Method, antibody set and kit for measuring viral antigen in sample

Abstract

The present invention aims to provide a novel antibody group for measuring viral antigens in a sample and a measuring method and a kit using the antibody group. The antibody group includes a capture antibody containing a CDR containing an amino acid sequence of SEQ ID NO: 1 to 6 and a detection antibody containing a CDR containing an amino acid sequence of SEQ ID NO: 7 to 12, and the viral antigens in a sample are measured by using the antibodies thereof, thereby solving the above-mentioned problems.

Description

[Technical field]

[0001] The present invention relates to a method for measuring viral antigens in a sample using a capture antibody and a detection antibody. The present invention relates to an antibody set for measuring viral antigens in a sample by an immunological assay. The present invention relates to a kit containing the capture antibody and the detection antibody. [Background technology]

[0002] In order to study whether a specified virus exists in a sample, it is known to measure the viral antigen in the sample. For example, non-patent documents 1 and 2 describe a kit for measuring the nucleoprotein of SARS-CoV-2 in a sample by an enzyme-linked immunosorbent assay (ELISA) using a capture antibody and a detection antibody.

[0003] [Prior art literature]

[0004]

Non-patent literature

[0005] [Non-patent document 1] SARS-CoV-2 (2019-nCoV) Nucleoprotein / NP ELISA Kit (Cat. No. KIT40588) Processing Instructions, Sino Biological

[0006] [Non-patent document 2] SARS-CoV-2 NP Antigen ELISA Kit (DEIA2020) Processing Instructions, Creative Diagnostics

[0007] [Summary of the Invention]

[0008] [Problems to be solved by the invention]

[0009] The present invention aims to provide a new antibody group capable of measuring the nucleoprotein of SARS-CoV-2 in a sample, and a measurement method and a kit using the antibody group.

[0010]

Methods for solving the problem

[0011] The present invention provides a method for measuring viral antigens, which is a method for measuring viral antigens in a sample using a capture antibody and a detection antibody, and comprises a step of forming a sandwich immune complex containing a capture antibody, a viral antigen, and a detection antibody, wherein the capture antibody comprises a heavy chain variable region comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6, and the detection antibody comprises a heavy chain variable region comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 12. The light chain variable region of CDR3 of the amino acid sequence of NO:12.

[0012] The present invention provides an antibody set for measuring viral antigens, which is an antibody set for measuring viral antigens in a sample by an immunological assay, the antibody set comprising a capture antibody and a detection antibody that form a sandwich immune complex with a viral antigen in the immunological assay, the capture antibody comprising a heavy chain variable region comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6, the detection antibody comprising a heavy chain variable region comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 7, a CDR2 comprising an amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 9, the detection antibody comprising a heavy chain variable region comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 12. The amino acid sequence of NO:12 is of the light chain variable region of CDR3.

[0013] The present invention provides a kit containing the capture antibody and the detection antibody.

[0014] [Effects of the invention]

[0015] According to the present invention, a novel antibody group capable of measuring the nucleoprotein of SARS-CoV-2 in a sample, and a measurement method and a kit using the antibody group are provided.

[0016] [Brief description of the attached drawings]

[0017]

Figure 1A

[0018]

Figure 1B

[0019]

Figure 1C

[0020]

Figure 2A

[0021]

Figure 2B

[0022]

Figure 3

[0023] In the method for measuring the viral antigen of the present embodiment (hereinafter also referred to as "measurement method"), the viral antigen in the sample is measured in vitro by an immunological assay using a capture antibody and a detection antibody containing a specified CDR. In the present embodiment, the capture antibody refers to an antibody that specifically binds to the test substance, and is used to capture the test substance on the solid phase by fixing itself to the solid phase. The detection antibody refers to an antibody that specifically binds to the test substance, and is used to provide a detectable signal by being labeled with a labeling substance itself. The detection antibody is usually not fixed to a solid phase, but there is a case where the labeling substance itself is a solid phase such as a particle.

[0024] As long as the immunological assay includes the process of forming a sandwich immune complex, it is not particularly limited. The sandwich immune complex refers to a complex containing a capture antibody, a test substance, and a detection antibody, and the capture antibody and the detection antibody are in a state of being bound to different parts of the test substance. As such an immunological assay, for example, a sandwich ELISA method, an immunochromatography method, etc. can be cited. Among them, the sandwich ELISA method is also particularly preferred. As a sandwich ELISA method, the immune complex transfer method described in the Special Publication No. 1-254868 can also be used.

[0025] In this specification, the term "determining viral antigens" includes obtaining the measured value of viral antigens, and determining the value of the amount or concentration of viral antigens in the sample. The measured value of viral antigens may be a value reflecting the amount or concentration of viral antigens in the sample. Among them, "the value reflecting the amount or concentration" refers to a value based on the type of labeling substance described later, for example, the measured value of luminescence intensity, the measured value of fluorescence intensity, the measured value of radiation intensity, the measured value of optical density, etc. The value of the amount or concentration of viral antigens in the sample can be determined, for example, based on the measured value of viral antigens and the measured value of a calibrator. A calibrator refers to a type of control sample, a quantitative sample of a test substance containing a test substance or a standard substance corresponding thereto at a known concentration.

[0026] In this specification, the term "antibody" includes full-length antibodies and fragments thereof. The class of the antibody may be any one of IgG, IgA, IgM, IgD and IgE, preferably IgG. The subclass of IgG is not particularly limited, and may be any one of IgG1, IgG2, IgG3 and IgG4. As fragments of the antibody, for example, reduced IgG (rIgG), Fab, Fab', F(ab')2, Fv, single-chain antibody (scFv), double antibody, triabody, etc. may be cited. The method of modulating these antibody fragments is well known per se. The antibody may be any one of a monoclonal antibody and a polyclonal antibody, preferably a monoclonal antibody. The antibody may also be an antibody derived from any one of mice, rats, hamsters, rabbits, goats, horses, camels, alpacas, chickens, etc. Preferably, the antibody is a monoclonal antibody of a mouse.

[0027] In this embodiment, the capture antibody and the detection antibody are identical or different from each other, and can be full-length antibodies or antibody fragments. When both the capture antibody and the detection antibody are antibody fragments, the types of the fragments can be identical or different between the capture antibody and the detection antibody. Both the capture antibody and the detection antibody are preferably monoclonal antibodies.

[0028] In this embodiment, the capture antibody includes a heavy chain variable region comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 1, a CDR2 comprising an amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 3. In addition, the capture antibody includes a light chain variable region comprising a CDR1 comprising an amino acid sequence of SEQ ID NO: 4, a CDR2 comprising an amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 6. Next, the amino acid sequence of each CDR of the capture antibody is shown. In the following, CDRH1, CDRH2, and CDRH3 each represent CDR1, CDR2, and CDR3 of the heavy chain. CDRL1, CDRL2, and CDRL3 each represent CDR1, CDR2, and CDR3 of the light chain. The amino acid sequence of each CDR is a sequence based on the Kabat method. In the present specification, "based on the Kabat method" means that the numbering of amino acid residues in CDRs and variable regions is based on the numbering scheme of Kabat et al. (see Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication No. 91-3242).

[0029] [Amino acid sequence of CDR of capture antibody]

[0030] ·CDRH1:TSGTGVS(SEQ ID NO:1)

[0031] ·CDRH2:HIYWDDDKRY NPSLKS(SEQ ID NO:2)

[0032] ·CDRH3:SNYGYDLDY(SEQ ID NO:3)

[0033] ·CDRL1:KASQNVGTNV V(SEQ ID NO:4)

[0034] ·CDRL2:SASYRYS(SEQ ID NO:5)

[0035] ·CDRL3:QQYNNYPLT(SEQ ID NO:6)

[0036] In this embodiment, the detection antibody includes a heavy chain variable region containing CDR1 containing the amino acid sequence of SEQ ID NO: 7, CDR2 containing the amino acid sequence of SEQ ID NO: 8, and CDR3 containing the amino acid sequence of SEQ ID NO: 9. In addition, the detection antibody includes a light chain variable region containing CDR1 containing the amino acid sequence of SEQ ID NO: 10, CDR2 containing the amino acid sequence of SEQ ID NO: 11, and CDR3 containing the amino acid sequence of SEQ ID NO: 12. Next, the amino acid sequence of each CDR of the detection antibody is shown. The amino acid sequence of each CDR is a sequence based on the Kabat method.

[0037] [Amino acid sequence of CDR of detection antibody]

[0038] CDRH1: DYYMY (SEQ ID NO: 7)

[0039] ·CDRH2:TISDGGSYTY YPDSVKG(SEQ ID NO:8)

[0040] ·CDRH3:AADYGGYFDY(SEQ ID NO:9)

[0041] ·CDRL1:SASQGISN(SEQ ID NO:10)

[0042] ·CDRL2:YTSSLHS(SEQ ID NO:11)

[0043] ·CDRL3:QQYSKLPYT(SEQ ID NO:12)

[0044] The capture antibody containing 6 CDRs each containing the amino acid sequence of SEQ ID NO: 1 to 6 and the detection antibody containing 6 CDRs each containing the amino acid sequence of SEQ ID NO: 7 to 12 specifically bind to the same viral antigen, and recognize different epitopes. Therefore, when the capture antibody and the detection antibody bind to the viral antigen, a sandwich immune complex is formed.

[0045] In one embodiment, the capture antibody may be a humanized antibody comprising six CDRs each comprising the amino acid sequence of SEQ ID NOs: 1 to 6. In addition, the detection antibody may be a humanized antibody comprising six CDRs each comprising the amino acid sequence of SEQ ID NOs: 7 to 12. A humanized antibody is an antibody obtained by transplanting a gene sequence of a CDR of a non-human antibody into a human antibody gene by a known CDR transplantation method.

[0046] The capture antibody may also be a heavy chain variable region containing CDRH1, CDRH2 and CDRH3 each containing an amino acid sequence of SEQ ID NO: 1 to 3, and a heavy chain variable region containing an amino acid sequence of SEQ ID NO: 13. In addition, the capture antibody may also be a light chain variable region containing CDRL1, CDRL2 and CDRL3 each containing an amino acid sequence of SEQ ID NO: 4 to 6, and a light chain variable region containing an amino acid sequence of SEQ ID NO: 14. Next, the amino acid sequences of the variable regions represented by SEQ ID NO: 13 and 14 are shown. The underlined portion represents CDR. The amino acid sequences of these variable regions are sequences based on the Kabat method.

[0047] [Amino acid sequence of the variable region of the capture antibody]

[0048] Heavy chain variable region

[0049] QVTLKESGPG ILQPSQTLSL TCSFSGFSLS TSGTGVS WIR QPSGKGLEWL A HIYWDDDKR YNPSLKS RLT VSKDTSGNQV FLKITSVDTA DTATYYCAR S NYGYDLDY WG QGTTLTVSS (SEQ ID NO: 13)

[0050] Light chain variable region

[0051] DIVMTQSQKF MSTSVGDRVS VTC KASQNVG TNVV WYQQKP GQSPKALIY S ASYRYS GVPDRFTGSGSGTD FTLTISNVQS EDLAEYFC QQ YNNYPLT FGS GTKLEIKRA (SEQ ID NO: 14)

[0052] The detection antibody may also be a heavy chain variable region containing CDRH1, CDRH2 and CDRH3 each containing the amino acid sequence of SEQ ID NO: 7 to 9, or a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15. In addition, the detection antibody may also be a light chain variable region containing CDRL1, CDRL2 and CDRL3 each containing the amino acid sequence of SEQ ID NO: 10 to 12, or a light chain variable region containing the amino acid sequence of SEQ ID NO: 16. Next, the amino acid sequences of the variable regions represented by SEQ ID NO: 15 and 16 are shown. The underlined portion represents CDR. The amino acid sequences of these variable regions are sequences based on the Kabat method.

[0053] [Amino acid sequence of the variable region of the detection antibody]

[0054] Heavy chain variable region

[0055] EVQLVESGGGLVKPGGSLKL SCAASGFTFS DYYMY WVRQT PEKRLEWVA T ISDGGSYTYY PDSVKG RFTI SRDNAKNNLY LQMSSLKSDD TAKYYCAR AA DYGGYFDY WG QGTTLTVSS (SEQ ID NO: 15)

[0056] Light chain variable region

[0057] DIQLTQTTSS LSASLGDRVT ISC SASQGIS N YLNWYQQKP DGTVKLLIY Y TSSLHS GVPSRFSGSGSGTD YSLTISNLEP EDIATYYC QQ YSKLPYT FGG GTKLEIKRA (SEQ ID NO: 16)

[0058] In one embodiment, the capture antibody may also be a chimeric antibody comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 14. In addition, the detection antibody may also be a chimeric antibody comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 16. A chimeric antibody refers to an antibody in which a variable region of an antibody derived from a certain species is linked to a constant region of an antibody of a different species.

[0059] In the present embodiment, the capture antibody and the detection antibody can also change the amino acid sequence in a manner that does not reduce the activity of binding to the viral antigen. As such a change in the amino acid sequence, the substitution, deletion, addition and / or insertion of amino acid residues can be cited. The position of the amino acid sequence of each antibody can be the constant region of the heavy chain or light chain, or it can be the variable region. When changing the variable region, it is preferred to change the framework region (FR). FR refers to the region outside the CDR that is present in the light chain variable region and the heavy chain variable region of the antibody. FR plays the role of the support connecting the three CDRs, and the structural stability of the CDR contributes. The change of the amino acid sequence of the antibody can be carried out by introducing a variation into the antibody gene by the known methods such as DNA recombination technology and other molecular biological techniques.

[0060] The number of amino acid residues changed is usually less than 10 residues, preferably less than 5 residues, and more preferably less than 3 residues. As a change in the amino acid sequence of the antibody, a conservative substitution is preferred. Conservative substitution refers to the substitution of an amino acid residue with an amino acid residue having a side chain having the same chemical properties as the side chain of the residue. The substitution of the preservation of the amino acid sequence itself is well known in the prior art. Alternatively, the amino acid sequence of the antibody can also be changed by a method for controlling the affinity of the antigen of the antibody containing the amino acid residue of the FR3 of the changed antibody as described in the specification of U.S. Patent Application Publication No. 2018 / 0179298. In addition, the amino acid sequence of the antibody can also be changed by a method for improving the thermal stability of the antibody by making the 80th amino acid residue of the variable region of the antibody and the 171st amino acid residue of the constant region cysteine ​​as described in the specification of U.S. Patent Application Publication No. 2019 / 0040119.

[0061] In this embodiment, the capture antibody may also be pre-fixed on a solid phase. The embodiment in which the capture antibody is fixed on a solid phase is not particularly limited. For example, the capture antibody and the solid phase may be directly bonded, or the capture antibody and the solid phase may be indirectly bound via another substance. As a direct binding, for example, physical adsorption may be cited. As an indirect binding, for example, a molecule that specifically binds to the antibody is immobilized on a solid phase, and the antibody is fixed on a solid phase via the binding of the molecule and the antibody. As molecules that specifically bind to the antibody, protein A or G, antibodies that specifically recognize antibodies (second antibodies), etc. may be cited. In addition, the capture antibody may also be fixed on a solid phase using a combination of substances between the antibody and the solid phase. As a combination of such substances, a combination of biotin and avidin, a hapten and an anti-hapten antibody, etc. may be cited. Biotin contains biotin, and biotin analogs such as desthiobiotin and oxybiotin. Avidin contains avidin, and avidin analogs such as streptavidin and Tamavidin (registered trademark). As a combination of a hapten and an anti-hapten antibody, a combination of a compound having a 2,4-dinitrophenyl (DNP) group and an anti-DNP antibody can be cited. For example, by using a capture antibody pre-modified with biotin (or a compound having a DNP group) and a solid phase pre-bound with avidins (or an anti-DNP antibody), the capture antibody can be fixed on the solid phase through the binding of biotin and avidins (or the binding of the DNP group and the anti-DNP antibody).

[0062] The raw material of the solid phase is not particularly limited, and can be selected from, for example, organic polymer compounds, inorganic compounds, biopolymers, etc. As organic polymer compounds, latex, polystyrene, polypropylene, etc. can be mentioned. As inorganic compounds, magnetic bodies (iron oxide, chromium oxide and ferrite, etc.), silicon oxide, aluminum oxide, glass, etc. can be mentioned. As biopolymers, insoluble agarose, insoluble dextran, gelatin, cellulose, etc. can be mentioned. It is also possible to use two or more of these in combination. The shape of the solid phase is not particularly limited, and can include, for example, particles, membranes, microplates, microtubes, test tubes, etc. Among them, particles and microplates are also preferred. As particles, magnetic particles are particularly preferred.

[0063] In the present embodiment, the detection antibody may also be pre-labeled with a labeling substance. The labeling substance is not particularly limited, and examples thereof include substances that generate signals themselves (hereinafter, also referred to as "signal generating substances"), and substances that generate signals by catalyzing reactions of other substances. Examples of signal generating substances include fluorescent substances, radioactive isotopes, and color-developing substances. Examples of substances that generate detectable signals by catalyzing reactions of other substances include enzymes. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor (registered trademark), and fluorescent proteins such as GFP. Examples of radioactive isotopes include125 I. 14 C. 32 As color developing material, metal colloids such as gold nano colloids can be enumerated. As enzyme, alkaline phosphatase, peroxidase, beta-galactosidase, glucosidase, polyphenol oxidase, tyrosinase, acid phosphatase, luciferase etc. can be enumerated. Preferred labeling material is enzyme, particularly preferably alkaline phosphatase.

[0064] In this embodiment, a labeled second antibody may also be used as a labeling substance. The labeled second antibody refers to an antibody labeled with the above-mentioned labeling substance, and is an antibody that specifically recognizes the detection antibody. By binding the labeled second antibody to the detection antibody, the detection antibody is indirectly labeled with the labeling substance.

[0065] Capture antibodies and detection antibodies can be prepared by known methods such as DNA recombination technology and other molecular biological techniques, for example, as follows. First, RNA is extracted from a hybridoma that produces any mouse antibody. Hybridomas can be obtained by known methods described in Kohler G. and Milstein C., Nature, vol.256, pp.495-497, 1975, etc. Using the extracted RNA, the respective polynucleotides encoding the heavy chain and light chain of the antibody are synthesized by reverse transcription reaction and RACE (Rapid Amplification of cDNA ends). Among these polynucleotides, by replacing the base sequences encoding CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 by the PCR method with the base sequences encoding the amino acid sequences of SEQ ID NO: 1 to 6 or SEQ ID NO: 7 to 12, the respective polynucleotides encoding the heavy chain and light chain of the capture antibody or the detection antibody can be obtained. Alternatively, in the polynucleotide encoding the heavy chain of any mouse antibody, the base sequence encoding the variable region can also be replaced with a base sequence encoding the amino acid sequence of SEQ ID NO: 13 or 15, and the base sequence of the region encoding the variable region in the polynucleotide encoding the light chain of the antibody can be replaced with a base sequence encoding the amino acid sequence of SEQ ID NO: 14 or 16. The obtained polynucleotide is integrated into a known expression vector. The polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain can be integrated into one expression vector in a manner that can be expressed independently, or they can be integrated into two expression vectors individually. Thus, an expression vector containing a polynucleotide encoding the heavy chain and light chain of a capture antibody or a detection antibody is obtained. The type of expression vector is not particularly limited, and it can be an expression vector for mammalian cells or an expression vector for Escherichia coli. The expression vector obtained by transforming or transfecting an appropriate host cell (e.g., a mammalian cell or Escherichia coli) can obtain a capture antibody or a detection antibody. Alternatively, the base sequences encoding the amino acid sequences of SEQ ID NOs: 1 to 6 and SEQ ID NOs: 7 to 12 can be used to prepare capture antibodies and detection antibodies as humanized antibodies by a known CDR transplantation method (see, for example, Jones PT et al., Nature, vol. 321, pp. 522-525, 1986, CoM.S. et al., Proc. Natl. Acad. Sci. USA, vol. 93, pp. 7843-7848, 1996, etc.).

[0066] In this embodiment, the viral antigen is not particularly limited as long as it is combined with the above-mentioned capture antibody and detection antibody to form a sandwich immune complex. As such viral antigens, for example, the nucleoprotein of SARS-CoV, the nucleoprotein of SARS-CoV-2, etc. can be cited. Nucleoprotein (hereinafter also referred to as "N protein") is a protein that exists together with the nucleic acid of the virus, also known as nucleocapsid protein.

[0067] When the capture antibody and / or the detection antibody do not specifically bind to the viral antigen and do not substantially form a sandwich immune complex, the viral antigen is not the test substance in the determination method of this embodiment. In one embodiment, the capture antibody and the detection antibody form a sandwich immune complex with the N protein of SARS-CoV or SARS-CoV-2, and do not substantially form a sandwich immune complex with the N protein of MERS-CoV, and the viral antigens of HCoV-NL63, HCoV-229E and HCoV-OC43 (e.g., viral lysates containing N protein).

[0068] In the present embodiment, the sample includes not only samples containing viral antigens or viruses containing the viral antigens, but also samples suspected of containing viral antigens or viruses containing the viral antigens. As such a sample, a biological sample is suitably used. A biological sample refers to a sample collected from a living body. As biological samples, nasopharyngeal swabs, saliva, nasal juice, sputum, whole blood, plasma, serum, bronchoalveolar washings, cerebrospinal fluid, lymph, etc. can be cited. In addition to biological samples, excrement, drainage, river water, seawater, soil, etc. can be used. As excrement, for example, urine, feces, etc. can be cited.

[0069] In the present embodiment, the sample is preferably in liquid form. Liquid samples are not limited to solutions, but include suspensions, sols, and the like. When the sample is not in liquid form, for example, a suitable aqueous medium may be added to the sample to make it liquid. Such an aqueous medium is not particularly limited as long as it does not interfere with the determination described below, and examples thereof include water, saline, and buffer solutions. The buffer solution is not particularly limited as long as it has a buffering effect at a pH near neutral (e.g., a pH of 6 or more and 8 or less). Such buffer solutions include Good's buffers such as ACES, HEPES, MES, and PIPES, triethanolamine hydrochloride buffer, Tris hydrochloride buffer, phosphate buffered saline (PBS), and Tris buffered saline (TBS). When the liquid sample contains insoluble impurities, the impurities may be removed from the sample by known means such as centrifugation and filtration. The liquid sample may also be diluted with the above-mentioned aqueous medium as needed.

[0070] When the sample includes a virus containing the above-mentioned viral antigen, an extraction reagent may be added to the sample in order to release the viral antigen from the virus. The extraction reagent refers to a reagent used to dissolve the virus in the sample and extract viral antigens such as N protein. As such a reagent, for example, a buffer solution containing a surfactant may be mentioned. The surfactant is not particularly limited as long as it does not interfere with the determination described below, and examples thereof include NP-40, Tween (registered trademark) -20, Triton (registered trademark) X-100, etc. Commercially available extraction reagents may also be used. The extraction reagent may also contain a chelating agent. As a chelating agent, for example, a sodium salt or potassium salt of ethylenediaminetetraacetic acid (EDTA) may be mentioned.

[0071] In the assay method of the present embodiment, a sandwich immune complex containing a capture antibody, a viral antigen, and a detection antibody is first formed. The sandwich immune complex can be formed by mixing a sample that may contain a viral antigen with a capture antibody and a detection antibody. In a preferred embodiment, a sandwich immune complex containing a capture antibody, a viral antigen, and a detection antibody is formed on a solid phase. For example, after forming a sandwich immune complex by mixing a sample that may contain a viral antigen with a capture antibody and a detection antibody, a solution containing the complex is contacted with a solid phase to which the capture antibody can be fixed, so that the complex can be formed on the solid phase. Alternatively, a capture antibody that is pre-fixed on a solid phase can also be used. That is, by mixing the capture antibody fixed on a solid phase with a sample that may contain a test substance and a detection antibody, the above-mentioned complex can be formed on the solid phase.

[0072] The determination method of the present embodiment includes a step of detecting a sandwich immune complex formed on a solid phase. By detecting the sandwich immune complex on the solid phase using a method known in the prior art, the viral antigen can be determined. For example, when the detection antibody is labeled with a labeling substance, the viral antigen can be determined by detecting a signal generated by the labeling substance. When a labeled second antibody for the detection antibody is used, the viral antigen can be determined by detecting a signal generated by the labeling substance of the labeled second antibody bound to the detection antibody. In the present embodiment, it is preferred to use a detection antibody that is pre-labeled with a labeling substance.

[0073] In this specification, "detection signal" includes the presence or absence of qualitative detection signal, quantification of signal intensity, and semi-quantitative detection signal intensity. Semi-quantitative detection refers to the intensity of the signal being expressed in stages such as "signal does not occur", "weak", "medium", "strong", etc. In this embodiment, the intensity of the signal is preferably quantitatively or semi-quantitatively detected.

[0074] The method of detecting the signal itself is well known in the prior art. In the present embodiment, it is appropriate to select a determination method corresponding to the type of the signal derived from the above-mentioned labeling substance. For example, when the labeling substance is an enzyme, the signal of light, color, etc. generated by reacting the substrate for the enzyme can be measured by using a known device such as a spectrophotometer.

[0075] The substrate of the enzyme can be appropriately selected from known substrates corresponding to the type of the enzyme. For example, when alkaline phosphatase is used as an enzyme, as a substrate, chemiluminescent substrates such as CDP-Star (registered trademark) (4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate, CSPD (registered trademark) (3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate, p-nitrophenyl phosphate, etc. can be cited. When peroxidase is used as the enzyme, examples of the substrate include chemiluminescent substrates such as LUMINOR and its derivatives, and colorimetric substrates such as 2,2'-azinobis(3-ethylbenzothiazoline-6-ammonium sulfonate) (ABTS), 1,2-phenylenediamine (OPD), and 3,3',5,5'-tetramethylbenzidine (TMB).

[0076] When the labeling substance is a radioisotope, the radiation as a signal can be measured using a known device such as a scintillation counter. When the labeling substance is a fluorescent substance, the fluorescence as a signal can be measured using a known device such as a fluorescent microplate reader. Furthermore, the excitation wavelength and the fluorescence wavelength can be appropriately determined according to the type of fluorescent substance used.

[0077] The detection result of the signal can be used as the measurement result of the viral antigen in the sample. For example, when the intensity of the signal is quantified, the measured value of the signal intensity itself or the value obtained from the measured value can be used as the measured value of the viral antigen. As the value obtained from the measured value of the signal intensity, for example, a value obtained by subtracting the measured value of the negative control or the value of the background from the measured value can be cited. The negative control can be appropriately selected, and for example, a buffer solution that does not contain viral antigen can be cited.

[0078] In the present embodiment, the measured value of the signal intensity can also be applied to a calibration curve to determine the amount or concentration of the viral antigen in the sample. The calibration curve can be made from the measured values ​​of multiple calibrators. The measured value of the calibrator is obtained by measuring the calibrator by the measurement method of the present embodiment in the same way as the sample. The calibration curve can be made by taking the concentration of the viral antigen in the calibrator on the X-axis and the XY plane of the measured value (such as signal intensity) on the Y-axis, plotting the measured values ​​of multiple calibrators, and obtaining a straight line or curve by a known method such as the least squares method. In the present embodiment, the calibrator can be prepared by, for example, adding a recombinant viral antigen to a buffer solution that does not contain viral antigens at an arbitrary concentration. As a calibrator that does not contain viral antigens, a buffer solution that does not contain viral antigens itself can also be used.

[0079] The determination method of the present embodiment can also be performed by a commercially available fully automatic immunoassay device. A fully automatic immunoassay device refers to a device that automatically performs the modulation of the determination sample and its immunoassay and outputs the determination result of the test substance when the user sets the sample and inputs the indication of the start of the determination. As such a fully automatic immunoassay device, HISCL series (Sysmex Co., Ltd.) or HI-1000 (Sysmex Co., Ltd.) such as HISCL (registered trademark)-5000 or HISCL-800 can be cited. The device of the HISCL series is measured by a sandwich ELISA method using magnetic particles as a solid phase.

[0080] In this embodiment, B / F (Bound / Free) separation can also be performed between the formation of the sandwich immune complex and the detection of the complex to remove unreacted free components that have not formed a complex. Unreacted free components refer to components that do not constitute the sandwich immune complex. For example, the remaining capture antibodies and detection antibodies that are not bound to the viral antigen can be cited. The means of B / F separation is not particularly limited. When the solid phase is a particle, B / F separation can be performed by recovering only the solid phase that captures the complex by centrifugal separation. When the solid phase is a container such as a microplate or a microtube, B / F separation can be performed by removing the liquid containing unreacted free components. In addition, when the solid phase is a magnetic particle, B / F separation can be performed by sucking out the liquid containing unreacted free components by a nozzle while the magnetic particles are magnetically constrained by a magnet, which is preferred from the perspective of automation. After removing the unreacted free components, the solid phase of the captured complex can also be washed with a suitable aqueous medium such as PBS.

[0081] A further embodiment is an antibody group (hereinafter also referred to as "antibody group") for measuring viral antigens in a sample by an immunological assay. The term "antibody group" in this specification refers to a combination of multiple antibodies containing at least a capture antibody and a detection antibody used in an immunological assay. The details of the immunological assay are the same as those described for the assay method of this embodiment. The antibody group of this embodiment contains a capture antibody and a detection antibody that form a sandwich immune complex with a viral antigen in the immunological assay. The details of the capture antibody and the detection antibody contained in the antibody group of this embodiment are the same as those described for the assay method of this embodiment. The antibody group of this embodiment is suitably used in the assay method of this embodiment described above.

[0082] A further embodiment is a kit (hereinafter also referred to as a "kit") containing a capture antibody and a detection antibody. The kit of this embodiment is used to measure viral antigens in a sample by an immunological assay. The kit of this embodiment contains one or more reagents. The details of the immunological assay, capture antibody, and detection antibody are the same as those described for the assay method of this embodiment.

[0083] In one embodiment, the kit includes a reagent containing a capture antibody and a reagent containing a detection antibody. The form of the antibody-containing reagent is not particularly limited, and may be a solid (e.g., powder, crystal, freeze-dried product, etc.), or a liquid (e.g., solution, suspension, emulsion, etc.). When the antibody-containing reagent is a liquid, the solvent is not particularly limited as long as it can dissolve the antibody and preserve it. As solvents, for example, water, saline, PBS, TBS, Good's buffer, etc. can be cited. As Good's buffer, for example, MES, Bis-Tris, ADA, PIPES, Bis-Tris-propane, ACES, MOPS, MOPSO, BES, TES, HEPES, HEPPS, Tricine, Tris, Bicine, TAPS, etc. can be cited.

[0084] The antibody-containing reagent may contain known additives, such as protein stabilizers such as bovine serum albumin (BSA), preservatives such as sodium azide, and inorganic salts such as sodium chloride.

[0085] In one embodiment, the kit may also include a reagent containing a capture antibody, a reagent containing a detection antibody, and a solid phase. Alternatively, the capture antibody may also be pre-immobilized on a solid phase. The details of the solid phase are the same as those described for the assay method of this embodiment. Preferably, the solid phase is a magnetic particle or a microplate.

[0086] The detection antibody may also be pre-labeled with a labeling substance. Alternatively, the kit may also include a reagent containing a capture antibody, a reagent containing a detection antibody, and a reagent containing a labeled second antibody. The details of the labeling substance and the labeled second antibody are the same as those described for the assay method of the present embodiment. A preferred labeling substance is an enzyme. As enzymes, alkaline phosphatase, peroxidase, β-galactosidase, glucosidase, polyphenol oxidase, tyrosinase, acid phosphatase, luciferase, etc. may be cited. A preferred enzyme is alkaline phosphatase. When the detection antibody is enzyme-labeled, or when the kit contains an enzyme-labeled second antibody, the kit may also contain a substrate for the enzyme. The details of the substrate are the same as those described for the assay method of the present embodiment.

[0087] The kit of this embodiment can also be provided to the user by packaging the containers containing each reagent in a box. The box can also contain accompanying documents. The accompanying documents can also record the composition of the kit, the composition of each reagent, the method of use, the method of storage, etc.

[0088] An example of the kit of this embodiment is shown in Figure 1A .exist Figure 1A In the figure, 11 represents a reagent kit, 12 represents a first container for containing a reagent containing a capture antibody, 13 represents a second container for containing a reagent containing a detection antibody, 14 represents a packing box, and 15 represents an accompanying document. In this example, the capture antibody may also be fixed to a solid phase (e.g., magnetic particles). In addition, the detection antibody may also be labeled with a labeling substance.

[0089] An example of a kit according to a further embodiment is shown in Figure 1B .exist Figure 1B In the figure, 21 indicates a reagent kit, 22 indicates a first container containing a reagent containing a capture antibody, 23 indicates a second container containing a reagent containing a detection antibody, 24 indicates a packing box, 25 indicates an accompanying document, and 26 indicates a microplate as a solid phase. In this example, the detection antibody may also be labeled with a labeling substance.

[0090] An example of a kit according to a further embodiment is shown in Figure 1C .exist Figure 1C In the figure, 31 indicates a reagent kit, 32 indicates a first container containing a reagent containing a capture antibody, 33 indicates a second container containing a reagent containing an enzyme-labeled detection antibody, 34 indicates a third container containing a reagent containing a substrate for the enzyme, 35 indicates a packaging box, and 36 indicates an accompanying document. In this example, the capture antibody may also be fixed to a solid phase (e.g., magnetic particles).

[0091] The kit of this embodiment may further include a calibrator. The calibrator may include, for example, a buffer solution containing no viral antigen (negative control) and a buffer solution containing a recombinant viral antigen at a known concentration.

[0092] Next, the present invention will be described in detail by way of examples, but the present invention is not limited to these examples.

[0093] [Example]

[0094] [Production Example: Preparation of an Antibody That Binds Specifically to the N Protein of SARS-CoV]

[0095] (1) Preparation of Antigen

[0096] Using the genomic RNA sequence of SARS-CoV (GenBank accession number: AY274119), the full-length cDNA encoding the N protein of SARS-CoV was synthesized by the method described in Fujimoto K. et al., 2008, J. Clin. Microbiol., vol. 46, p. 302-310. The obtained cDNA was integrated into the downstream of the sequence encoding the His tag (6 histidine residues) of the pQE30 vector (QIAGEN) to obtain the SARS-CoVNP expression vector. Using the obtained expression vector, the His tag-attached recombinant N protein was expressed in Escherichia coli by conventional methods. By conventional methods, Escherichia coli was dissolved to obtain a soluble fraction, and the recombinant N protein was purified using a His-Trap HP column (QIAGEN).

[0097] (2) Preparation and screening of hybridomas

[0098] Balb / c mice (7 weeks old, female) were immunized with a mixture of the prepared recombinant N protein and complete Freund's adjuvant, and hybridomas producing antibodies against the N protein of SARS-CoV were prepared by the method described in Kohler G. and Milstein C., Nature, vol. 256, pp. 495-497, 1975. For the obtained hybridomas, strains that produce antibodies that show reactivity to the antigen are selected by the ELISA method. The selected hybridomas are cloned by the limiting dilution method, and strains that stably produce antibodies are also selected. Thus, two hybridomas that produce antibodies that specifically bind to the N protein of SARS-CoV are obtained. Hereinafter, these hybridomas are also referred to as "clone 1" and "clone 2".

[0099] (3) Purification of monoclonal antibodies

[0100] The culture supernatant (100 mL) of each hybridoma obtained was filtered with a 0.22 μm filter to remove insoluble matter. The filtered culture supernatant was passed through a column filled with 1 mL of protein G-sepharose 4B (GE Healthcare) to adsorb the antibody to the column. After removing the nonspecific adsorbed fraction from the column, the monoclonal antibody was freed by placing the column under acidic conditions. The recovered monoclonal antibody was dialyzed with 100 times the amount of phosphate buffered saline (PBS) and purified. Hereinafter, the monoclonal antibody purified from the culture of the hybridoma of clone 1 is also referred to as the "first antibody", and the monoclonal antibody purified from the culture of the hybridoma of clone 2 is also referred to as the "second antibody".

[0101] (4) Analysis of the amino acid sequence of antibodies

[0102] Total RNA was extracted from each hybridoma obtained, and cDNA encoding the site containing the variable region of the heavy chain and light chain was synthesized by the 5'-RACE method. The obtained cDNA was integrated into a plasmid vector, and the base sequence encoding the variable region of the heavy chain and light chain of each antibody was analyzed by sequencing. The amino acid sequence was predicted from the obtained base sequence, and the amino acid sequence of the variable region of the heavy chain and light chain of each antibody based on the Kabat method was obtained. In addition, the amino acid sequence of each CDR was obtained.

[0103] The amino acid sequences of the variable regions of the heavy chain and light chain of the first antibody are as follows.

[0104] Heavy chain variable region

[0105] QVTLKESGPG ILQPSQTLSL TCSFSGFSLS TSGTGVSWIR QPSGKGLEWL AHIYWDDDKRYNPSLKSRLT VSKDTSGNQV FLKITSVDTA DTATYYCARS NYGYDLDYWG QGTTLTVSS(SEQ ID NO:13)

[0106] Light chain variable region

[0107] DIVMTQSQKF MSTSVGDRVS VTCKASQNVG TNVVWYQQKP GQSPKALIYS ASYRYSGVPDRFTGSGSGTD FTLTISNVQS EDLAEYFCQQ YNNYPLTFGS GTKLEIKRA(SEQ ID NO:14)

[0108] The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain and heavy chain of the first antibody are as follows. The amino acid sequences of these CDRs are based on the Kabat method.

[0109] ·CDRH1:TSGTGVS(SEQ ID NO:1)

[0110] ·CDRH2:HIYWDDDKRY NPSLKS(SEQ ID NO:2)

[0111] ·CDRH3:SNYGYDLDY(SEQ ID NO:3)

[0112] ·CDRL1:KASQNVGTNV V(SEQ ID NO:4)

[0113] ·CDRL2:SASYRYS(SEQ ID NO:5)

[0114] ·CDRL3:QQYNNYPLT(SEQ ID NO:6)

[0115] The amino acid sequences of the variable regions of the heavy chain and light chain of the second antibody are as follows.

[0116] Heavy chain variable region

[0117] EVQLVESGGG LVKPGGSLKL SCAASGFTFS DYYMYWVRQT PEKRLEWVAT ISDGGSYTYYPDSVKGRFTI SRDNAKNNLY LQMSSLKSDD TAKYYCARAA DYGGYFDYWG QGTTLTVSS(SEQ ID NO:15)

[0118] Light chain variable region

[0119] DIQLTQTTSS LSASLGDRVT ISCSASQGIS NYLNWYQQKP DGTVKLLIYY TSSLHSGVPSRFSGSGSGTD YSLTISNLEP EDIATYYCQQ YSKLPYTFGG GTKLEIKRA(SEQ ID NO:16)

[0120] The amino acid sequences of CDR1, CDR2, and CDR3 of the light chain and heavy chain of the second antibody are as follows. The amino acid sequences of these CDRs are based on the Kabat method.

[0121] CDRH1: DYYMY (SEQ ID NO: 7)

[0122] ·CDRH2:TISDGGSYTY YPDSVKG(SEQ ID NO:8)

[0123] ·CDRH3:AADYGGYFDY(SEQ ID NO:9)

[0124] ·CDRL1:SASQGISN(SEQ ID NO:10)

[0125] ·CDRL2:YTSSLHS(SEQ ID NO:11)

[0126] ·CDRL3:QQYSKLPYT(SEQ ID NO:12)

[0127] [Example 1: Immunological determination of N protein of SARS-CoV-2]

[0128] Reagents for a fully automatic immunoassay device were prepared using the first antibody and the second antibody obtained in the preparation example, and the N protein of SARS-CoV-2 was measured using these reagents.

[0129] (1) Preparation of antigen solution

[0130] As a viral antigen, SARS-CoV-2 nucleocapsid-His recombinant protein (Sino Biological Company), which is a recombinant N protein of SARS-CoV-2, was used. This antigen was diluted with an extraction reagent or a dilution buffer to prepare an antigen solution containing the viral antigen at a concentration of 100 pg / mL, 1,000 pg / mL, or 10,000 pg / mL. The composition of the extraction reagent was 0.3% NP-40 (polyoxyethylene (9) octylphenyl ether), 15 mM EDTA·2Na, 60 mM NaOH, 6 mM ACES (N-(2-acetamide)-2-aminoethanesulfonic acid), 0.22 M NaCl, and 15 mM NaN3, and the pH was 7.0. The composition of the dilution buffer was 80 mM triethanolamine hydrochloride, 1% BSA, and 0.1% NaN3, and the pH was 7.5.

[0131] (2) Preparation of reagents

[0132] The R1 reagent (agent containing the capture antibody) is prepared by dissolving the first antibody or the second antibody with biotin by a conventional method in a buffer for the R1 reagent (100mM HEPES, 150mM NaCl, 25mM EDTA·2Na, 2.5% casein Na, 1% BSA, 0.09% NaN3, 0.015% defoaming agent SI, pH 7.5). The concentration of each antibody in the R1 reagent is 1μg / mL. As the R2 reagent (solid phase), the HISCL R2 reagent (Sysmex Co., Ltd.) containing streptavidin-immobilized magnetic particles was used. R3 reagent (reagent containing detection antibody) is prepared by labeling the first antibody or the second antibody with alkaline phosphatase (ALP) by a conventional method and dissolving it in R3 reagent buffer (100mM MES, 150mM NaCl, 1mM MgCl2, 0.1mM ZnCl2, 1% BSA, 50mg / L scavenger ALP, 0.09% NaN3, 0.015% defoaming agent SI, pH6.5). The concentration of each antibody in R3 reagent is 400ng / mL. As R4 reagent (determination buffer), HISCL R4 reagent (Sysmex Co., Ltd.) is used. As R5 reagent (substrate solution), HISCL R5 reagent (Sysmex Co., Ltd.) containing CDP-Star (registered trademark) is used. As a cleaning solution for magnetic particles, HISCL cleaning solution (Sysmex Co., Ltd.) is used.

[0133] (3) Determination

[0134] The above-mentioned R1 to R5 reagents were used to perform the measurement by the fully automatic immunoassay device HISCL-800 (Sysmex Corporation). First, the first antibody was used as the capture antibody, and the second antibody was used as the detection antibody for measurement. The specific operation is as follows. After adding the antigen solution (10 μL) to the R1 reagent (60 μL) containing the first antibody and mixing, the R2 reagent (30 μL) was added and mixed. The magnetic particles in the obtained mixed solution were magnetized and the supernatant was removed, and the HISCL cleaning solution (300 μL) was added to wash the magnetic particles. After removing the supernatant, the R3 reagent (60 μL) containing the second antibody was added to the magnetic particles and mixed. The magnetic particles in the obtained mixed solution were magnetized and the supernatant was removed, and the HISCL cleaning solution (300 μL) was added to wash the magnetic particles. After removing the supernatant, the R4 reagent (50 μL) and the R5 reagent (100 μL) were added to the magnetic particles, and the mixture was fully mixed to measure the chemiluminescence intensity. Next, the second antibody is used as the capture antibody, and the first antibody is used as the detection antibody to perform the measurement. This measurement is performed in the same manner as above, except that the R1 reagent containing the second antibody is used instead of the R1 reagent containing the first antibody, and the R3 reagent containing the first antibody is used instead of the R3 reagent containing the second antibody. In addition, in order to measure the background, the measurement is performed in the same manner as above, except that an extraction reagent or dilution buffer not containing an antigen is used instead of the antigen solution. The measured SN ratio is calculated from the measured value of each antigen solution and the background.

[0135] (4) Results

[0136] The SN ratio of each measurement is shown in Table 1. In the table, "Measurement 1" refers to a measurement using the first antibody as a capture antibody and the second antibody as a detection antibody, and "Measurement 2" refers to a measurement using the second antibody as a capture antibody and the first antibody as a detection antibody. "Antigen solution A" refers to an antigen solution prepared using a dilution buffer, and "Antigen solution B" refers to an antigen solution prepared using an extraction reagent.

[0137]

Table 1

[0138]

[0139] As can be seen from Table 1, the SN ratio of assay 1 is significantly higher than that of assay 2. Therefore, the first antibody represents a capture antibody suitable for immunological assays, and the second antibody represents a detection antibody suitable for immunological assays. That is, it is shown that the N protein of SARS-CoV-2 in a sample can be well measured by a sandwich ELISA method using the first antibody as a capture antibody and the second antibody as a detection antibody. In addition, in assay 1, the SN ratio of either antigen solution A or B was high, indicating that the surfactant did not particularly affect the assay.

[0140] [Example 2: Reactivity with N proteins of SARS-CoV, SARS-CoV-2 and MERS-CoV]

[0141] The present invention investigates whether the N proteins of SARS-CoV and MEAS-CoV can be detected by immunological assays using the first antibody and the second antibody as capture antibody and detection antibody, respectively. For comparison, the N protein of SARS-CoV-2 was also detected.

[0142] (1) Preparation of antigen solution

[0143] As the viral antigen, the recombinant N protein of SARS-CoV prepared in the preparation example, the commercially available recombinant N protein of SARS-CoV-2 similar to Example 1, and the recombinant N protein of MERS-CoV were used. The recombinant N protein of MERS-CoV was prepared by a conventional method using a known silkworm expression system. These recombinant N proteins were diluted with the dilution buffer of Example 1 to prepare antigen solutions containing each viral antigen at a concentration of 100 pg / mL.

[0144] (2) Determination

[0145] The reagents used were the R1 reagent containing the first antibody and the R3 reagent containing the second antibody prepared in Example 1, as well as the HISCL R2 reagent, the HISCL R4 reagent, and the HISCL R5 reagent (all Sysmex Co., Ltd.). Using these reagents, each antigen solution was measured by HISCL-800 (Sysmex Co., Ltd.) in the same manner as in Example 1. In addition, as a background, a dilution buffer solution containing no antigen was used instead of the antigen solution, and the measurement was performed in the same manner as above. The luminescence intensity (HISCL count value) of each measurement is shown in Table 2.

[0146]

Table 2

[0147] SARS-CoV-2 SARS-CoV MERS-CoV background 40,946 31,709 1,711 1,648

[0148] (3) Results

[0149] As can be seen from Table 2, the luminescence intensity when measuring the N protein of SARS-CoV-2 and SARS-CoV is significantly higher than the background. On the other hand, the luminescence intensity when measuring the N protein of MERS-CoV is about the same as the background. These suggest that the first antibody and the second antibody form a sandwich immune complex with the N protein of SARS-CoV-2 and SARS-CoV, but not with the N protein of MERS-CoV.

[0150] [Example 3: Reactivity with viral antigens of HCoV-OC43, HCoV-NL63 and HCoV-229]

[0151] The presence or absence of viral antigens of HCoV-OC43, HCoV-NL63 and HCoV-229 can be determined by immunological assays using the first antibody and the second antibody as the capture antibody and the detection antibody, respectively.

[0152] (1) Preparation of antigen solution

[0153] As viral antigens, coronavirus strain: OC43 lysate (1 mg), coronavirus strain: NL63 lysate (1 mg) and coronavirus strain: 229E lysate (1 mg) (all from ZeptoMetrix) were used as inactivated solutions of HCoV-OC43, HCoV-NL63 and HCoV-229E. These inactivated solutions were prepared by dissolving the purified virus in a solution containing a surfactant and inactivating it by heating, and contained 1 mg of protein. These inactivated solutions were diluted with the dilution buffer of Example 1 to prepare antigen solutions containing protein at a concentration of 10 ng / mL, 100 ng / mL or 1,000 ng / mL.

[0154] (2) Determination

[0155] Each antigen solution was measured by HISCL-800 (Sysmex Corporation) in the same manner as in Example 1 using the same R1 to R5 reagents as in Example 2. In addition, the measurement was performed in the same manner as above except that a dilution buffer containing no antigen was used instead of the antigen solution as a background. The luminescence intensity (HISCL count value) of each measurement is shown in Table 3.

[0156]

Table 3

[0157]

[0158] (3) Results

[0159] As can be seen from Table 3, the luminescence intensity when measuring the proteins contained in the inactivated solutions of HCoV-OC43, HCoV-NL63 and HCoV-229E is the same as the background. Therefore, it is suggested that the first antibody and the second antibody do not form a sandwich immune complex with the viral antigens of HCoV-OC43, HCoV-NL63 and HCoV-229E.

[0160] [Example 4: Discussion of detection limit]

[0161] The detection limit of an immunological assay using the first antibody and the second antibody as a capture antibody and a detection antibody, respectively, was investigated using an antigen solution containing the SARS-CoV-2 N protein at a low concentration.

[0162] (1) Preparation of antigen solution

[0163] As a viral antigen, SARS-CoV-2 nucleocapsid-His recombinant protein (Sino Biological Company) was used. This antigen was added to the calibrant dilution buffer or the extraction reagent of Example 1 to prepare an antigen solution containing the viral antigen at a concentration of 1pg / mL, 2pg / mL, 3pg / mL, 4pg / mL, 5pg / mL, or 10pg / mL. For each concentration, 10 antigen solutions were prepared (n=10). The calibrant dilution buffer refers to a buffer that does not contain a surfactant and is used to dilute the standard substance solution used to prepare the calibration curve. The composition of the calibrant dilution buffer is 80mM triethanolamine hydrochloride, 1% BSA and 0.1% NaN3, and the pH is 7.5.

[0164] (2) Determination

[0165] Using the same R1 to R5 reagents as in Example 2, each antigen solution was measured by HISCL-800 (Sysmex Co., Ltd.) in the same manner as in Example 1. In addition, the measurement was performed in the same manner as above, except that a calibrant dilution buffer or extraction reagent that does not contain an antigen was used instead of the antigen solution as a background. The average value (Av), standard deviation (SD) and coefficient of variation (CV) of the luminescence intensity (HISCL count value) of each antigen solution are shown in Table 4. In the table, "antigen solution C" refers to an antigen solution prepared using a calibrant dilution buffer, and "antigen solution D" refers to an antigen solution prepared using an extraction reagent. In addition, a coordinate diagram plotting the average value of the luminescence intensity relative to the antigen concentration is shown in Figure 2A and B.

[0166]

Table 4

[0167]

[0168] (3) Results

[0169] As from Figure 2A From Table 4, it can be seen that the detection limit (LoD) is above 1 pg / mL for the antigen solution prepared with the buffer for calibrant dilution, and above 2 pg / mL for the antigen solution prepared with the extraction reagent.

[0170] [Example 5: Correlation between immunological assay and PCR method]

[0171] Commercially available biological samples containing SARS-CoV-2 were measured using the measurement method of the present embodiment and the PCR method, and the measurement values ​​obtained by each method were compared to investigate whether a correlation was confirmed between the measurement method of the present embodiment and the PCR method.

[0172] (1) Pretreatment of biological samples

[0173] Nasopharyngeal swabs (36 subjects) from patients infected with SARS-CoV-2 were purchased from Cantor Bioconnect. Each subject (500 μL) was mixed with the extraction reagent (500 μL) of Example 1 to prepare an antigen solution. The obtained antigen solution was used in immunological assays. In addition, RNA was extracted from each of the above subjects using the QIAamp viral RNA mini kit (QIAGEN). The obtained RNA solution was used in the assay by the PCR method.

[0174] (2) Immunological assay

[0175] The antigen solution was measured by HISCL-800 (Sysmex Corporation) in the same manner as in Example 1 using the same R1 to R5 reagents as in Example 2. The measurement was performed in the same manner as above except that a calibrator dilution buffer containing no antigen was used instead of the antigen solution as a background.

[0176] (3) Determination by PCR

[0177] The determination by the PCR method was carried out according to the procedure of “Detection of 2019-nCoV by real-time one-step RT-PCR using TaqMan probes” described in the “Pathogen Detection Manual 2019-nCoVVer.2.9.1” (March 19, Reiwa 2 (2020)) prepared by the National Institute of Infectious Diseases. As a real-time RT-PCR reagent, QuantiTect (registered trademark) Probe RT-PCR kit (QIAGEN) was used. The amplification reaction was carried out using the conditions described in the manual attached to the kit. The sequences of the primer sets and probes used in real-time RT-PCR are as follows. Below, “FAM” represents a fluorescent dye (fluorescein phosphoramidite), and “BHQ” represents a black hole quencher.

[0178] Forward primer: 5'-CACATTGGCA CCCGCAATC-3' (SEQ ID NO: 17)

[0179] Reverse primer: 5'-GAGGAACGAG AAGAGGCTTG-3' (SEQ ID NO: 18)

[0180] Probe: 5'-FAM-ACTTCCTCAA GGAACAACAT TGCCA-BHQ-3'(SEQ ID NO:19)

[0181] (4) Results

[0182] The XY plane with the luminescence intensity (HISCL count value) on the X-axis and the viral RNA concentration (copy / μL) on the Y-axis is plotted against the values ​​measured by the measurement method of this embodiment and the PCR method for each subject. The resulting coordinate diagram is shown in Figure 3 . As from Figure 3 It was found that there was a correlation between the measurement method of the present embodiment and the PCR method.

[0183]

Explanation of symbols

[0184] 11, 21, 31: Test kit

[0185] 12, 22, 32: Container 1

[0186] 13, 23, 33: Second container

[0187] 34: Container 3

[0188] 14, 24, 35: Bale boxes

[0189] 15, 25, 36: Accompanying documents Sequence Listing <110> Sysmex Co., Ltd. <120> Method, antibody set and kit for measuring viral antigen in sample <130> 20-015JP <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> House mouse (Mus musculus) <400> 1 Thr Ser Gly Thr Gly Val Ser 1 5 <210> 2 <211> 16 <212> PRT <213> House mouse (Mus musculus) <400> 2 His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 3 <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> 3 Ser Asn Tyr Gly Tyr Asp Leu Asp Tyr 1 5 <210> 4 <211> 11 <212> PRT <213> House mouse (Mus musculus) <400> 4 Lys Ala Ser Gln Asn Val Gly Thr Asn Val Val 1 5 10 <210> 5 <211> 7 <212> PRT <213> House mouse (Mus musculus) <400> 5 Ser Ala Ser Tyr Arg Tyr Ser 1 5 <210> 6 <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> 6 Gln Gln Tyr Asn Asn Tyr Pro Leu Thr 1 5 <210> 7 <211> 5 <212> PRT <213> House mouse (Mus musculus) <400> 7 Asp Tyr Tyr Met Tyr 1 5 <210> 8 <211> 17 <212> PRT <213> House mouse (Mus musculus) <400> 8 Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly <210> 9 <211> 10 <212> PRT <213> House mouse (Mus musculus) <400> 9 Ala Ala Asp Tyr Gly Gly Tyr Phe Asp Tyr 1 5 10 <210> 10 <211> 8 <212> PRT <213> House mouse (Mus musculus) <400> 10 Ser Ala Ser Gln Gly Ile Ser Asn 1 5 <210> 11 <211> 7 <212> PRT <213> House mouse (Mus musculus) <400> 11 Tyr Thr Ser Ser Leu His Ser 1 5 <210> 12 <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> 12 Gln Gln Tyr Ser Lys Leu Pro Tyr Thr 1 5 <210> 13 <211> 119 <212> PRT <213> Mus musculus <400> 13 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Thr Gly Val Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Val Ser Lys Asp Thr Ser Gly Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ser Asn Tyr Gly Tyr Asp Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 14 <211> 109 <212> PRT <213> Mus musculus <400> 14 Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30 Val Val Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala 100 105 <210> 15 <211> 119 <212> PRT <213> Mus musculus <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Asp Asp Thr Ala Lys Tyr Tyr Cys 85 90 95 Ala Arg Ala Ala Asp Tyr Gly Gly Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 16 <211> 109 <212> PRT <213> Mus musculus <400> 16 Asp Ile Gln Leu Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala 100 105 <210> 17 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 17 cacattggca cccgcaatc 19 <210> 18 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <400> 18 gaggaacgag aagaggcttg 20 <210> 19 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Probe <400> 19 acttcctcaa ggaacaacat tgcca 25

Claims

1. A virus antigen assay antibody set for use in assaying a virus antigen in a sample by an immunological assay, wherein The antibody group comprises a capture antibody and a detection antibody that form a sandwich immune complex with the viral antigen in the immunological assay, The viral antigen is a nucleoprotein of SARS-CoV or SARS-CoV-2, The capture antibody and the detection antibody are antibodies that can specifically bind to the nucleoprotein of SARS-CoV or SARS-CoV-2 to form a sandwich immune complex. The capture antibody comprises: The heavy chain variable region comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and A CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and The light chain variable region comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 6; and The detection antibody contains: The heavy chain variable region comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 7, CDR2 consisting of the amino acid sequence of SEQ ID NO: 8, and A CDR3 consisting of the amino acid sequence of SEQ ID NO: 9, and The light chain variable region comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 10, CDR2 consisting of the amino acid sequence of SEQ ID NO: 11, and CDR3 consisting of the amino acid sequence of SEQ ID NO:

12.

2. The antibody panel of claim 1, wherein the capture antibody and the detection antibody do not form a sandwich immune complex with the nucleoprotein of MERS-CoV, the viral antigen of HCoV-OC43, HCoV-NL63 or HCoV-229.

3. The antibody panel according to claim 1, wherein The capture antibody comprises: A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 13, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 14, The detection antibody contains: A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 15, and The light chain variable region consists of the amino acid sequence of SEQ ID NO:

16.

4. The antibody panel of claim 1, wherein the capture antibody or the detection antibody is IgG, Fab, Fab', F(ab')2, Fv, scFv, diabody or triabody. The antibody panel according to claim 1 , wherein the sample is a biological sample. The antibody panel of claim 1 , wherein the capture antibody is immobilized on a solid phase.

7. The antibody panel of claim 6, wherein the solid phase is a magnetic particle or a microplate. The antibody panel according to claim 1 , wherein the detection antibody is labeled with a labeling substance. 9 . The antibody panel according to claim 8 , wherein the labeling substance is at least one enzyme selected from the group consisting of alkaline phosphatase, peroxidase, β-galactosidase, glucosidase, polyphenol oxidase, tyrosinase, acid phosphatase, and luciferase.

10. A kit comprising a capture antibody and a detection antibody, wherein The capture antibody and the detection antibody are antibodies that can specifically bind to the nucleoprotein of SARS-CoV or SARS-CoV-2 to form a sandwich immune complex. The capture antibody comprises: The heavy chain variable region comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and A CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and The light chain variable region comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 6; and The detection antibody contains: The heavy chain variable region comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 7, CDR2 consisting of the amino acid sequence of SEQ ID NO: 8, and A CDR3 consisting of the amino acid sequence of SEQ ID NO: 9, and The light chain variable region comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 10, CDR2 consisting of the amino acid sequence of SEQ ID NO: 11, and CDR3 consisting of the amino acid sequence of SEQ ID NO:

12.

11. The kit of claim 10, wherein the capture antibody and the detection antibody do not form a sandwich immune complex with the nucleoprotein of MERS-CoV, the viral antigen of HCoV-OC43, HCoV-NL63 or HCoV-229.

12. The kit of claim 10, wherein The capture antibody comprises: A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 13, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 14, The detection antibody contains: A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 15, and The light chain variable region consists of the amino acid sequence of SEQ ID NO:

16.

13. The kit of claim 10, wherein the capture antibody or the detection antibody is IgG, Fab, Fab', F(ab')2, Fv, scFv, diabody or triabody.

14. The kit according to any one of claims 10 to 13, wherein the capture antibody is immobilized on a solid phase.

15. The kit of claim 14, wherein the solid phase is a magnetic particle or a microplate.

16. The kit according to any one of claims 10 to 13, wherein the detection antibody is labeled with a labeling substance.

17. The kit according to claim 16, wherein the labeling substance is at least one enzyme selected from the group consisting of alkaline phosphatase, peroxidase, β-galactosidase, glucosidase, polyphenol oxidase, tyrosinase, acid phosphatase and luciferase. The kit according to claim 17 , further comprising a substrate for the enzyme.

19. Use of a capture antibody and a detection antibody in the manufacture of a kit for measuring a viral antigen in a sample, wherein In the assay, a sandwich immune complex is formed containing the capture antibody, the viral antigen and the detection antibody, The viral antigen is a nucleoprotein of SARS-CoV or SARS-CoV-2, The capture antibody and the detection antibody are antibodies that can specifically bind to the nucleoprotein of SARS-CoV or SARS-CoV-2 to form a sandwich immune complex. The capture antibody comprises: The heavy chain variable region comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and A CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and The light chain variable region comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 6; and The detection antibody contains: The heavy chain variable region comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 7, CDR2 consisting of the amino acid sequence of SEQ ID NO: 8, and A CDR3 consisting of the amino acid sequence of SEQ ID NO: 9, and The light chain variable region comprises: CDR1 consisting of the amino acid sequence of SEQ ID NO: 10, CDR2 consisting of the amino acid sequence of SEQ ID NO: 11, and CDR3 consisting of the amino acid sequence of SEQ ID NO:

12.

20. The use of claim 19, wherein the capture antibody and the detection antibody do not form a sandwich immune complex with the nucleoprotein of MERS-CoV, the viral antigen of HCoV-OC43, HCoV-NL63 or HCoV-229.

21. The use of claim 19, wherein The capture antibody comprises: A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 13, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 14, The detection antibody contains: A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 15, and The light chain variable region consists of the amino acid sequence of SEQ ID NO:

16.

22. The use of claim 19, wherein the capture antibody or the detection antibody is IgG, Fab, Fab', F(ab')2, Fv, scFv, diabody or triabody.

23. The use according to claim 19, wherein the sample is a biological sample.

24. The use of claim 19, wherein the capture antibody is immobilized on a solid phase.

25. The use according to claim 24, wherein the solid phase is a magnetic particle or a microplate. The use according to claim 19 , wherein the detection antibody is labeled with a labeling substance.

27. The use according to claim 26, wherein the labeling substance is at least one enzyme selected from the group consisting of alkaline phosphatase, peroxidase, β-galactosidase, glucosidase, polyphenol oxidase, tyrosinase, acid phosphatase and luciferase.

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