTD), and further these form tetramers. Furthermore, it is considered that the genomic RNA binds to the N-terminal domain (NTD) of the nucleocapsid protein (N protein) to form a nucleocapsid. The nucleocapsid has a structure covered by an envelope composed of a lipid bilayer. The envelope has the S protein, E protein, and M protein bound by their transmembrane regions. The N protein binds to the M protein that constitutes the envelope to form an enveloped virus particle.

[0004] There is no report on the molecular properties of the N protein in a specimen, but generally, (i) those contained in the nucleocapsid of the virus particle and (ii) those released when virus-infected cells are disrupted by virus growth or the immune response of the infected host are considered to exist.

[0005] Regarding the immunological measurement method of SARS coronavirus, methods of treating SARS-CoV-containing specimens with nonionic surfactants (e.g., Triton X100, NP40) or anionic surfactants (e.g., SDS) have been reported (Patent Documents 1, 2).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a technique useful for the immunological detection of SARS-CoV-2.

Means for Solving the Problems

[0008] As a result of intensive studies, the present inventors have found that SARS-CoV-2 can be immunologically detected with high sensitivity by mixing a specimen with an amphoteric surfactant containing a hydrocarbon chain having 12 to 18 carbon atoms.

[0009] Patent Documents 1 and 2 are techniques that enable the immunological detection of SARS-CoV, but do not disclose the immunological detection of SARS-CoV-2 with high sensitivity. Also, Patent Documents 1 and 2 do not teach or suggest the use of the above amphoteric surfactant in immunological detection.

[0010] Based on the above findings, the present inventors have successfully developed an immunological detection method and reagent for SARS-CoV-2 and have completed the present invention.

[0011] That is, the present invention is as follows. 〔1〕A method for immunological detection of SARS-CoV-2, which comprises mixing a specimen collected from a subject with a solution containing an amphoteric surfactant containing a hydrocarbon chain having 12 to 18 carbon atoms. 〔2〕The method according to 〔1〕, wherein the hydrocarbon chain has a chain length of 14 to 18 carbon atoms. 〔3〕The method according to 〔1〕 or 〔2〕, wherein the hydrocarbon chain is an alkyl chain. 〔4〕The method according to any one of 〔1〕 to 〔3〕, wherein the amphoteric surfactant is an amphoteric surfactant containing an ammonium group having the hydrocarbon chain. 〔5〕The method according to any one of 〔1〕 to 〔4〕, wherein the amphoteric surfactant containing an ammonium group having the hydrocarbon chain is 3-(N,N-dimethyldodecylammonio)propanesulfonate (C12APS), 3-(N,N-dimethylmyristylammonio)propanesulfonate (C14APS), 3-(N,N-dimethylpalmitylammonio)propanesulfonate (C16APS), or 3-(N,N-dimethylstearylammonio)propanesulfonate (C18APS). 〔6〕The method according to any one of 〔1〕 to 〔5〕, wherein the solution further contains an anionic surfactant or an amphoteric surfactant having a steroid skeleton. 〔7〕The method according to 〔6〕, wherein the anionic surfactant or the amphoteric surfactant having a steroid skeleton is a bile acid or a derivative thereof retaining a steroid skeleton, or a salt thereof. 〔8〕The method according to 〔7〕, wherein the bile acid or the derivative thereof retaining a steroid skeleton is one or more compounds selected from the group consisting of deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, hyodeoxycholic acid, cholic acid, glycocholic acid, taurocholic acid, hyocholic acid, 5α-cyprinol, lithocholic acid, taurodeoxycholic acid, taurocholic acid, CHAPS, and CHAPSO. 〔9〕The method according to any one of 〔1〕 to 〔8〕, wherein the solution further contains a nonionic surfactant. 〔10〕In immunological detection, the method according to any one of 〔1〕 to 〔9〕, wherein one or more antibodies against the SARS-CoV-2 nucleocapsid protein (N protein) are used. 〔11〕The method according to any one of 〔1〕 to 〔10〕, wherein the immunological detection is performed by the sandwich method. 〔12〕An immunological detection reagent for SARS-CoV-2, comprising the following components (1) and (2): (1) An amphoteric surfactant containing a hydrocarbon chain having 12 to 18 carbon atoms; and (2) One or more antibodies against the target molecule constituting SARS-CoV-2. 〔13〕The reagent according to 〔12〕, wherein the reagent further comprises the following component (3) and / or (4): (3) An anionic surfactant or an amphoteric surfactant having a steroid skeleton; and / or (4) A nonionic surfactant. 〔14〕The reagent according to 〔12〕 or 〔13〕, wherein the reagent is a reagent used in the sandwich method.

Advantages of the Invention

[0012] According to the present invention, SARS-CoV-2 can be detected with high sensitivity.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Embodiments for Carrying Out the Invention

[0014] The present invention provides a method for immunologically detecting SARS-CoV-2, which comprises mixing a specimen collected from a subject with a solution containing an amphoteric surfactant containing a hydrocarbon chain having 12 to 18 carbon atoms.

[0015] As the subject from which the specimen is obtained, any subject that can be infected with SARS-CoV-2 can be used. 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; 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.

[0016] As the specimen, any biological sample that may contain SARS-CoV-2 can be used. Examples of such specimens include saliva, sputum, nasal discharge, swabs (e.g., swabs of mucosal sites such as nasal swabs and pharyngeal swabs), washing solutions (e.g., nasal washing solutions, oral cavity washing solutions, bronchial washing solutions, lung washing solutions), blood (e.g., whole blood, plasma, serum), feces, and other specimens (e.g., specimens containing infected cells). From the perspective of obtaining a specimen that may contain a large amount of SARS-CoV-2 in a minimally invasive, rapid, and simple manner, the specimen is preferably saliva, sputum, nasal discharge, or a swab. The specimen may be pretreated. Such treatments may include, for example, centrifugation, extraction, dilution, filtration, precipitation, heating, freezing, refrigeration, and agitation, as well as treatment with components such as surfactants.

[0017] As the solution to be mixed with the sample, an aqueous solution can be used. Examples of the aqueous solution include water (e.g., distilled water, sterilized water, sterilized distilled water, and pure water), and buffer solutions, with buffer solutions being preferred. Examples of buffer solutions include phosphate buffer, phosphate buffered saline (PBS), tartrate buffer, citrate buffer, acetate buffer, glycine buffer, carbonate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, borate buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) buffer, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer, and imidazole buffer. The solution may contain a small amount of an organic solvent (e.g., alcohol). The volume of the solution can be appropriately set according to factors such as the volume of the sample, and may be, for example, 1 μL to 10 mL, preferably 5 μL to 5 mL, and more preferably 10 μL to 1 mL. The pH of the buffer solution is preferably neutral. More specifically, such a pH may preferably be 5.0 or higher, more preferably 5.5 or higher, and even more preferably 6.0 or higher. The pH may also preferably be 9.0 or lower, more preferably 8.5 or lower, and even more preferably 8.0 or lower. The pH can be measured using known methods in the art. Preferably, the pH can be the value measured at 25 °C using a pH meter having a glass electrode.

[0018] The above amphoteric surfactant contains a hydrocarbon chain having 12 to 18 carbon atoms. Preferably, the number of carbon atoms may be 13 or more, 14 or more, 15 or more, or 16 or more. The number of carbon atoms may also be 17 or less, or 16 or less. The hydrocarbon chain may be linear or branched, with a linear chain being preferred. The hydrocarbon chain may also be an alkyl chain, an alkenyl chain, or an alkynyl chain, with an alkyl chain being preferred. By mixing such an amphoteric surfactant with a specimen, proteins in the specimen can be detected immunologically with high sensitivity. One or more than one amphoteric surfactant can be used.

[0019] The amphoteric surfactant containing the above hydrocarbon chain is also a surfactant containing both a cationic moiety (e.g., ammonium, phosphonium, sulfonium) and an anionic moiety (e.g., sulfate group, sulfonic acid group, carboxylic acid group, phosphate group). Therefore, in the present invention, a surfactant containing a hydrocarbon chain having 12 to 18 carbon atoms, as well as such a cationic moiety and anionic moiety, can be used.

[0020] The amphoteric surfactant containing the above hydrocarbon chain may be in a free form or in a salt form. Examples of the salt include metal salts (e.g., monovalent metal salts such as sodium salt and potassium salt, and divalent metal salts such as calcium salt and magnesium salt), inorganic salts (e.g., halide salts such as fluoride, chloride, bromide, and iodide, and ammonium salts), organic salts (e.g., ammonium salts substituted with an alkyl group), and acid addition salts (e.g., salts with inorganic acids such as sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, and phosphoric acid, and salts with organic acids such as acetic acid, oxalic acid, lactic acid, citric acid, trifluoromethanesulfonic acid, and trifluoroacetic acid).

[0021] In a specific embodiment, the zwitterionic surfactant containing the hydrocarbon chain may contain an ammonium group having the hydrocarbon chain. Examples of such zwitterionic surfactants include quaternary ammonium-sulfonic acid type surfactants having the hydrocarbon chain, and quaternary ammonium-carboxylic acid type surfactants having the hydrocarbon chain.

[0022] Examples of the quaternary ammonium-sulfonic acid type surfactant having the hydrocarbon chain include 3-(N,N-dimethyldodecylammonio)propanesulfonate (C12APS), 3-(N,N-dimethylmyristylammonio)propanesulfonate (C14APS), 3-(N,N-dimethylpalmitylammonio)propanesulfonate (C16APS), 3-(N,N-dimethylstearylammonio)propanesulfonate (C18APS), and salts thereof.

[0023] Examples of the quaternary ammonium-carboxylic acid type surfactant having the hydrocarbon chain include C12-C18 alkyldimethylaminoacetic acid betaines such as lauryl (C12) dimethylaminoacetic acid betaine, myristyl (C14) dimethylaminoacetic acid betaine, palmityl (C16) dimethylaminoacetic acid betaine, stearyl (C18) dimethylaminoacetic acid betaine, and salts thereof.

[0024] As the zwitterionic surfactant containing the hydrocarbon chain, the quaternary ammonium-sulfonic acid type surfactant having the hydrocarbon chain is preferred.

[0025] The concentration of the zwitterionic surfactant containing the hydrocarbon chain (final concentration in the mixed solution containing the sample) is not particularly limited as long as it is a concentration sufficient for mixing with the sample, and varies depending on factors such as the type of the sample and the target molecule to be immunologically detected (e.g., target protein such as N protein). However, for example, it may be a concentration of 0.001 to 10% by weight, preferably 0.005 to 5% by weight, more preferably 0.01 to 2% by weight, even more preferably 0.02 to 1.8% by weight, and particularly preferably 0.04 to 0.5% by weight.

[0026] The solution to be mixed with the sample may further contain an anionic surfactant or zwitterionic surfactant having a steroid skeleton. By using an anionic surfactant or zwitterionic surfactant having a steroid skeleton in combination with the zwitterionic surfactant containing the hydrocarbon chain, it is effective for treating the envelope of SARS-CoV-2 and can improve the extraction efficiency of the target protein (e.g., N protein). As the anionic surfactant or zwitterionic surfactant having a steroid skeleton, one or more kinds can be used.

[0027] The anionic surfactant having a steroid skeleton is typically a bile acid or its derivative retaining the steroid skeleton, or a salt thereof. More specifically, such anionic surfactants include, for example, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, hyodeoxycholic acid, cholic acid, glycocholic acid, taurocholic acid, hyocholic acid, 5α-cyprinol, lithocholic acid, taurodeoxycholic acid, and taurocholic acid, and salts thereof. The salts are the same as those described above.

[0028] The zwitterionic surfactant having a steroid skeleton is typically a derivative of a bile acid derivatized to have a cationic moiety, or a salt thereof retaining the steroid skeleton. More specifically, such zwitterionic surfactants include, for example, CHAPS, CHAPSO, and salts thereof. The salts are the same as those described above.

[0029] As the anionic surfactant or zwitterionic surfactant having a steroid skeleton, an anionic surfactant having a steroid skeleton is preferred.

[0030] The concentration of the anionic surfactant or zwitterionic surfactant having a steroid skeleton (final concentration in the mixed solution containing the sample) is not particularly limited as long as it is a concentration sufficient for mixing with the sample, and although it varies depending on factors such as the sample and the type of the target molecule to be immunologically detected, for example, it may be a concentration of 0.001 to 10% by weight, preferably 0.01 to 5% by weight, more preferably 0.05 to 4% by weight, and even more preferably 0.12 to 2.0% by weight.

[0031] The solution to be mixed with the sample may further contain a nonionic surfactant. Depending on the type, the surfactant having a steroid skeleton may form micelles when used at a high concentration. The nonionic surfactant can suppress such micelle formation and can also suppress nonspecific reactions in immunological detection. One or more nonionic surfactants can be used.

[0032] Examples of the nonionic surfactant include polyoxyethylene sorbitan fatty acid esters [e.g., TWEEN (registered trademark) series (e.g., TWEEN20, TWEEN40, TWEEN80)], polyoxyethylene octylphenyl ethers [e.g., TRITON (registered trademark) series (e.g., Triton X-100, Triton X-114, Triton X-305, Triton X-405, Triton X-705)], N-D-gluco-N-methylalkane amides [e.g., MEGA series (e.g., MEGA 8, MEGA 10)], and nonionic surfactants containing a polyoxyethylene alcohol structure (e.g., alcohol ethoxylate, polyoxyethylene-polyoxyalkylene block polymer).

[0033] The concentration of the nonionic surfactant (final concentration in the mixed solution containing the sample) is not particularly limited as long as it is a concentration sufficient for mixing with the sample, and although it varies depending on factors such as the sample and the type of target molecule to be immunologically detected, for example, it may be a concentration of 0.001 to 10% by weight, preferably 0.01 to 5% by weight, more preferably 0.02 to 1% by weight, and preferably 0.04 to 0.5% by weight.

[0034] In immunological detection, one or more antibodies against the target molecule constituting SARS-CoV-2 are used to detect the target molecule. One or more antibodies against the target molecule may be mixed as described above by being contained in the above solution. Alternatively, one or more antibodies against the target molecule may be contained in a solution (second solution) different from the above solution (first solution). In such a case, the other solution containing one or more antibodies against the target molecule may be mixed with the sample simultaneously with the above solution (first solution), or may be mixed with the mixed solution generated after mixing the above solution (first solution) and the sample. By using such antibodies, the target molecule can be detected.

[0035] The target molecule is a substance that can be retained by SARS-CoV-2 and is not particularly limited as long as it can be detected using an antibody, but a protein is preferred. Such proteins are not particularly limited as long as they are proteins that can be retained by SARS-CoV-2, and examples include nucleocapsid protein (N protein), S protein, E protein, and M protein. Preferably, the protein is the N protein. Therefore, as one or more antibodies against the target molecule constituting SARS-CoV-2, one or more antibodies against the N protein are preferred.

[0036] The SARS-CoV-2 N protein detected in the present invention is the N protein (i.e., natural or mutant N protein) in any strain of SARS-CoV-2. Such strains include, for example, the major L and S strains, as well as their substrains. A large number of strains of SARS-CoV-2 have been reported. For example, for such a large number of strains, reference can be made to the influenza virus gene database GISAID (Global Initiative on Sharing All Influenza Data). Regarding SARS-CoV-2, its genomic sequence information has been disclosed (for example, for the SARS-CoV-2 Wuhan-Hu-1 strain, refer to GenBank accession number: MN908947). Therefore, as the SARS-CoV-2 N protein, reference can be made to the N protein encoded by such a genomic sequence. Also, for the natural N protein of SARS-CoV-2, reference may be made to the amino acid sequence of SEQ ID NO: 4.

[0037] The antibody may be either a polyclonal antibody or a monoclonal antibody. The antibody may be of any isotype of immunoglobulin (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 that includes heavy and light chains each containing a variable region and a constant region (e.g., an antibody containing 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 part 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.

[0038] The antibody can be prepared using previously known methods. For example, the antibody can be efficiently prepared by using a desired epitope as an antigen.

[0039] 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 the solid phase include a solid phase that can be suspended or dispersed in a liquid phase (e.g., a solid-phase carrier such as particles or beads), and a solid phase that can accommodate or carry a liquid phase (e.g., a support such as a plate, membrane, test tube, etc., and a container such as a well plate, microchannel, glass capillary, nanopillar, monolithic column, etc.). Examples of the material of the solid phase include glass, silica, polymer compounds (e.g., polystyrene, plastic), metal, and carbon. As the material of the solid phase, a non-magnetic material or a magnetic material can also be used. The immobilization of the antibody on the solid phase can be performed by any method. Such methods include, for example, a covalent bonding method, a method using an affinity substance (e.g., biotin, streptavidin), an ionic bonding method, and a physical adsorption method. In the covalent bonding method, for example, periodic acid, glutaraldehyde, maleimide, or N-hydroxysuccinimide can be used.

[0040] 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 nucleic acids of a sense strand and an antisense strand complementary to each other), 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, and pigments). Labeling of the antibody with the labeling substance can be performed by any method. Examples of such methods include the methods described for immobilization of the antibody on a solid phase.

[0041] The method of the present invention can detect the target molecule by an immunological method using the above antibody. Examples of such immunological methods include direct competitive method, indirect competitive method, sandwich method, Western blot method, and immunohistochemical staining method. Such immunoassays may preferably be the sandwich method. In addition, examples of such immunoassays include chemiluminescent immunoassay (CLIA) (e.g., chemiluminescent enzyme immunoassay (CLEIA)), immunoturbidimetry (TIA), enzyme immunoassay (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), radioimmunoassay (RIA), latex agglutination reaction method, fluorescence immunoassay (FIA), and immunochromatography method, and any measurement system based on the principle of specifically detecting the binding of an antigen and an antibody is applicable. The method of the present invention may be a qualitative method or a quantitative method.

[0042] The detection of the label can be carried out based on a method appropriately selected according to the type of the label. For example, when the label is an enzyme, the label can be detected by detecting the enzyme activity using a signal generating substrate (e.g., a fluorescent substrate, a luminescent substrate, a chromogenic substrate). When the label is an affinity substance, the label can be detected by detecting the enzyme or the signal generating substance bound to the affinity substance using an enzyme or a signal generating substance having the ability to bind to the affinity substance. Such an enzyme or signal generating substance having the ability to bind to the affinity substance may be an enzyme or a signal generating substance bound to a substance having the ability to bind 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 these labels. When a coloring substance is used as the labeling substance, the label can be detected visually.

[0043] In a specific embodiment, the method of the present invention may be carried out by the sandwich method. The sandwich method is excellent in sensitivity and specificity.

[0044] In the sandwich method, the detection of the target molecule may be performed, for example, by a step of treating the sample with a solid-phase antibody to bind the target molecule in the sample to the solid-phase antibody (e.g., a step of contacting the sample with the solid-phase antibody), and a step of detecting the target molecule bound to the solid-phase antibody with a labeled antibody. The detection of the target molecule may further include a step of removing the target molecule not bound to the solid-phase antibody (e.g., a (B / F separation or washing step). Also, in the sandwich method, the detection of the target molecule may be performed, for example, by a step of treating the sample with a labeled antibody to bind the target molecule in the sample to the labeled antibody (e.g., a step of contacting the sample with the labeled antibody), a step of further treating the target molecule with a solid-phase antibody to bind the target molecule to the solid-phase antibody, and a step of detecting the target molecule bound to the solid-phase antibody and the labeled antibody with the labeled antibody. As the solid-phase antibody, an antibody previously immobilized on a solid phase or an antibody immobilized on a solid phase when detecting an N protein may be used. For example, a step of treating the sample with an antibody capable of being immobilized on a solid phase to bind the N protein in the sample to the antibody (e.g., a step of contacting the sample with the solid-phase antibody), a step of binding the N protein bound to the antibody capable of being immobilized on a solid phase to the labeled antibody, a step of immobilizing the antibody capable of being immobilized on a solid phase on the solid phase, and a step of detecting the N protein bound to the antibody immobilized on the solid phase (solid-phase antibody) and the labeled antibody with the labeled antibody may be performed. As a method for binding the antibody immobilized on the solid phase to the solid phase, the above-described affinity substance can be used.

[0045] In another specific embodiment, it may be performed by an immunochromatography method. The immunochromatography method is preferable as a rapid and simple qualitative method. The immunochromatography method and the instruments used therefor are well-known. As such an instrument, for example, an immunochromatography cartridge including a zone containing a solid-phase antibody and a zone containing a labeled antibody (which may further include a storage portion for a developing solution) can be used. Hereinafter, as a preferred example of immunochromatography, an outline of a lateral flow type immunochromatography method and instrument will be described with reference to FIG. 1.

[0046] In FIG. 1, an immunochromatography cartridge 1 includes a detection zone 3 (zone containing a solid-phase antibody) in which a capture antibody is immobilized linearly on a matrix 2 made of a porous material such as a nitrocellulose membrane, and a labeled reagent zone 4 (zone containing a labeled antibody) carrying a detection antibody upstream thereof (upstream in the direction in which the developing solution described later flows). The matrix 2 is usually formed in a strip shape. The labeled reagent zone 4 is composed of a porous pad spotted with a labeled antibody. A developing solution tank 5 for storing the developing solution is provided at the upstream end of the matrix. Further, a developing confirmation section 6 for confirming that the developing solution has flowed is provided downstream of the detection zone, and further downstream thereof, a developing solution absorption zone 7 provided with a porous absorption pad for absorbing the developing solution is provided. In the developing confirmation section, a probe (for example, an antibody) having an affinity for a substance other than the detection substance (target molecule) that flows with the developing solution, such as an anti-labeled antibody, is immobilized linearly. Further, when the label is an enzyme, a substrate zone 8 carrying the substrate of the enzyme is provided upstream of the labeled reagent zone. By previously providing a member (not described), such as a pushing-in portion having a protrusion, near the developing solution tank 5, the developing solution tank can be easily broken by pushing it, and the developing solution can be supplied to the matrix. Also, a developing solution pad 9 may be provided so as to cover the developing solution tank and the upper end of the matrix so that the developing solution in the broken developing solution tank is easily supplied to the upper end of the matrix.

[0047] In use, a sample is added to the labeling reagent zone 4, the developing liquid tank 5 is broken, and the developing liquid is supplied by bringing it into contact with the upper end of the matrix. The developing liquid supplied to the matrix flows downstream due to the capillary action of the matrix. When the developing liquid passes through the substrate zone, the substrate is eluted into the developing liquid, and the developing liquid containing the substrate flows. Subsequently, when the developing liquid passes through the labeling reagent zone, the labeled antibody and the sample are eluted into the developing liquid, and the developing liquid containing the substrate, the labeled antibody, and the sample flows. When the target molecule is contained in the sample, the target molecule and the labeled antibody bind by an antigen-antibody reaction. When these reach the detection zone, in the detection zone, the solid-phase antibody and the target molecule bind by an antigen-antibody reaction. As a result, the labeled antibody is immobilized on the detection zone via the target molecule. By detecting the label thus immobilized on the detection zone, the target molecule will be detected. When the target molecule is not contained in the sample, nothing binds to the immobilized antibody, so the labeled antibody is not immobilized on the detection zone. The developing liquid continues to flow downstream and when it reaches the developing liquid confirmation part, for example, if an anti-label antibody is immobilized on the developing liquid confirmation part, the labeled antibody that did not bind to the target molecule and the anti-label antibody bind by an antigen-antibody reaction, and as a result, the labeled antibody is immobilized on the developing liquid confirmation part. When a label is detected in the developing liquid confirmation part, it can be confirmed that the developing liquid has been correctly developed up to the developing confirmation part. The developing liquid is further absorbed by the absorption pad downstream thereof.

[0048] In certain embodiments, the following antibodies (1) and (2) may be used to detect the SARS-CoV-2 N protein: (1) A first antibody against the first epitope in the amino acid region 260 - 305 of the SARS-CoV-2 N protein; and (2) A second antibody against the second epitope in the amino acid region 365 - 419 of the SARS-CoV-2 N protein.

[0049] The first epitope is present in the amino acid region 260 - 305 of the SARS-CoV-2 N protein.

[0050] The second epitope is present in the amino acid region from position 365 to 419 in the SARS-CoV-2 N protein.

[0051] In one embodiment, the method of the present invention may further include using a third antibody against a third epitope in the amino acid region from position 120 to 147 in the SARS-CoV-2 N protein. By using such a third antibody in further combination with the antibodies of (1) and (2) above, the SARS-CoV-2 N protein can be detected with high sensitivity. The third epitope is present in the amino acid region from position 120 to 147 in the SARS-CoV-2 N protein.

[0052] In another embodiment, the method of the present invention may further include using a fourth antibody against a fourth epitope in the amino acid region from position 44 to 78 in the SARS-CoV-2 N protein. By using such a fourth antibody in further combination with the antibodies of (1) and (2) above, the SARS-CoV-2 N protein can be detected with high sensitivity. The fourth epitope is present in the amino acid region from position 44 to 78 in the SARS-CoV-2 N protein.

[0053] In yet another embodiment, the method of the present invention may further include using a fifth antibody against a fifth epitope in the amino acid region from position 243 to 259 in the SARS-CoV-2 N protein. By using such a fifth antibody in further combination with the antibodies of (1) and (2) above, the SARS-CoV-2 N protein can be detected with high sensitivity. The fifth epitope is present in the amino acid region from position 243 to 259 in the SARS-CoV-2 N protein.

[0054] In yet another embodiment, the method of the present invention may further comprise using a sixth antibody against the sixth epitope in the amino acid region 306 - 339 of the SARS-CoV-2 N protein. By using such a sixth antibody in further combination with the antibodies of (1) and (2) above, the SARS-CoV-2 N protein can be detected with high sensitivity. The sixth epitope is present in the amino acid region 306 - 339 of the SARS-CoV-2 N protein.

[0055] The present invention may also use the following combinations of antibodies: (1) A combination of the third antibody and the first antibody; (2) A combination of the third antibody and the second antibody; (3) A combination of the fourth antibody and the first antibody; (4) A combination of the fourth antibody and the 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.

[0056] In a particular embodiment, both the first antibody and the second antibody may be used as solid-phase antibodies or labeled antibodies. By using both the first antibody and the second antibody together as solid-phase antibodies or labeled antibodies, the SARS-CoV-2 N protein can be detected with higher sensitivity, and there is an advantage that it is easier to avoid the binding competition often seen between solid-phase antibodies and labeled antibodies. More preferably, both the first antibody and the second antibody may be used as solid-phase antibodies.

[0057] 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, the SARS-CoV-2 N protein can be detected with higher sensitivity.

[0058] In another specific embodiment, the fourth antibody and the fifth antibody may be used in different forms. 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 forms, the SARS-CoV-2 N protein can be detected with higher sensitivity.

[0059] In another specific embodiment, the sixth antibody may be used as a labeled antibody or a solid-phase antibody.

[0060] The present invention also provides an immunological detection reagent for SARS-CoV-2, which comprises the following antibodies (1) and (2): (1) An amphoteric surfactant containing a hydrocarbon chain having a chain length of 12 to 18 carbon atoms; and (2) One or more antibodies against the target molecule constituting SARS-CoV-2.

[0061] The reagent of the present invention may further contain the following component (3) and / or (4): (3) An anionic surfactant or an amphoteric surfactant having a steroid skeleton; and / or (4) A nonionic surfactant.

[0062] Details (e.g., definitions, exemplifications, and preferred exemplifications) of terms such as the zwitterionic surfactant containing the hydrocarbon chain and the above one or more antibodies in the reagent of the present invention are the same as those described in the method of the present invention.

[0063] The reagent of the present invention can contain the zwitterionic surfactant containing the hydrocarbon chain and the above one or more antibodies in the form of a mixture (e.g., in the form of a mixed solution contained in the same container), or in a form isolated from each other (e.g., in a form contained in different containers). The reagent of the present invention can also contain, in addition to the zwitterionic surfactant containing the hydrocarbon chain and the above one or more antibodies, the components of the above (3) and / or (4) in the form of a mixture or in a form isolated from each other. The reagent of the present invention may be provided in the form of a kit. The reagent of the present invention may also be provided in the form of a device. For example, all of the above one or more antibodies may be contained in the device. Alternatively, a part of the above one or more antibodies may be contained in the device, and the rest may not be contained in the device (e.g., in a form contained in different containers). In this case, the antibody not contained in the device may be injected into the device during detection.

[0064] The reagent of the present invention can contain a solid-phase antibody and / or a labeled antibody as the above one or more antibodies. Alternatively, such a reagent may contain a solid phase and / or a labeling substance when it does not contain a solid-phase antibody and / or a labeled antibody. The solid phase and the labeling substance are the same as those described above. When the labeling substance is an enzyme, the reagent of the present invention may contain a substrate of the enzyme (e.g., a substrate that generates a detection signal, or a substrate that is converted into a product that generates a detection signal by the enzyme, or a substrate that can be conjugated with another enzyme reaction that utilizes a substrate that generates a detection signal or a substrate that is converted into a product that generates a detection signal by the enzyme).

[0065] The reagent of the present invention may further contain the above combination of antibodies.

[0066] The reagent of the present invention is not particularly limited as long as it can be used in immunological measurement methods, and can have a configuration according to the type of immunological measurement method. Preferably, the reagent of the present invention may be a reagent used in the sandwich method. Therefore, the reagent of the present invention may contain a solid phase (e.g., particles such as magnetic particles, well plates, membranes) used in the sandwich method, or a solid-phase antibody immobilized on such a solid phase. The reagent of the present invention may also contain an instrument containing a solid phase used in the immunochromatography method (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 contain a standard of the target molecule (e.g., a protein standard such as an N-protein standard).

Examples

[0067] Next, examples are shown to explain the present invention in more detail, but the present invention is not limited to the following examples. As far as the amounts described in the following examples are concerned, % corresponds to % by weight.

[0068] Example 1: Production of Antibody An antibody was produced according to the method described in International Publication No. 2005 / 042579. Specifically, it is as follows.

[0069] (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 plasmid pWS-N. Using this, Escherichia coli was transformed to obtain ampicillin-resistant transformant Escherichia coli. The nucleotide sequence and amino acid sequence of the N protein are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0070] The obtained transformant was cultured at 37°C in 2 ml of LB medium containing 50 μg / ml of ampicillin. The transformant was grown by preculture, and after the OD at 600 nm reached a density of about 0.7, 0.4 mM IPTG was added to induce expression. After culturing for 18 hours, centrifugation was performed to collect Escherichia coli. 20 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM PMSF was added to the collected Escherichia coli, and ultrasonic disruption treatment was performed under ice cooling. After centrifugation, ammonium sulfate was added to the soluble fraction, and the 20-40% ammonium sulfate fraction was collected. This ammonium sulfate fraction was applied to SP Sepharose Fast Flow (manufactured by Amersham) equilibrated with 0.1 M NaCl, 8 M urea, and 20 mM phosphate buffer (pH 6.9), and eluted and purified 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 and 20 mM Tris-HCl buffer (pH 8.0) to obtain a purified recombinant N protein (CoV) solution. When the purity of the recombinant N protein was confirmed by SDS-PAGE and Western blot, a single band was shown.

[0071] (2) Preparation of monoclonal antibody The recombinant N protein (CoV) prepared above was used to immunize mice, and anti-N protein monoclonal antibodies were prepared by fusing the spleen lymphocytes and myeloma cells of the same mice. That is, BALB / C mice were initially immunized with 50 - 100 μg / mouse of the recombinant N protein emulsified with Freund's complete adjuvant, and 2 - 3 weeks later, boosted with 50 - 100 μg / mouse of the same antigen emulsified with Freund's incomplete adjuvant. The antibody titer was checked by solid-phase ELISA using a 96-well ELISA plate coated with the recombinant N protein. Mice with increased antibody titers were intravenously administered 25 - 100 μg of free recombinant N protein, and 3 - 4 days later, the spleens were removed from the mice and spleen cells were prepared. Mouse myeloma cells (P3U1) that had been cultured in RPMI-1640 medium beforehand were mixed with spleen cells at a ratio of 1:2 - 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 CO 2 incubator at 37°C.

[0072] Antibody screening was performed by the solid-phase ELISA shown above. That is, 50 μL / well of the recombinant N protein was dispensed into a 96-well ELISA plate at a concentration of 1 μg / mL and adsorbed by leaving it overnight at 4°C. After blocking the wells with 1% skim milk, they were washed three times with a washing buffer (PBS containing 0.05% Tween), 50 μL of the culture supernatant of the plate on which cell fusion had been performed was added, and the reaction was carried out at 37°C for 1 hour. After washing three times with the washing buffer in the same manner, a POD-labeled anti-mouse immunoglobulin antibody (manufactured by DACO) was added, and the reaction was further carried out at 37°C for 1 hour. After washing four times with the washing buffer, substrate ABTS was added, and wells showing color development were selected. Next, the cells in the selected wells were transferred to a 24-well culture plate and cultured in a CO 2 incubator at 37°C, and then made into single clones by the limiting dilution method to obtain each monoclonal antibody.

[0073] In addition, instead of the recombinant N protein (CoV), a KLH conjugate of a synthetic peptide (N5 peptide: GQTVTKKSAAEASKKPRC; SEQ ID NO: 3) was used as an immunogen to immunize mice, and anti-N protein monoclonal antibodies were similarly obtained.

[0074] (3) Confirmation of Reactivity of Monoclonal Antibodies The reactivity of each established monoclonal antibody against the native antigen (N protein derived from SARS-CoV) was confirmed by Western blot (WB) using a concentrated virus suspension as a specimen. Vero E6 cells were infected with the SARS virus Hanoi strain and cultured in a CO 2 incubator for 48 hours, then centrifuged at 2,000 rpm for 15 minutes to prepare a virus culture supernatant (TCID50 was 7.95×10 6 / mL). This culture supernatant was inactivated at 56°C for 90 minutes, and then 31.5 mL of it was centrifuged at 30,000 rpm for 3 hours using a Hitachi ultracentrifuge (40T rotor). 0.3 mL of Tris-NaCl-EDTA buffer was added to the obtained precipitate and pipetted to prepare a concentrated virus suspension. An equal volume of sample treatment solution for electrophoresis was added to this suspension, and then heat treatment was performed to obtain a specimen for analysis. After performing SDS-PAGE using a 12.5% gel, the specimen was transferred to a nitrocellulose membrane to prepare a transfer membrane for WB (antigen transfer WB membrane). After blocking the transfer membrane with skim milk, each monoclonal antibody was shaken with the antigen transfer WB membrane at room temperature for 1 hour, subjected to the reaction, and then washed 3 times with a washing buffer (shaking wash for 5 minutes). Next, a POD-labeled anti-mouse immunoglobulin antibody was added and reacted at room temperature for another 1 hour. After washing 4 times with a washing buffer (shaking wash for 5 minutes), a 4-chloronaphthol solution was added as a substrate to confirm the band, and monoclonal antibodies with a band confirmed at a position corresponding to an N protein slightly less than 50 kD in molecular weight were selected.

[0075] The selected monoclonal antibody was further subjected to WB using the recombinant N protein of SARS-CoV-2 prepared in the same manner as in (1) above and the N protein of common coronavirus, and those that bound to the recombinant N protein of SARS-CoV-2 but not to the N protein of common coronavirus were used as anti-N protein monoclonal antibodies. In the following examples, unless otherwise specified, the N protein of SARS-CoV-2 was used as the N protein.

[0076] Example 2: Identification of the reaction region of the antibody (Part 1) Regarding the plurality of anti-N protein monoclonal antibodies obtained in Example 1 above, their epitopes were identified by the WB method using a recombinant antigen containing a partial sequence of the N protein.

[0077] First, an equal amount of sample treatment solution for electrophoresis was added to a solution containing recombinant antigens of the full-length N protein of SARS-CoV-2 (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, and then heat treatment was performed to obtain 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.

[0078] As a result, it was confirmed that six antibodies (anti-N protein antibodies N1 to N6) reacted with the full-length antigen and the NTD antigen, and six antibodies (anti-N protein antibodies C1 to C6) reacted with the full-length antigen and the CTD antigen, and a plurality of antibodies reacting with each of NTD and CTD were obtained. In addition, antibodies (anti-N protein antibodies C12, C13) that recognize the region contained in the amino acid sequence at positions 365 to 419, which react with the full-length antigen and the C-terminal peptide but do not react with the NTD or CTD antigen, were also obtained. Furthermore, as an immunogen, a plurality of anti-N protein monoclonal antibodies (P1, P2) that recognize the amino acid region at positions 243 to 259 of the N protein, obtained by immunizing mice with a KLH conjugate of a synthetic peptide (N5 peptide: GQTVTKKSAAEASKKPRC: SEQ ID NO: 3), were also obtained.

[0079] Example 3: Preparation of solid-phase antibody (antibody-immobilized magnetic particles) Using a carboxyl-amine cross-linking agent (carbodiimide, Thermo-Fisher Scientific), according to the product manual, the anti-N protein monoclonal antibody obtained above was chemically bonded to magnetic particles (manufactured by Fuji Rebio) to obtain a solid-phase antibody (antibody-immobilized magnetic particles).

[0080] Example 4: Preparation of labeled antibody (alkaline phosphatase-labeled anti-N protein monoclonal antibody) The anti-N protein monoclonal antibody obtained above and pepsin were mixed in 0.1 M citrate buffer (pH 3.5) and allowed to stand at 37°C for 1 hour for pepsin digestion. After stopping the reaction, gel filtration purification was performed to obtain an antibody from which the Fc region was removed. Next, 2-mercaptoethylamine hydrochloride (2-MEA) was added for thiolation. Furthermore, this was desalted to obtain a Fab' fragment.

[0081] Alkaline phosphatase treated with N-(4-maleimidobutyryloxy)-succinimide (GMBS) and Fab’ fragments prepared from each anti-N protein monoclonal antibody were mixed at a molar ratio of 1 - 3:1 for coupling. 2-Mercaptoethylamine hydrochloride and iodoacetamide were added to the coupling solution to stop the reaction. Further, the peaks of the molecular weights at which Fab’ and ALP were in a ratio of 1 - 3:1 were pooled by gel filtration to obtain a labeled antibody (ALP-labeled anti-N protein monoclonal antibody).

[0082] Example 5: Sandwich immunoassay using particles 100 μL of a solution containing recombinant N protein or a specimen was added to 50 μL of a particle suspension diluent (50 mM Tris, 1% BSA, 150 mM NaCl, 1 mM EDTA, 0.1% NaN 3 ) containing 0.03% of the antibody-immobilized magnetic particles prepared in Example 3 above, and reacted at 37 °C for 8 minutes.

[0083] After the reaction, B / F separation was performed with a magnet and washed with a Lumipulse (registered trademark) washing solution (manufactured by Fujirebio Inc.). Then, 50 μL of a 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 reacted at 37 °C for 8 minutes. B / F separation was performed with a magnet and washed with the Lumipulse washing solution. Then, 200 μL of a Lumipulse (registered trademark) substrate solution (manufactured by Fujirebio Inc.) containing 3-(2’-spiroadamantane)-4-methoxy-4-(3’’-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD) was added, and an enzymatic reaction was carried out at 37 °C for 4 minutes, and the luminescence amount at a wavelength of 463 nm was measured.

[0084] Example 6: Immunochromatography method As shown in Fig. 1, 0.5 μL of an aqueous solution containing an anti-N protein monoclonal antibody was spotted on a nitrocellulose membrane and dried at a position 15 mm from the end on the developing solution absorption zone side of a matrix made of a nitrocellulose membrane with a width of 3.7 mm and a length of 50 mm to create a detection zone. Further, an anti-alkaline phosphatase (ALP) antibody solution (sodium carbonate buffer containing 0.15 M NaCl) was spotted and dried at a position 12 mm from the end on the developing solution absorption zone side to create a development confirmation section. Also, 0.9 μL of a 5-bromo-4-chloro-3-indolyl phosphate disodium salt (BCIP) solution as a substrate was spotted linearly 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 composed of an enzyme-labeled reagent pad.

[0085] After overlapping the matrix, the developing solution pad, the enzyme-labeled reagent pad, and the absorption pad (high water-retentive filter paper) as shown in Fig. 1 and fixing them with an adhesive tape, they were fixed in a plastic case having a developing solution tank 11 to manufacture an immunochromatography cartridge for detecting SARS-N protein.

[0086] A solution containing recombinant N protein or 20 μL of a specimen was mixed with 100 μL of a treatment solution and allowed to stand at room temperature for about 5 minutes. The N protein in the obtained mixed solution was detected using the manufactured immunochromatography cartridge. Specifically, 20 μL of the mixed solution was dropped onto the labeled antibody zone of the immunochromatography cartridge, and then the developing solution tank was broken to supply the developing solution to the developing solution pad and the upper end of the matrix to start the measurement. After 30 minutes from the start of the measurement, after confirming the development of the developing solution by the color development of the development confirmation section 10, the color development of the detection zone was visually measured.

[0087] Example 7: Identification of the reaction region of the antibody (Part 2) According to the sandwich immunoassay method of Example 5 above, recombinant N protein (SARS-CoV-2 or SARS-CoV) was measured, and grouping was performed according to the reaction regions of each N protein monoclonal antibody. Specifically, N1-N6 antibodies, C1-C6 antibodies, and C12 antibody were used as antibody-immobilized magnetic particles (solid-phase antibody) and ALP-labeled antibody, respectively, to measure recombinant N protein.

[0088] As a result, when recombinant N protein (0, 10 ng / mL) was measured using N1 antibody and N2 antibody as the solid-phase antibody and the labeled antibody, respectively, the luminescence ratio of 10 ng / mL to the luminescence amount (background value) of 0 ng / mL was very low, and it was confirmed that both reacted with overlapping regions (Table 1). Similarly, when recombinant N protein (0, 1 ng / mL) was measured using N4, N5, N6 antibodies (N6 antibody group) as the solid-phase antibody and the labeled antibody, respectively, the luminescence difference obtained by subtracting the luminescence amount of 0 ng / mL from the luminescence amount of 1 ng / mL was very low, and it was confirmed that these antibodies recognized overlapping regions (Table 2). On the other hand, when N1 antibody and N3 antibody were used as the solid-phase antibody and the labeled antibody, respectively, it was confirmed that there was partial competition because the luminescence difference was smaller than that of combinations with other antibodies. Since the reactivity of N1 antibody was higher than that of N3 antibody in the sandwich immunoassay with other NTD antibodies, in the sandwich immunoassay using multiple antibodies, the N1 antibody group (N1, N2 antibodies) was considered to be more useful than N3 antibody.

[0089]

Table 1

[0090]

Table 2

[0091] When C1 to C6 antibodies were similarly examined, it was confirmed that the C1 antibody and the C2 antibody recognize overlapping regions, the C3 antibody and the C4 antibody recognize overlapping regions, and the C5 antibody and the C6 antibody recognize overlapping regions (Tables 3 to 5). However, since the luminescence amount decreased in any combination of the C5 and C6 antibodies, it was confirmed that the antibodies against the regions recognized by the C5 antibody group (C5 and C6 antibodies) have low reactivity in the measurement by the sandwich immunoassay.

[0092]

Table 3

[0093]

Table 4

[0094]

Table 5

[0095] Example 8: Examination of Antibody Combinations (Part 1) For the antibodies grouped in Example 7 above and the C12 antibody, combinations of the solid-phase antibody and the labeled antibody were examined. In the same manner as the method of the sandwich immunoassay in Example 5 above, recombinant N protein was measured using each combination of the antibodies shown in Table 6. The measurement was performed with N = 2 for each.

[0096] The results are shown in Table 6. Table 6 shows the ratio (S / N ratio) of the average luminescence amount from a 1 ng / mL sample to the average luminescence amount from a 0 ng / mL sample.

[0097]

Table 6

[0098] As a result, as combinations of the solid-phase antibody and the labeled antibody in the sandwich immunoassay, combinations of the N1 antibody and the C3 antibody, the N1 antibody and the C12 antibody, the N4 antibody and the C3 antibody, the N4 antibody and the C12 antibody, and the C3 antibody and the C12 antibody, each showing a high S / N ratio, were confirmed to be preferable.

[0099] Example 9: Examination of Antibody Combinations (Part 2) Regarding the antibodies grouped in Example 7 above and the P1 antibody and the C12 antibody, combinations of the solid-phase antibody and the labeled antibody were examined. Using each combination of the antibodies shown in Tables 7 and 8 by the immunochromatography method of Example 6 above, a specimen containing 10 ng / mL of recombinant N protein was measured. Also, using each combination of the antibodies shown in Tables 7 and 8, after confirming the development of the developing solution by the color development of the development confirmation section 10 30 minutes (Table 7) or 60 minutes (Table 8) after the start of measurement, the color development of the detection zone was visually measured. The results are shown in Tables 7 and 8. In Tables 7 and 8, the color development intensity of the detection zone under each condition is shown. The larger the numerical value, the darker the color development, and "w" indicates weaker. That is, as the color development intensity, it becomes weaker in the order of 4.5>4>3>2>1>w. A blank indicates not carried out.

[0100]

Table 7

[0101]

Table 8

[0102] As a result, similar to the sandwich immunoassay using particles, strong luminescence intensities were confirmed for combinations of the N1 antibody·N2 antibody and the C3 antibody, the N1 antibody and the C12 antibody, the N4 antibody and the C3 antibody, the N4 antibody and the C12 antibody, and the C3 antibody and the C12 antibody. Furthermore, the N1 antibody·N2 antibody and the N4 antibody also showed relatively high luminescence intensities.

[0103] Furthermore, when evaluating the combinations of the C1 antibody and the P1 antibody with other antibodies, the C1 antibody showed relatively strong luminescence intensity in combination with the C3 antibody. On the other hand, the P1 antibody showed strong luminescence intensity in combination with the C3 antibody and relatively strong luminescence intensity in combination with the N1 antibody and the N4 antibody.

[0104] Example 10: Examination of Antibody Combinations (Part 3) In Examples 8 and 9 above, the combination of a single solid-phase antibody and a single labeled antibody was examined. Although a highly sensitive measurement system could be constructed even with such a combination, it is more preferable to use a combination of solid-phase antibodies that recognize different regions and / or labeled antibodies that recognize different regions. Therefore, an immunoassay system was constructed in which one of the antibodies that showed an effect alone in Examples 8 and 9 above was used as the labeled antibody, and a plurality of other antibodies were used as the solid-phase antibodies, and a sample containing 10 ng / mL of recombinant N protein (SARS-CoV) was evaluated in the same manner as in Example 8 above. The solid-phase antibody was prepared by simultaneously chemically bonding a plurality of antibodies and particles according to the production method of Example 3 above. The results are shown in Table 9.

[0105] As a result, when a mixture of the C3 antibody and the C12 antibody was used, excellent improvements in the luminescence amount (count at 1000 pg / ml) and the S / N ratio were observed. In particular, when the N1 antibody or the N4 antibody was used as the labeled antibody, when a mixture of the C3 antibody and the C12 antibody was used as the solid-phase antibody, it was confirmed that the luminescence amount increased and the S / N ratio also increased compared to the case where the C3 antibody and the C12 antibody were used alone as the solid-phase antibody. On the other hand, when the C3 antibody or the C12 antibody was used as the labeled antibody, even when a mixture of the N1 antibody and the N4 antibody was used as the solid-phase antibody, neither the count value nor the S / N ratio increased. From this, it was confirmed that it is effective for improving the sensitivity in a sandwich immunoassay to use the C3 antibody and the C12 antibody in combination, rather than simply using a plurality of antibodies for the solid-phase antibody.

[0106]

Table 9

[0107] Example 11: Identification of the reaction region of the antibody (Part 3) For the antibodies confirmed to be effective in Examples 8 to 10 above, the epitopes of the N protein (SARS-CoV-2) were analyzed.

[0108] For the N1 antibody group (N1, N2 antibodies) and N6 antibody group (N4 - N6 antibodies) that recognize the NTD, the recombinant antigens consisting of the N-terminal region (amino acids 44 - 112), central region (amino acids 79 - 147), and C-terminal region (amino acids 113 - 180) of the NTD, and the recombinant antigens (trNP1, trNP2, trNP3, trNP4 antigens) consisting of the amino acid regions 1 - 119, 110 - 229, 220 - 339, and 330 - 419 of the N protein, prepared in the same manner as in Example 1 above, were used for WB to analyze the reaction region. As a result, the N1 antibody group was shown to recognize the amino acid region 44 - 78 of the N protein. Also, the N6 antibody group was shown to recognize the amino acid region 120 - 147 of the N protein.

[0109] Similarly, for the C3 antibody group and C1 antibody group that recognize the CTD, the recombinant antigens consisting of the N-terminal region (amino acids 247 - 305), central region (amino acids 276 - 334), and C-terminal region (amino acids 306 - 364) of the CTD, prepared in the same manner as in Example 1 above, and trNP1, trNP2, trNP4, and trNP4 antigens were used for WB, sandwich assay, and analysis of the reaction region based on the reactivity with synthetic peptides and other antibodies. As a result, the C3 antibody group was shown to recognize the amino acid region 260 - 305 of the N protein. Also, the C1 antibody group was shown to recognize the amino acid region 306 - 339 of the N protein.

[0110] In summary, the epitopes of the analyzed antibodies were as follows.

Table A

[0111] Example 12: Examination of Antibody Combinations (Part 4) In Example 10 above, when using the N1 antibody or the N4 antibody, it was shown that it is useful to use a mixture of the C3 antibody and the C12 antibody as the solid-phase antibody. To confirm that these antibody combinations are similarly useful for SARS-CoV-2, a solution containing 100 pg / mL of recombinant N protein was measured in the same manner as in Example 5 using the combinations of solid-phase antibodies and labeled antibodies described in Table 10.

[0112] Specifically, 100 μL of a solution containing recombinant N protein, 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 diluent containing the solid-phase antibody, and reacted at 37°C for 8 minutes. After the reaction, B / F separation was performed with a magnet, and after washing with a Lumipulse washing solution, 50 μL of a labeled body fluid containing an ALP-labeled antibody was added and reacted at 37°C for 8 minutes. B / F separation was performed with a magnet, and after washing with a Lumipulse washing solution, 200 μL of a Lumipulse substrate solution containing AMPPD was added and an enzymatic reaction was carried out at 37°C for 4 minutes, and the amount of luminescence at a wavelength of 463 nm was measured to measure the N protein of each sample. The solid-phase antibody was prepared by chemically bonding each antibody and particles according to Example 3 above and then mixing them at a ratio of 1:1. The labeled antibody was prepared by simultaneously mixing GMBS-treated ALP and Fab’ fragments prepared from a plurality of anti-N protein monoclonal antibodies so that the molar ratio of each Fab’ fragment to ALP was 1:1, performing coupling, and preparing an ALP-labeled antibody according to Example 4 above.

[0113] The results are shown in Table 10. As a result, when a mixture of the C3 antibody and the C12 antibody was used, excellent improvements in the luminescence amount (count at 100 pg / ml) and the S / N ratio were observed. It was confirmed that the combined use of the C3 antibody and the C12 antibody is also effective in improving the sensitivity in the sandwich immunoassay for SARS-CoV-2.

[0114]

Table 10

[0115] Example 13: Examination of Antibody Combinations (Part 5) In Examples 10 and 12 above, when the N1 antibody or the N4 antibody was used, it was shown that it is useful to use a mixture of the C3 antibody and the C12 antibody as the solid-phase antibody. As described above, since 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, antibodies in a group that are less reactive with the groups of N1 / N2 antibodies, N4 / N6 antibodies, C3 / C4 antibodies, and C12 / C13 antibodies were evaluated for their combinations with the P1 / P2 antibodies, which are highly reactive with the N1 antibody, the N4 antibody, and the C3 antibody in Example 10 above. Combinations with the C1 / C2 antibodies were also evaluated.

[0116] Specifically, combinations of the solid-phase antibody and the labeled antibody described in Tables 11-1 to 11-3 were used and evaluated in the same manner as in Example 8 above. The solid-phase antibody was prepared by simultaneously chemically bonding a plurality of antibodies and particles according to Example 3 above. The labeled antibody was prepared by simultaneously mixing GMBS-treated ALP and Fab' fragments prepared from a plurality of anti-N protein monoclonal antibodies so that the molar ratio of each Fab' fragment to alkaline phosphatase (ALP) was 1:1, followed by coupling to prepare an ALP-labeled antibody.

[0117] As the sample, a SARS-CoV-2 negative saliva sample to which recombinant N protein or 1 - 5 nasal swabs from SARS-CoV-2 positive patients (purchased sample: Boca Biolistics, LLC) were added was used as the sample. To 100 μL of the sample, 170 μL of a sample treatment solution (Tris buffer, 150 mM NaCl, 1% BSA, 0.1% NaN 3 , 0.25% C16APS) was added and used.

[0118] The results are shown in Tables 11 - 1 to 11 - 3. In Tables 11 - 1 to 11 - 3, the luminescence ratio (S / N ratio) of the luminescence amount when measuring 10 ng / mL of recombinant N protein to the luminescence amount when measuring 0 ng / mL of recombinant N protein, and the luminescence ratio of the luminescence amount when measuring each SARS-CoV-2 positive sample to the luminescence amount when measuring 0 ng / mL of recombinant N protein are shown.

[0119]

Table 11 - 1

[0120]

Table 11 - 2

[0121]

Table 11 - 3

[0122] As a result, when using the C3 antibody and the C12 antibody as the solid-phase antibody or the labeled antibody, and using the N6 antibody on the other hand, SARS-CoV-2 in the sample could be measured with very high sensitivity. That is, when using the antibody that recognizes the amino acid region from the 260th to the 305th and the antibody that recognizes the amino acid region from the 365th to the 419th as the solid-phase antibody or the labeled antibody, and using the antibody that recognizes the amino acid region from the 120th to the 147th on the other hand, it was confirmed that SARS-CoV-2 in the sample could be measured with very high sensitivity.

[0123] Furthermore, by combining the N1 antibody and the P1 antibody, it was possible to measure SARS-CoV-2 in a sample with higher sensitivity. That is, in addition to the above antibody combination, it was confirmed that by further combining an antibody that recognizes the amino acid region from the 44th to the 78th and an antibody that recognizes the amino acid region from the 243rd to the 259th, SARS-CoV-2 in a sample can be measured with higher sensitivity.

[0124] In addition, even when the C1 antibody was further added to these antibody combinations, it was possible to measure SARS-CoV-2 in a sample with sufficiently high sensitivity. That is, it was confirmed that further combining an antibody that recognizes a CTD region different from these antibodies is also effective.

[0125] Example 14: Examination of Surfactants Used in the Reaction Solution A sample was prepared by adding nasal swab fluid (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 (Tris buffer, 150 mM NaCl, 1% BSA, 0.1% NaN 3 , with a concentration of each surfactant in the treatment solution of 2.5% and a concentration after mixing the sample and the particle suspension of 0.19%) were added to 150 μL of a particle suspension diluent containing a solid-phase antibody, and reacted at 37°C for 8 minutes. After the reaction, B / F separation was performed with a magnet, and after washing with a Lumipulse washing solution (manufactured by Fujirebio), 150 μL of a labeled body fluid containing the ALP (alkaline phosphatase) labeled antibody prepared in Example 4 above was added and reacted at 37°C for 8 minutes. B / F separation was performed with a magnet, and after washing with a Lumipulse washing solution, 200 μL of a Lumipulse substrate solution containing AMPPD (3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane disodium salt) was added and an enzymatic reaction was carried out at 37°C for 4 minutes, and the amount of luminescence at a wavelength of 463 nm was measured to measure the N protein of each sample. Note that the N6 antibody and the P1 antibody were used as the solid-phase antibodies, and the N1 antibody, the C3 antibody, and the C12 antibody were used as the labeled antibodies.

[0126] The results are shown in Table 12. Table 12 also shows the ratio (%) of the luminescence value under each surfactant addition condition to the luminescence value under the condition without adding a surfactant, in addition to the measured luminescence value.

[0127]

Table 12

[0128] As a result, when using zwitterionic surfactants (C12APS and C14APS) containing a hydrocarbon chain as the hydrophobic part, very high luminescence values were obtained for positive specimens, and the N protein could be detected with high sensitivity. From the above, it was shown that zwitterionic surfactants containing a hydrocarbon chain as the hydrophobic part are effective for measuring the SARS-CoV-2 N protein contained in patient specimens.

[0129] Example 15: Examination of surfactants used in specimen processing solution (Part 1) In Example 14 above, it was found that mixing a zwitterionic surfactant containing a hydrocarbon chain as the hydrophobic part with the specimen is useful in measuring the SARS-CoV-2 N protein. Therefore, using the specimen treated with a zwitterionic surfactant containing a hydrocarbon chain as the hydrophobic part, the N protein was detected by the immunochromatography method of Example 6 above.

[0130] As the specimens, purchased nasopharyngeal specimens (negative specimen, positive specimen, Boca Biolistics, LLC) were used. As the specimen processing solution, a Tris buffer solution (150 mM NaCl, 1% BSA, 0.1% NaN 3 ) containing each surfactant was used. Also, P1 antibody and N6 antibody were used as the solid-phase antibodies, and N1 antibody, C3 antibody and C12 antibody were used as the labeled antibodies. The coloring states of the detection zones under each condition are shown in Table 13. The larger the numerical value, the darker the coloring, and "w" indicates weaker. That is, as the coloring intensity, it becomes weaker in the order of 1>1w>1ww. As a result, as shown in Table 13, it was confirmed that the N protein could be detected with sufficient sensitivity.

[0131]

Table 13

[0132] Example 16: Examination of Surfactants in Specimen Processing Solution (Part 2) In the above Examples 14 and 15, it was found that in the measurement of SARS-CoV-2 N protein, it is useful to process the specimen with an amphoteric surfactant containing a hydrocarbon chain as the hydrophobic part.

[0133] Next, combinations of amphoteric surfactants containing a hydrocarbon chain as the hydrophobic part and other surfactants were examined by a sandwich immunoassay method using particles.

[0134] As the specimen, a sample obtained by adding 6 μL of a SARS-CoV-2 positive nasopharyngeal specimen (Boca Biolistics, LLC) to 3 mL of the viral transport fluid SGVTM-3R (Sugiyama Ken Co., Ltd.) was used. Among the purchased SARS-CoV-2 positive specimens (Boca Biolistics, LLC), specimens with different virus amounts determined by RT-PCR were selected and used as low-value specimens and medium-value specimens.

[0135] The surfactant shown in Table 14 and a specimen processing solution containing 0.25% C16APS (Tris buffer, 150 mM NaCl, 1% BSA, 0.1% NaN 3) 20 μL was added to 100 μL of the sample and incubated at 37°C for 6.5 minutes. Then, 50 μL of a particle suspension diluent containing a solid-phase antibody was added to the treated sample and reacted at 37°C for 8 minutes. After the reaction, B / F separation was performed with a magnet and washed with a Lumipulse washing solution (manufactured by Fujirebio). Then, 50 μL of a labeled body fluid containing the ALP-labeled antibody prepared in Example 4 above was added and reacted at 37°C for 8 minutes. After B / F separation with a magnet and washing with a Lumipulse washing solution, 200 μL of a 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 luminescence amount at a wavelength of 463 nm. Also, standard solutions (0, 100, 5000, 10000 pg / mL) containing recombinant N protein of known concentrations were measured in the same manner and a standard curve was created. From the luminescence amount of each sample and the standard curve, the concentration of the N protein in each sample was determined. The results are shown in Table 14.

[0136] As a result, by further adding deoxycholic acid or cholic acid to the treatment solution, an increase in the luminescence amount was observed for both the low-value sample (LS) and the medium-value sample (MS). In particular, by additionally adding deoxycholic acid, the luminescence amount increased significantly at all added concentrations.

[0137] Also, when components such as deoxycholic acid are added, the solution gels. Although sample treatment is possible with the gelled solution, it was found that when a nonionic surfactant such as Tween 80, Tween 20, or NP-40 is further added to the treatment solution so that handling becomes easier, a high luminescence amount can be obtained without the solution gelling.

[0138]

Table 14

[0139] Example 17: Destruction of Capsid by Sample Treatment The destruction efficiency of the capsid by sample treatment was examined.

[0140] As a method, the quantification of the extracted RNA was performed by semi-quantifying the genomic RNA extracted from virus particles using the RT-PCR method. At that time, by adding RNase in advance to the specimen processing solution or the specimen diluent used as a control, when the capsid of the virus is destroyed by the specimen processing solution, the RNA is decomposed before the RNA extraction process. By comparing with the amount of RNA before the RNase treatment, the amount of RNA extracted from the particles by the processing solution can be estimated. Thereby, the destruction efficiency of the capsid can be estimated.

[0141] Specifically, a specimen diluent (Tris buffer, 150 mM NaCl, 1% BSA) and a specimen processing solution containing 0.25% C16APS (Tris buffer, 150 mM NaCl, 1% BSA, 0.1% NaN 3 ) to which RNase A (final concentration 200 μg / mL) was added to each were added in an equivalent amount to a specimen prepared by adding the purchased nasopharyngeal swab (Boca Biolistics, LLC) to the specimen diluent. After mixing, it was allowed to stand at room temperature for 5 minutes. Then, RNA was extracted using an RNA extraction kit (GeneJET Viral DNA / RNA Purification kit (Thermo Fisher)) according to the attached manual. The obtained RNA was measured using an RT-PCR kit (SARS-CoV-2 Direct Detection RT-qPCR Kit (TaKaRa)), and the Ct value of RT-PCR was calculated. The larger the Ct value, the more the RNA is decomposed, indicating a higher capsid destruction efficiency. The results are shown in Table 15 and Table 16.

[0142]

Table 15

[0143]

Table 16

[0144] As a result, when the sample was treated with the sample treatment solution (without addition), the Ct value increased by 4.9 for the sample diluent. That is, since the RNA amount decreased to about 1 / 30, it was found that the RNA extraction efficiency increased 30-fold.

[0145] Also, when 0.5% deoxycholic acid was added to the sample treatment solution, although there was no significant change in the Ct value, as the addition amount increased to 1% and 2%, the Ct value increased by 3.8 and 4.9, respectively. That is, it was found that the RNA amount decreased to about 1 / 14 and 1 / 30, respectively, and the RNA extraction efficiency increased 14-fold and 30-fold, respectively.

[0146] Furthermore, when a nonionic surfactant (Tween 20) was added to the sample treatment solution, the Ct value increased by 1. That is, since the RNA amount decreased to 1 / 2, the RNA extraction efficiency increased.

[0147] From these results, it was found that C16APS is effective for the extraction of the capsid, and further, when 1% or more of deoxycholic acid and a nonionic surfactant are added thereto, the RNA extraction effect, that is, the capsid destruction efficiency is enhanced.

Explanation of Symbols

[0148] 1 Immunochromatography cartridge 2 Matrix 3 Detection zone 4 Labeling reagent zone 5 Developing solution tank 6 Development confirmation section 7 Developing solution absorption zone 8 Substrate zone 9 Developing solution pad

Claims

**Claim 1** A method for immunologically detecting SARS-CoV-2, comprising mixing a sample collected from a subject with a buffer solution having a pH of 5.0 to 9.0 containing an amphoteric surfactant containing a hydrocarbon chain having 12 to 18 carbon atoms, and immunologically detecting SARS-CoV-2 using one or more antibodies against target molecules constituting SARS-CoV-2. **Claim 2** The method according to claim 1, wherein the hydrocarbon chain has a chain length of 14 to 18 carbon atoms. **Claim 3** The method according to claim 1 or 2, wherein the hydrocarbon chain is an alkyl chain. **Claim 4** The method according to any one of claims 1 to 3, wherein the amphoteric surfactant containing a hydrocarbon chain having 12 to 18 carbon atoms is an amphoteric surfactant containing an ammonium group having the hydrocarbon chain. **Claim 5** The method according to any one of claims 1 to 4, wherein the amphoteric surfactant containing an ammonium group having the hydrocarbon chain is 3-(N,N-dimethyldodecylammonio)propanesulfonate (C12APS), 3-(N,N-dimethylmyristylammonio)propanesulfonate (C14APS), 3-(N,N-dimethylpalmitylammonio)propanesulfonate (C16APS), or 3-(N,N-dimethylstearylammonio)propanesulfonate (C18APS). **Claim 6** The method according to any one of claims 1 to 5, wherein the amphoteric surfactant containing a hydrocarbon chain having 12 to 18 carbon atoms is used at a final concentration of 0.01 to 2% by weight in the mixed solution after mixing the sample and the buffer solution. **Claim 7** The method according to any one of claims 1 to 6, wherein the buffer solution further contains an anionic surfactant or an amphoteric surfactant having a steroid skeleton. **Claim 8** The method according to claim 7, wherein the anionic surfactant or amphoteric surfactant having a steroid skeleton is a bile acid or a derivative thereof retaining a steroid skeleton, or a salt thereof. **Claim 9** The method according to claim 8, wherein the bile acid or its derivative retaining the steroid skeleton is one or more compounds selected from the group consisting of deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, hyodeoxycholic acid, cholic acid, glycocolic acid, taurocholic acid, hyocholic acid, 5α-cyprinol, lithocholic acid, taurodeoxycholic acid, CHAPS, and CHAPSO.

10. The method according to claim 8, wherein the bile acid or its derivative retaining the steroid skeleton is one or more compounds selected from the group consisting of deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, hyodeoxycholic acid, cholic acid, glycocolic acid, taurocholic acid, hyocholic acid, 5α-cyprinol, lithocholic acid, and taurodeoxycholic acid.

11. The method according to claim 8, wherein the bile acid or its derivative retaining the steroid skeleton is one or more compounds selected from the group consisting of CHAPS and CHAPSO.

12. The method according to any one of claims 7 to 11, wherein the anionic surfactant or zwitterionic surfactant having the steroid skeleton is used at a final concentration of 0.01 to 5% by weight in the mixed solution after mixing the sample and the buffer solution.

13. The method according to any one of claims 1 to 12, wherein the buffer solution further contains a nonionic surfactant.

14. The method according to claim 13, wherein the nonionic surfactant is used at a final concentration of 0.01 to 5% by weight in the mixed solution after mixing the sample and the buffer solution.

15. The method according to any one of claims 1 to 14, wherein in the immunological detection, one or more antibodies against the SARS-CoV-2 nucleocapsid protein (N protein) are used.

16. The method according to any one of claims 1 to 15, wherein the immunological detection is performed by the sandwich method.

17. The method according to any one of claims 1 to 16, wherein the sample is saliva, sputum, nasal discharge, nasal swab, or throat swab.

18. An immunological detection reagent for SARS-CoV-2 for a sample collected from a subject, comprising the following components (1) and (2): (1) A buffer solution having a pH of 5.0 to 9.0 containing a zwitterionic surfactant containing a hydrocarbon chain having 12 to 18 carbon atoms; and (2) One or more antibodies against the target molecule constituting SARS-CoV-2.

19. The reagent according to claim 18, wherein the zwitterionic surfactant containing a hydrocarbon chain having a chain length of 12 to 18 carbon atoms is used at a final concentration of 0.01 to 2% by weight in the mixture after mixing the sample and the buffer solution.

20. The reagent according to claim 18 or 19, wherein the reagent further contains the following component (3) and / or (4): (3) An anionic surfactant or a zwitterionic surfactant having a steroid skeleton; and / or (4) A nonionic surfactant.

21. The reagent according to any one of claims 18 to 20, wherein the anionic surfactant or zwitterionic surfactant having a steroid skeleton is used at a final concentration of 0.01 to 5% by weight in the mixture after mixing the sample and the buffer solution.

22. The reagent according to claim 20 or 21, wherein the nonionic surfactant is used at a final concentration of 0.01 to 5% by weight in the mixture after mixing the sample and the buffer solution.

23. The reagent according to any one of claims 18 to 22, wherein the reagent is a reagent used in the sandwich method.

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