WO2023167317A1 - ANTI-SARS-CoV-2 ANTIBODY AGAINST SARS-CoV-2 ANTIGEN IN BODY FLUID, INCLUDING MUTANT; METHOD FOR DETECTING SARS-CoV-2 USING ANTIBODY; AND KIT CONTAINING ANTIBODY - Google Patents
ANTI-SARS-CoV-2 ANTIBODY AGAINST SARS-CoV-2 ANTIGEN IN BODY FLUID, INCLUDING MUTANT; METHOD FOR DETECTING SARS-CoV-2 USING ANTIBODY; AND KIT CONTAINING ANTIBODY Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/08—RNA viruses
- C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
Definitions
- the present invention relates to anti-SARS-CoV-2 antibodies against SARS-CoV-2 antigens in body fluids, including mutant strains, methods for detecting SARS-CoV-2 using the antibodies, kits containing the antibodies, and the like. .
- a method for detecting a protein that constitutes a SARS-CoV-2-derived nucleocapsid from a sample wherein the sample is the antibody or fragment thereof according to any one of [1] to [6], or a combination thereof The above method, comprising the step of contacting.
- the method of [7] wherein the sample is a biological sample.
- the kit of [11] which is in the form of an immunochromatographic strip.
- FIG. 8 shows the results of detection by sandwich ELISA using cultured virus-derived NP of each mutant strain.
- FIG. 9 shows SDS-PAGE and CBB staining of NPs mixed with saliva specimens.
- Left is a sample of raw saliva mixed with NP or PBS
- Light is a sample of heat-treated saliva mixed with NP or PBS.
- FIG. 10 shows detection by Western blot using each antibody of NP mixed with a saliva sample.
- FIG. 11 shows the results of detection by sandwich ELISA using each antibody for NP added to a saliva sample.
- the epitope recognized by the antibody of the present invention, a fragment thereof, or a combination thereof is the amino acid sequence represented by SEQ ID NO: 1 to 50, 51 to 105, 106 to 160, or 161 to It is in the 207th position.
- the antibodies of the invention, or fragments thereof, or combinations thereof specifically bind to proteins that make up the SARS-CoV-2-derived nucleocapsid.
- An antibody of the invention is, for example, a complete immunoglobulin molecule, preferably IgM, IgD, IgE, IgA or IgG, more preferably IgG1, IgG2a, IgG2b, IgG3 or IgG4.
- Antibodies of the present invention can also be modified and/or altered antibodies, such as chimeric and humanized antibodies.
- Antibodies of the invention can also be monoclonal or polyclonal antibodies, modified or altered monoclonal or polyclonal antibodies, or recombinantly or synthetically produced or synthesized antibodies.
- the antibody of the present invention is preferably a monoclonal antibody from the viewpoint of accurate, convenient, and rapid detection of SARS-CoV-2-derived NPs.
- Antibodies or fragments thereof, or combinations thereof of the present invention may also be antibody derivatives such as bifunctional antibodies, or antibody constructs such as single-chain Fvs (scFv), bispecific scFvs or antibody fusion proteins. obtain.
- scFv single-chain Fvs
- scFv single-chain Fvs
- a combination of fragments although the position of the epitope is not particularly limited, 1 to 207, especially 1 to 50, 51 to 105, 106 to 160 on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1 , or a combination of two or more antibodies or fragments that recognize epitopes 161 to 207, or 1 to 207 on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1, especially 1 to 50, 51 to 105
- a combination of an antibody or fragment that recognizes the 106th, 106th to 160th, or 161st to 207th epitopes and an existing antibody or fragment that recognizes other epitopes is preferred.
- the antibody of the present invention may specifically bind to a protein that constitutes a SARS-CoV-2-derived nucleocapsid.
- Specifically binding means in a general sense having a high binding affinity compared to other proteins, and in a preferred form, for example compared to the NPs of SARS, MERS, the binding affinity may mean that the
- the method of the present invention detects a protein that constitutes a SARS-CoV-2-derived nucleocapsid by at least one immunological test selected from the group consisting of ELISA, CLEIA, immunochromatography, and filter antigen assay. further comprising the step of
- the method of the present invention comprises the step of conducting an antigen-antibody reaction between an antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NPs. In one aspect, the method of the invention comprises multiple steps of conducting an antigen-antibody reaction between an antibody of the invention, or a fragment thereof, or a combination thereof, and SARS-CoV-2-derived NPs. In one embodiment, the antibodies of the invention or fragments thereof, or combinations thereof, differ in epitope each time through the steps. In one aspect, an antibody or fragment thereof, or combination thereof, of the present invention is labeled with a labeling substance.
- the method of the present invention includes a step of performing an antigen-antibody reaction between an unlabeled antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NPs, and labeled with a labeling substance.
- Antigen-antibody reaction between the antibody of the present invention or a fragment thereof, or a combination thereof (the epitope is different from the unlabeled antibody of the present invention, a fragment thereof, or a combination thereof) and SARS-CoV-2-derived NPs including the step of performing
- the method of the present invention includes the step of conducting an antigen-antibody reaction between the antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NPs, and/or the antibody of the present invention or It further comprises the step of conducting an antigen-antibody reaction between a fragment thereof, or a combination thereof, and an antibody or a fragment thereof, or a combination thereof, which recognizes the antibody, a fragment thereof, or a combination thereof.
- the antibody of the present invention or a fragment thereof, or a combination thereof, and/or an antibody or a fragment thereof, or a combination thereof that recognizes the antibody or a fragment thereof, or a combination thereof is labeled with a labeling substance.
- sample is any sample that can contain SARS-CoV-2-derived NPs, and is not particularly limited, but includes, for example, biological samples.
- sample is used interchangeably with “specimen.”
- biological sample refers to human or animal blood, plasma, serum, urine, semen, cerebrospinal fluid, saliva, oral mucosa, nasal discharge, sweat, tears, ascites, amniotic fluid, feces, organs such as blood vessels or liver Any sample that can contain SARS-CoV-2-derived NPs, such as , tissues, cells, or extracts thereof, preferably from oral cavity, tonsil, nasal cavity, pharynx, larynx, trachea that is easy to collect.
- Non-Patent Document 6 put 1 to 2 ml of serum in a plastic tube that can be sealed after separation, cap it, and seal it with Parafilm. It may contain a coagulant, and if a blood collection tube containing a serum separator is used, transfer 1 to 2 ml of the serum after centrifugation into a plastic tube (preferably a sterilized tube), cap it, and seal it with Parafilm. .
- Non-Patent Document 6 whole blood was collected in a blood collection tube containing an anticoagulant (EDTA-Na or K), and 1 to 2 ml was dispensed into a plastic tube that can be sealed and capped. Then seal with Parafilm. If possible, blood cells are separated and peripheral blood mononuclear cells are suspended in a cell preservation medium and cryopreserved. Isolation of peripheral blood mononuclear cells is facilitated using BD Vacutainer® CPTTM mononuclear cell isolation blood collection tubes. In addition, when it is not possible to perform dispensing or blood cell separation after blood collection, blood may be collected using a PAXgene (registered trademark) RNA collection tube and stored frozen as it is.
- PAXgene registered trademark
- Non-Patent Document 6 sputum is collected when sputum is produced. If on mechanical ventilation, tracheal aspirate should be obtained using a sterile catheter under aseptic technique. Consider collecting bronchoalveolar lavage if not clinically contraindicated. The collected sputum or aspirate is placed in a plastic tube with a screw cap, capped and sealed with Parafilm.
- a nasopharyngeal swab is prepared by inserting a sterile cotton swab (thin swab for nasal cavity, such as a floss swab or a cotton swab containing rayon or polyester in the material) from the nostril, and thoroughly cleaning the nasopharynx. Place wipes and swabs into sterile spitz tubes containing 1-3 ml of viral transport fluid (VTM/UTM), cap and seal with Parafilm. If no virus transport solution is available, use PBS, physiological saline, or the like.
- VTM/UTM viral transport fluid
- Non-Patent Document 6 put 1 to 2 ml of saliva in a sterilized plastic tube, cap it, and seal it with Parafilm.
- a saliva collection kit such as Salivet (registered trademark) is used to suck saliva into cotton, and then the saliva is separated and collected by centrifugation.
- Non-Patent Document 6 1 to 2 ml of urine is placed in a test tube (Falcon tube, etc.), capped, and sealed with Parafilm.
- a test tube Fecon tube, etc.
- Parafilm For example, about 0.1 g (adzuki bean size) of stool is collected in a hermetically sealed plastic tube, capped, and then sealed with Parafilm.
- the “labeling substance” refers to the antibody of the present invention or a fragment thereof, or a combination thereof, and/or an antibody or a fragment thereof, or a combination thereof that recognizes the antibody or a fragment thereof, or a combination thereof. Any substance capable of detecting an antigen-antibody reaction is not particularly limited. From the viewpoint of accurate, convenient, and rapid detection of SARS-CoV-2-derived NPs, the labeling substance is one or more selected from enzymes, dyes, colloidal metal particles, latex particles, cellulose particles, and the like. preferable.
- Antibodies that recognize the antibodies of the invention or fragments thereof, or combinations thereof can also be modified and/or altered antibodies, such as chimeric and humanized antibodies.
- Antibodies that recognize the antibodies of the present invention or fragments thereof, or combinations thereof may also be monoclonal or polyclonal antibodies, modified or altered monoclonal or polyclonal antibodies, or recombinantly or synthetically produced or synthesized antibodies. .
- An antibody of the invention or a fragment thereof, or a fragment of an antibody that recognizes a combination thereof can be a Fab fragment or part of such immunoglobulin molecules, such as the VL-, VH- or CDR-regions, although the antibody of the invention Or a fragment thereof, or a fragment of an antibody that recognizes a combination thereof, is also as specific for an antibody of the invention, or a fragment thereof, or a combination thereof, as an antibody that recognizes an antibody of the invention, or a fragment thereof, or a combination thereof. can be recognized and bound to each other.
- Fragments of antibodies that recognize antibodies or fragments thereof, or combinations thereof, of the present invention include antibody fragments and separate light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab') 2 and so on.
- a fragment of an antibody that recognizes an antibody of the invention or a fragment thereof, or a combination thereof is a Fab, Fab/c, Fv, Fab' or F(ab') 2 fragment.
- the antibody or fragment thereof of the present invention may be an antibody derivative such as a bifunctional antibody, or a single-chain Fv (scFv), bispecific It may be an antibody construct such as a sex scFv or an antibody fusion protein.
- an antibody derivative such as a bifunctional antibody, or a single-chain Fv (scFv), bispecific It may be an antibody construct such as a sex scFv or an antibody fusion protein.
- SARS-CoV-2-derived NPs are detected by any immunological test that can detect an antigen-antibody reaction between the antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NPs. .
- Another aspect of the present invention is a method for diagnosing SARS-CoV-2 infection comprising the step of contacting a sample with an antibody or fragment thereof of the present invention, or a combination thereof (hereinafter referred to as "diagnostic method of the present invention"). (as may be the case).
- the diagnostic method of the present invention identifies proteins that make up the SARS-CoV-2-derived nucleocapsid by at least one immunological test selected from the group consisting of ELISA, CLEIA, immunochromatography, and filter antigen assay. Further comprising the step of detecting.
- the diagnostic method of the present invention includes a step of performing an antigen-antibody reaction between the unlabeled antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NPs, and labeled with a labeling substance.
- Antigen antibody between the antibody of the present invention or a fragment thereof, or a combination thereof (the epitope is different from the unlabeled antibody of the present invention or a fragment thereof, or a combination thereof) and the SARS-CoV-2-derived NP including the step of conducting the reaction.
- the diagnostic method of the present invention includes the step of conducting an antigen-antibody reaction between the antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NPs, and/or the antibody of the present invention. or a fragment thereof, or a combination thereof, and an antibody or fragment thereof, or a combination thereof, which recognizes the antibody, a fragment thereof, or a combination thereof, and a step of conducting an antigen-antibody reaction.
- the antibody of the present invention or a fragment thereof, or a combination thereof, and/or an antibody or a fragment thereof, or a combination thereof that recognizes the antibody or a fragment thereof, or a combination thereof is labeled with a labeling substance.
- the DNA sequence represented by SEQ ID NO: 4 was artificially synthesized, and by PCR using the DNA sequence as a template, the amino acid sequence represented by SEQ ID NO: 1 corresponded to the 1st to 207th regions (Fig. 1). A region from 1 to 621 of the DNA sequence represented by SEQ ID NO:4 was amplified.
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Abstract
The present invention addresses the problem of providing a highly sensitive and highly specific means for detecting a protein constituting a nucleocapsid derived from SARS-CoV-2. The above problem has been solved by, for example, an antibody binding to a protein constituting the nucleocapsid derived from SARS-CoV-2 or a fragment thereof, or a combination thereof; a method for detecting, from a sample, a protein constituting the nucleocapsid derived from SARS-CoV-2 using said antibody or a fragment thereof, or a combination thereof; and a kit for detecting a protein constituting the nucleocapsid derived from SARS-CoV-2, the kit containing said antibody or a fragment thereof, or a combination thereof.
Description
本発明は、変異株を含む、体液中のＳＡＲＳ－ＣｏＶ－２抗原に対する抗ＳＡＲＳ－ＣｏＶ－２抗体、該抗体を用いてＳＡＲＳ－ＣｏＶ－２を検出する方法、および該抗体を含むキット等に関する。
The present invention relates to anti-SARS-CoV-2 antibodies against SARS-CoV-2 antigens in body fluids, including mutant strains, methods for detecting SARS-CoV-2 using the antibodies, kits containing the antibodies, and the like. .
２０１９年に発生が確認された新型コロナウイルス（ＳＡＲＳ－ＣｏＶ－２）は新型コロナウイルス感染症（ＣＯＶＩＤ－１９）の原因となるウイルスである。同ウイルス感染を判定する方法として、ウイルス遺伝子を検出するリアルタイムＰＣＲ法等の遺伝子検査と、抗原抗体反応を利用して、ウイルス抗原タンパク質を検出する抗原検査法がある（非特許文献１）。抗原検査法は遺伝子検査法と比べ、コストおよび検出時間の面でメリットがあるものの、感度に劣るという課題がある。
The new coronavirus (SARS-CoV-2), which was confirmed to occur in 2019, is the virus that causes the new coronavirus infection (COVID-19). Methods for determining viral infection include genetic testing such as real-time PCR for detecting viral genes, and antigen testing for detecting viral antigen proteins using antigen-antibody reactions (Non-Patent Document 1). Antigen testing methods have advantages over genetic testing methods in terms of cost and detection time, but they have the problem of being inferior in sensitivity.
抗原抗体反応にて検出するウイルスの標的タンパク質としてはウイルス粒子内部に存在し、構成タンパク質の中で最も量の多い、ヌクレオカプシドタンパク質（ＮＰ）が主に用いられる。ＮＰは他のウイルスタンパク質と比べて、比較的変異は少ないものの、一部のアミノ酸配列には変異が認められており、ウイルスの変異によって抗原検査試薬の検出能が低下することや、ウイルスの株間で検出感度が異なることが報告されている。（非特許文献２、３）
The nucleocapsid protein (NP), which exists inside the virus particle and has the highest amount among the constituent proteins, is mainly used as the target protein of the virus to be detected by the antigen-antibody reaction. Although NP has relatively few mutations compared to other viral proteins, some amino acid sequences have mutations, and viral mutations may reduce the detectability of antigen test reagents, have been reported to have different detection sensitivities. (Non-Patent Documents 2 and 3)
また、抗原検査試薬に適用される検体として、鼻咽頭拭い液、鼻腔拭い液、唾液検体が挙げられる。これらの体液中では、ＮＰが分解されるため、感度が落ちることが問題とされる。この内、唾液検体は侵襲を伴わずに採取できるというメリットがあるが、鼻咽頭拭い液検体と比べるとさらに感度に劣るという課題が挙げられる（非特許文献４）。
In addition, nasopharyngeal swabs, nasal swabs, and saliva samples are examples of samples that can be applied to antigen test reagents. Since NP is decomposed in these body fluids, the sensitivity is lowered. Of these, the saliva sample has the advantage of being able to be collected without invasion, but has the problem of being even less sensitive than the nasopharyngeal swab sample (Non-Patent Document 4).
その他、ＳＡＲＳ－ＣｏＶ－２のＮＰに対する抗体の作製に関する論文が公開されている（非特許文献５）。しかしながら、本発明の抗体とはエピトープが異なっており、感度が十分でなく、また、ＭＥＲＳやＳＡＲＳとの交差反応が認められており特異性が十分とは言えない。
In addition, a paper on the production of antibodies against SARS-CoV-2 NP has been published (Non-Patent Document 5). However, the epitope is different from that of the antibody of the present invention, the sensitivity is not sufficient, and cross-reactivity with MERS and SARS is observed, so the specificity is not sufficient.
本発明は、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出するための、感度が高くかつ特異性が高い手段を提供することを課題とする。
An object of the present invention is to provide highly sensitive and highly specific means for detecting proteins that constitute SARS-CoV-2-derived nucleocapsids.
本発明者らは、ＳＡＲＳ－ＣｏＶ－２のヌクレオカプシドタンパク質（ＮＰ）のＣ末端側（２０８－４１９）を除いた人工タンパク質を作製した。ここで、本発明者らは、ＮＰのＮ末端側が、唾液等の体液中でＣ末端側より安定であることを見出し、これに着目することによって、次に、作製した人工タンパク質をマウスに免疫し、常法に従ってＮＰのＮ末端側（アミノ酸配列：１－２０７）をエピトープとするモノクローナル抗体を作製した。そして、開発した抗体を用いて、ウイルスの抗原タンパク質を、検出したところ、驚くべきことに、ウイルスの株の変異によらずに、そして、既存の抗体よりも高感度かつ特異性高く、検出する方法を見出した。
The present inventors created an artificial protein by removing the C-terminal side (208-419) of the SARS-CoV-2 nucleocapsid protein (NP). Here, the present inventors found that the N-terminal side of NP is more stable than the C-terminal side in body fluids such as saliva. Then, a monoclonal antibody having the N-terminal side of NP (amino acid sequence: 1-207) as an epitope was prepared according to a conventional method. Then, when the developed antibody was used to detect the antigenic protein of the virus, it was surprisingly detected without the mutation of the virus strain and with higher sensitivity and specificity than existing antibodies. found a way.
さらに、唾液検体中においてＮＰがＣ末端側から分解を受けることを確認し、上述したエピトープの抗体を用いることで、唾液検体等の体液中であってもＮＰを高感度に検出できることを発見し、本発明を完成するに至った。
すなわち、本発明は以下に関する。 Furthermore, they confirmed that NP is degraded from the C-terminal side in saliva specimens, and discovered that NP can be detected with high sensitivity even in bodily fluids such as saliva specimens by using antibodies with the above-mentioned epitopes. , have completed the present invention.
That is, the present invention relates to the following.
すなわち、本発明は以下に関する。 Furthermore, they confirmed that NP is degraded from the C-terminal side in saliva specimens, and discovered that NP can be detected with high sensitivity even in bodily fluids such as saliva specimens by using antibodies with the above-mentioned epitopes. , have completed the present invention.
That is, the present invention relates to the following.
［１］ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質に結合する抗体もしくはその断片、またはそれらの組み合わせであって、該抗体もしくはその断片、またはそれらの組み合わせの認識するエピトープが、配列番号１で表されるアミノ酸配列の１～２０７番目にある、前記抗体もしくはその断片、またはそれらの組み合わせ。
[1] An antibody, fragment thereof, or a combination thereof that binds to a protein that constitutes a SARS-CoV-2-derived nucleocapsid, wherein the epitope recognized by the antibody, fragment, or combination thereof is SEQ ID NO: 1 Said antibody or fragment thereof, or a combination thereof, at positions 1-207 of the amino acid sequence represented.
［２］エピトープが、配列番号１で表されるアミノ酸配列の１～５０番目、５１～１０５番目、１０６～１６０番目、または１６１～２０７番目にある、［１］に記載の抗体もしくはその断片、またはそれらの組み合わせ。
[2] The antibody or fragment thereof according to [1], wherein the epitope is located at positions 1 to 50, 51 to 105, 106 to 160, or 161 to 207 of the amino acid sequence represented by SEQ ID NO: 1, or a combination thereof.
［３］エピトープが、配列番号１で表されるアミノ酸配列の５１～１０５番目にある、［１］に記載の抗体またはその断片と、１０６～１６０番目にある、［１］または［２］に記載の抗体またはその断片との組み合わせ。
[3] The antibody or fragment thereof according to [1], wherein the epitope is located at positions 51-105 of the amino acid sequence represented by SEQ ID NO: 1, and [1] or [2], which is located at positions 106-160 Combination with the described antibodies or fragments thereof.
［４］ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質に特異的に結合する、［１］～［３］のいずれかに記載の抗体もしくはその断片、またはそれらの組み合わせ。
［５］抗体が、モノクローナル抗体である、［１］～［４］のいずれかに記載の抗体もしくはその断片、またはそれらの組み合わせ。 [4] The antibody or fragment thereof according to any one of [1] to [3], which specifically binds to a protein that constitutes a SARS-CoV-2-derived nucleocapsid, or a combination thereof.
[5] The antibody or fragment thereof according to any one of [1] to [4], or a combination thereof, wherein the antibody is a monoclonal antibody.
［５］抗体が、モノクローナル抗体である、［１］～［４］のいずれかに記載の抗体もしくはその断片、またはそれらの組み合わせ。 [4] The antibody or fragment thereof according to any one of [1] to [3], which specifically binds to a protein that constitutes a SARS-CoV-2-derived nucleocapsid, or a combination thereof.
[5] The antibody or fragment thereof according to any one of [1] to [4], or a combination thereof, wherein the antibody is a monoclonal antibody.
［６］その断片が、Ｆａｂフラグメント、Ｆａｂ／ｃフラグメント、Ｆｖフラグメント、Ｆａｂ’フラグメントまたはＦ（ａｂ’）２フラグメントである、［１］～［５］のいずれかに記載の抗体もしくはその断片、またはそれらの組み合わせ。
［７］ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を試料から検出する方法であって、試料を、［１］～［６］のいずれかに記載の抗体もしくはその断片、またはそれらの組み合わせと接触させる工程を含む、前記方法。
［８］試料が、生体試料である、［７］に記載の方法。
［９］試料が、体液中の試料である、［７］または［８］に記載の方法。 [6] The antibody or fragment thereof according to any one of [1] to [5], wherein the fragment is a Fab fragment, Fab/c fragment, Fv fragment, Fab' fragment or F(ab') 2 fragment; or a combination thereof.
[7] A method for detecting a protein that constitutes a SARS-CoV-2-derived nucleocapsid from a sample, wherein the sample is the antibody or fragment thereof according to any one of [1] to [6], or a combination thereof The above method, comprising the step of contacting.
[8] The method of [7], wherein the sample is a biological sample.
[9] The method of [7] or [8], wherein the sample is a sample in body fluid.
［７］ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を試料から検出する方法であって、試料を、［１］～［６］のいずれかに記載の抗体もしくはその断片、またはそれらの組み合わせと接触させる工程を含む、前記方法。
［８］試料が、生体試料である、［７］に記載の方法。
［９］試料が、体液中の試料である、［７］または［８］に記載の方法。 [6] The antibody or fragment thereof according to any one of [1] to [5], wherein the fragment is a Fab fragment, Fab/c fragment, Fv fragment, Fab' fragment or F(ab') 2 fragment; or a combination thereof.
[7] A method for detecting a protein that constitutes a SARS-CoV-2-derived nucleocapsid from a sample, wherein the sample is the antibody or fragment thereof according to any one of [1] to [6], or a combination thereof The above method, comprising the step of contacting.
[8] The method of [7], wherein the sample is a biological sample.
[9] The method of [7] or [8], wherein the sample is a sample in body fluid.
［１０］ＥＬＩＳＡ法、ＣＬＥＩＡ法、イムノクロマト法およびフィルター抗原アッセイ法からなる群から選択される少なくとも１つの免疫学検査によりＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出する工程をさらに含む、［７］～［９］のいずれかに記載の方法。
［１１］［１］～［６］のいずれかに記載の抗体もしくはその断片、またはそれらの組み合わせを含む、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出するためのキット。
［１２］イムノクロマトストリップの形態である、［１１］に記載のキット。 [10] Further comprising the step of detecting proteins that make up the SARS-CoV-2-derived nucleocapsid by at least one immunological test selected from the group consisting of ELISA, CLEIA, immunochromatography and filter antigen assay, [ 7] The method according to any one of [9].
[11] A kit for detecting proteins constituting a SARS-CoV-2-derived nucleocapsid, comprising the antibody or fragment thereof according to any one of [1] to [6], or a combination thereof.
[12] The kit of [11], which is in the form of an immunochromatographic strip.
［１１］［１］～［６］のいずれかに記載の抗体もしくはその断片、またはそれらの組み合わせを含む、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出するためのキット。
［１２］イムノクロマトストリップの形態である、［１１］に記載のキット。 [10] Further comprising the step of detecting proteins that make up the SARS-CoV-2-derived nucleocapsid by at least one immunological test selected from the group consisting of ELISA, CLEIA, immunochromatography and filter antigen assay, [ 7] The method according to any one of [9].
[11] A kit for detecting proteins constituting a SARS-CoV-2-derived nucleocapsid, comprising the antibody or fragment thereof according to any one of [1] to [6], or a combination thereof.
[12] The kit of [11], which is in the form of an immunochromatographic strip.
本発明のＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質に結合する抗体もしくはその断片、またはそれらの組み合わせ、該抗体もしくはその断片、またはそれらの組み合わせを用いてＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を試料から検出する方法、および該抗体もしくはその断片、またはそれらの組み合わせを含むＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出するためのキット等により、変異株に対しても反応性を有し、偽陰性と判定されるリスクが低下する。また、従来の抗体よりも高感度にウイルスタンパク質を検出可能である。さらに、唾液検体等の体液中のウイルスタンパク質の検出能が向上する。
An antibody that binds to a protein that constitutes the SARS-CoV-2-derived nucleocapsid of the present invention, or a fragment thereof, or a combination thereof, and constructing a SARS-CoV-2-derived nucleocapsid using the antibody, a fragment thereof, or a combination thereof A method for detecting a protein from a sample, a kit for detecting a protein that constitutes a SARS-CoV-2-derived nucleocapsid containing the antibody or a fragment thereof, or a combination thereof, etc., can also enhance reactivity against mutant strains. and reduce the risk of false-negative results. In addition, viral proteins can be detected with higher sensitivity than conventional antibodies. Furthermore, the ability to detect viral proteins in bodily fluids such as saliva specimens is improved.
すなわち本発明により、遺伝子検査法と比べ、コストおよび検出時間の面でメリットがあり、タンパク質の変異にかかわらず感度および特異性に優れ、かつ、鼻咽頭拭い液、鼻腔拭い液、咽頭拭い液、唾液検体等の体液中においても高い感度を有し、特に侵襲を伴わずに採取できるというメリットがある唾液検体においても、高い感度を維持できる抗原検査試薬を提供することが可能である。
That is, the present invention has advantages in terms of cost and detection time compared to genetic testing methods, is excellent in sensitivity and specificity regardless of protein mutation, and has a nasopharyngeal swab, nasal swab, pharyngeal swab, It is possible to provide an antigen test reagent that has high sensitivity even in body fluids such as saliva specimens and that can maintain high sensitivity even in saliva specimens, which have the advantage of being able to be collected without invasion.
本明細書において別様に定義されない限り、本明細書で用いる全ての技術用語および科学用語は、当業者が通常理解しているものと同じ意味を有する。本明細書中で参照する全ての特許、出願および他の出版物や情報は、その全体を参照により本明細書に援用する。
Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications and other publications and information referenced herein are hereby incorporated by reference in their entirety.
［ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質に結合する抗体もしくはその断片、またはそれらの組み合せ］
本発明の一側面は、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質に結合する抗体もしくはその断片、またはそれらの組み合わせであって、該抗体もしくはその断片、またはそれらの組み合わせの認識するエピトープが、配列番号１で表されるアミノ酸配列の１～２０７番目にある、前記抗体もしくはその断片、またはそれらの組み合わせ（以下、「本発明の抗体もしくはその断片、またはそれらの組み合わせ」と記す場合がある）に関する。一態様において、本発明の抗体もしくはその断片、またはそれらの組み合わせの認識するエピトープは、配列番号１で表されるアミノ酸配列の１～５０番目、５１～１０５番目、１０６～１６０番目、または１６１～２０７番目にある。一態様において、本発明の抗体もしくはその断片、またはそれらの組み合わせは、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質に特異的に結合する。 [Antibodies that bind to proteins constituting SARS-CoV-2-derived nucleocapsids, fragments thereof, or combinations thereof]
One aspect of the present invention is an antibody or fragment thereof, or a combination thereof, that binds to a protein that constitutes a SARS-CoV-2-derived nucleocapsid, wherein the epitope recognized by the antibody, fragment, or combination thereof is 1 to 207 of the amino acid sequence represented by SEQ ID NO: 1, the antibody or fragment thereof, or a combination thereof (hereinafter sometimes referred to as "the antibody of the present invention, a fragment thereof, or a combination thereof") Regarding. In one aspect, the epitope recognized by the antibody of the present invention, a fragment thereof, or a combination thereof is the amino acid sequence represented by SEQ ID NO: 1 to 50, 51 to 105, 106 to 160, or 161 to It is in the 207th position. In one aspect, the antibodies of the invention, or fragments thereof, or combinations thereof, specifically bind to proteins that make up the SARS-CoV-2-derived nucleocapsid.
本発明の一側面は、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質に結合する抗体もしくはその断片、またはそれらの組み合わせであって、該抗体もしくはその断片、またはそれらの組み合わせの認識するエピトープが、配列番号１で表されるアミノ酸配列の１～２０７番目にある、前記抗体もしくはその断片、またはそれらの組み合わせ（以下、「本発明の抗体もしくはその断片、またはそれらの組み合わせ」と記す場合がある）に関する。一態様において、本発明の抗体もしくはその断片、またはそれらの組み合わせの認識するエピトープは、配列番号１で表されるアミノ酸配列の１～５０番目、５１～１０５番目、１０６～１６０番目、または１６１～２０７番目にある。一態様において、本発明の抗体もしくはその断片、またはそれらの組み合わせは、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質に特異的に結合する。 [Antibodies that bind to proteins constituting SARS-CoV-2-derived nucleocapsids, fragments thereof, or combinations thereof]
One aspect of the present invention is an antibody or fragment thereof, or a combination thereof, that binds to a protein that constitutes a SARS-CoV-2-derived nucleocapsid, wherein the epitope recognized by the antibody, fragment, or combination thereof is 1 to 207 of the amino acid sequence represented by SEQ ID NO: 1, the antibody or fragment thereof, or a combination thereof (hereinafter sometimes referred to as "the antibody of the present invention, a fragment thereof, or a combination thereof") Regarding. In one aspect, the epitope recognized by the antibody of the present invention, a fragment thereof, or a combination thereof is the amino acid sequence represented by SEQ ID NO: 1 to 50, 51 to 105, 106 to 160, or 161 to It is in the 207th position. In one aspect, the antibodies of the invention, or fragments thereof, or combinations thereof, specifically bind to proteins that make up the SARS-CoV-2-derived nucleocapsid.
本発明において、「ＳＡＲＳ－ＣｏＶ－２」は、中国武漢市付近で２０１９年に発生が初めて確認された、ＳＡＲＳ関連コロナウイルスに属するコロナウイルスを指し、「２０１９新型コロナウイルス」と互換的に用いられる。ＳＡＲＳ－ＣｏＶ－２は、ヒトに対して病原性があり、急性呼吸器疾患（ＣＯＶＩＤ－１９）を引き起こす。ＳＡＲＳ－ＣｏＶ－２は、他のコロナウイルスと同様に、スパイク、ヌクレオカプシド、内在性膜タンパク質、エンベロープタンパク質、およびＲＮＡより構成されている。このうちヌクレオカプシドがＲＮＡと結合してＮＰを形成し、脂質と結合したスパイク、内在性膜タンパク質、エンベロープタンパク質がその周りを取り囲んでエンベロープを形成する。なお、ＳＡＲＳ－ＣｏＶ－２のゲノム配列はＧｅｎＢａｎｋアクセッション番号ＭＮ９０８９４７．３で公開されている。
In the present invention, "SARS-CoV-2" refers to a coronavirus belonging to SARS-related coronaviruses, which was first confirmed to occur in the vicinity of Wuhan, China in 2019, and is used interchangeably with "2019 novel coronavirus". be done. SARS-CoV-2 is pathogenic to humans and causes acute respiratory disease (COVID-19). SARS-CoV-2, like other coronaviruses, is composed of a spike, a nucleocapsid, integral membrane proteins, envelope proteins, and RNA. Of these, the nucleocapsid binds to RNA to form NP, which is surrounded by lipid-bound spikes, integral membrane proteins, and envelope proteins to form an envelope. The genome sequence of SARS-CoV-2 is published under GenBank accession number MN908947.3.
本発明において、「ヌクレオカプシドを構成するタンパク質」は、ヌクレオカプシドおよびＲＮＡからなる複合体を構成するタンパク質を指し、「ＮＰ」と互換的に用いられる。ＳＡＲＳ－ＣｏＶ－２由来ＮＰのアミノ酸配列は配列番号１で表される。
In the present invention, "a protein that constitutes a nucleocapsid" refers to a protein that constitutes a complex consisting of a nucleocapsid and RNA, and is used interchangeably with "NP". The amino acid sequence of SARS-CoV-2-derived NP is represented by SEQ ID NO:1.
本発明において、「ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片」は、ＳＡＲＳ－ＣｏＶ－２由来ＮＰのアミノ酸配列（配列番号１）における、唾液の影響を受けやすいＣ末端側（２０８－４１９）を除いた領域（配列番号１で表されるアミノ酸配列の１～２０７番目の領域）からなるタンパク質の断片を指す。ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片は、配列番号２で表されるアミノ酸配列に対して、９０％以上の配列同一性を有する。正確、簡便、かつ迅速にＳＡＲＳ－ＣｏＶ－２由来ＮＰを検出する観点から、ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片は、配列番号２で表されるアミノ酸配列に対して、９５％以上の配列同一性を有することが好ましく、９８％以上の配列同一性を有することが特に好ましく、１００％の配列同一性を有することがさらに好ましい。一態様において、ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片は、配列番号２で表されるアミノ酸配列からなるものである。ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片は、ＳＡＲＳ－ＣｏＶ－２由来ＮＰのアミノ酸配列と他のコロナウイルス由来ＮＰのアミノ酸配列との間で同一性が低いＣ末端側とは異なる部分を認識する抗体を得るために該断片を作製し、これを免疫原として常法に従って本発明の抗体またはその断片を得ることができる。
In the present invention, the "SARS-CoV-2-derived NP fragment" is the amino acid sequence (SEQ ID NO: 1) of the SARS-CoV-2-derived NP, excluding the C-terminal side (208-419) that is susceptible to saliva. A protein fragment consisting of a region (1-207th region of the amino acid sequence represented by SEQ ID NO: 1). The SARS-CoV-2-derived NP fragment has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO:2. From the viewpoint of accurate, convenient, and rapid detection of SARS-CoV-2-derived NPs, the fragment of SARS-CoV-2-derived NPs has a sequence identity of 95% or more to the amino acid sequence represented by SEQ ID NO: 2. Having a sequence identity of 98% or more is particularly preferable, and having a sequence identity of 100% is even more preferable. In one aspect, the fragment of SARS-CoV-2-derived NP consists of the amino acid sequence represented by SEQ ID NO:2. A fragment of SARS-CoV-2-derived NP is an antibody that recognizes a portion different from the C-terminal side with low identity between the amino acid sequence of SARS-CoV-2-derived NP and the amino acid sequence of other coronavirus-derived NP. The antibody of the present invention or a fragment thereof can be obtained according to a conventional method by using the fragment as an immunogen.
本発明の抗体は、例えば、完全な免疫グロブリン分子、好ましくはＩｇＭ、ＩｇＤ、ＩｇＥ、ＩｇＡまたはＩｇＧであり、より好ましくはＩｇＧ１、ＩｇＧ２ａ、ＩｇＧ２ｂ、ＩｇＧ３またはＩｇＧ４である。また、本発明の抗体は、キメラのおよびヒト化した抗体等の、修飾および／または変化した抗体で有り得る。また、本発明の抗体は、モノクローナルまたはポリクローナル抗体、修飾または変化したモノクローナルまたはポリクローナル抗体、または組換えもしくは合成的に産生したまたは合成した抗体であり得る。本発明の抗体は、正確、簡便、かつ迅速にＳＡＲＳ－ＣｏＶ－２由来ＮＰを検出する観点から、モノクローナル抗体であることが好ましい。
An antibody of the invention is, for example, a complete immunoglobulin molecule, preferably IgM, IgD, IgE, IgA or IgG, more preferably IgG1, IgG2a, IgG2b, IgG3 or IgG4. Antibodies of the present invention can also be modified and/or altered antibodies, such as chimeric and humanized antibodies. Antibodies of the invention can also be monoclonal or polyclonal antibodies, modified or altered monoclonal or polyclonal antibodies, or recombinantly or synthetically produced or synthesized antibodies. The antibody of the present invention is preferably a monoclonal antibody from the viewpoint of accurate, convenient, and rapid detection of SARS-CoV-2-derived NPs.
本発明の抗体の断片は、ＦａｂフラグメントまたはＶＬ－、ＶＨ－またはＣＤＲ－領域等の、かかる免疫グロブリン分子の部分であり得るが、本発明の抗体の断片も、本発明の抗体と同程度にＳＡＲＳ－ＣｏＶ－２由来ＮＰを特異的に認識し結合し得るものである。本発明の抗体の断片は、抗体フラグメント、ならびに、分離した軽鎖および重鎖、Ｆａｂ、Ｆａｂ／ｃ、Ｆｖ、Ｆａｂ’、Ｆ（ａｂ’）２等の、それらの部分であり得る。一態様において、本発明の抗体の断片は、Ｆａｂフラグメント、Ｆａｂ／ｃフラグメント、Ｆｖフラグメント、Ｆａｂ’フラグメントまたはＦ（ａｂ’）２フラグメントである。
Fragments of antibodies of the invention may be Fab fragments or portions of such immunoglobulin molecules, such as the VL-, VH- or CDR-regions, although fragments of antibodies of the invention may also be similar to antibodies of the invention. It is capable of specifically recognizing and binding SARS-CoV-2-derived NPs. Fragments of antibodies of the invention can be antibody fragments and portions thereof, such as separate light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab') 2 . In one aspect, the fragment of the antibody of the invention is a Fab, Fab/c, Fv, Fab' or F(ab') 2 fragment.
また、本発明の抗体もしくはその断片、またはそれらの組み合わせは、二機能性抗体等の抗体派生物、または、単鎖Ｆｖ（ｓｃＦｖ）、二重特異性ｓｃＦｖまたは抗体融合タンパク質等の抗体構築物であり得る。
Antibodies or fragments thereof, or combinations thereof of the present invention may also be antibody derivatives such as bifunctional antibodies, or antibody constructs such as single-chain Fvs (scFv), bispecific scFvs or antibody fusion proteins. obtain.
本発明の、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質に結合する抗体もしくはその断片の組み合わせとは、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質のいずれかのエピトープを認識する２以上の抗体もしくは断片の組み合わせであって、エピトープの位置は特に限定されないが、配列番号１で表されるアミノ酸配列のＮ末端側の１～２０７番目、特に１～５０番目、５１～１０５番目、１０６～１６０番目、または１６１～２０７番目のエピトープを認識する抗体もしくは断片の２以上の組み合わせ、または配列番号１で表されるアミノ酸配列のＮ末端側の１～２０７番目、特に１～５０番目、５１～１０５番目、１０６～１６０番目、または１６１～２０７番目のエピトープを認識する抗体もしくは断片と、それ以外のエピトープを認識する既存の抗体もしくは断片の組み合わせが好ましい。より好ましくは配列番号１で表されるアミノ酸配列の１～５０番目のエピトープを認識する抗体またはその断片または、５１～２０７番目のエピトープを認識する抗体またはその断片と、５１～１６０番目のエピトープを認識する抗体またはその断片の組み合わせ、さらにより好ましくは、配列番号１で表されるアミノ酸配列の１～５０番目のエピトープを認識する抗体またはその断片と、５１～１６０番目のエピトープを認識する抗体またはその断片の組み合わせである。
The combination of antibodies or fragments thereof that bind to a protein that constitutes a SARS-CoV-2-derived nucleocapsid of the present invention is two or more antibodies that recognize any epitope of a protein that constitutes a SARS-CoV-2-derived nucleocapsid. Or a combination of fragments, although the position of the epitope is not particularly limited, 1 to 207, especially 1 to 50, 51 to 105, 106 to 160 on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1 , or a combination of two or more antibodies or fragments that recognize epitopes 161 to 207, or 1 to 207 on the N-terminal side of the amino acid sequence represented by SEQ ID NO: 1, especially 1 to 50, 51 to 105 A combination of an antibody or fragment that recognizes the 106th, 106th to 160th, or 161st to 207th epitopes and an existing antibody or fragment that recognizes other epitopes is preferred. More preferably, an antibody or a fragment thereof that recognizes the 1st to 50th epitopes of the amino acid sequence represented by SEQ ID NO: 1, or an antibody or a fragment thereof that recognizes the 51st to 207th epitopes, and the 51st to 160th epitopes A combination of recognizing antibodies or fragments thereof, still more preferably, an antibody or fragment thereof recognizing the 1st to 50th epitopes of the amino acid sequence represented by SEQ ID NO: 1, and an antibody recognizing the 51st to 160th epitopes or It is a combination of the fragments.
本発明の抗体もしくはその断片、またはそれらの組み合わせは、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質に特異的に結合するものであってもよい。特異的に結合するとは、一般的な意味において、他のタンパク質に比較して、結合親和性が高いことを意味し、好ましい形態において、たとえば、ＳＡＲＳ、ＭＥＲＳのＮＰと比較して、結合親和性が高いことを意味する場合がある。
The antibody of the present invention, a fragment thereof, or a combination thereof may specifically bind to a protein that constitutes a SARS-CoV-2-derived nucleocapsid. Specifically binding means in a general sense having a high binding affinity compared to other proteins, and in a preferred form, for example compared to the NPs of SARS, MERS, the binding affinity may mean that the
［ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を試料から検出する方法］
本発明の別の側面は、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を試料から検出する方法であって、試料を、本発明の抗体もしくはその断片、またはそれらの組み合わせと接触させる工程を含む、前記方法（以下、「本発明の方法」と記す場合がある）に関する。一態様において、本発明の方法において、試料は生体試料である。一態様において、本発明の方法は、ＥＬＩＳＡ法、ＣＬＥＩＡ法、イムノクロマト法およびフィルター抗原アッセイ法からなる群から選択される少なくとも１つの免疫学検査によりＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出する工程をさらに含む。 [Method for detecting proteins constituting SARS-CoV-2-derived nucleocapsid from sample]
Another aspect of the invention is a method of detecting from a sample proteins that make up the SARS-CoV-2-derived nucleocapsid comprising contacting the sample with an antibody of the invention or a fragment thereof, or a combination thereof. , the method (hereinafter sometimes referred to as “the method of the present invention”). In one aspect, in the methods of the invention, the sample is a biological sample. In one aspect, the method of the present invention detects a protein that constitutes a SARS-CoV-2-derived nucleocapsid by at least one immunological test selected from the group consisting of ELISA, CLEIA, immunochromatography, and filter antigen assay. further comprising the step of
本発明の別の側面は、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を試料から検出する方法であって、試料を、本発明の抗体もしくはその断片、またはそれらの組み合わせと接触させる工程を含む、前記方法（以下、「本発明の方法」と記す場合がある）に関する。一態様において、本発明の方法において、試料は生体試料である。一態様において、本発明の方法は、ＥＬＩＳＡ法、ＣＬＥＩＡ法、イムノクロマト法およびフィルター抗原アッセイ法からなる群から選択される少なくとも１つの免疫学検査によりＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出する工程をさらに含む。 [Method for detecting proteins constituting SARS-CoV-2-derived nucleocapsid from sample]
Another aspect of the invention is a method of detecting from a sample proteins that make up the SARS-CoV-2-derived nucleocapsid comprising contacting the sample with an antibody of the invention or a fragment thereof, or a combination thereof. , the method (hereinafter sometimes referred to as “the method of the present invention”). In one aspect, in the methods of the invention, the sample is a biological sample. In one aspect, the method of the present invention detects a protein that constitutes a SARS-CoV-2-derived nucleocapsid by at least one immunological test selected from the group consisting of ELISA, CLEIA, immunochromatography, and filter antigen assay. further comprising the step of
一態様において、本発明の方法は、本発明の抗体もしくはその断片、またはそれらの組み合わせとＳＡＲＳ－ＣｏＶ－２由来ＮＰとの間で抗原抗体反応を行う工程を含む。一態様において、本発明の方法は、本発明の抗体もしくはその断片、またはそれらの組み合わせとＳＡＲＳ－ＣｏＶ－２由来ＮＰとの間で抗原抗体反応を行う工程を複数回含む。一態様において、該工程の各回毎に本発明の抗体もしくはその断片、またはそれらの組み合わせはエピトープが異なる。一態様において、本発明の抗体もしくはその断片、またはそれらの組み合わせは、標識物質により標識されている。例えば、本発明の方法は、無標識の本発明の抗体もしくはその断片、またはそれらの組み合わせとＳＡＲＳ－ＣｏＶ－２由来ＮＰとの間で抗原抗体反応を行う工程、および標識物質により標識されている本発明の抗体もしくはその断片、またはそれらの組み合わせ（前記無標識の本発明の抗体もしくはその断片、またはそれらの組み合わせとはエピトープが異なる）とＳＡＲＳ－ＣｏＶ－２由来ＮＰとの間で抗原抗体反応を行う工程を含む。
In one aspect, the method of the present invention comprises the step of conducting an antigen-antibody reaction between an antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NPs. In one aspect, the method of the invention comprises multiple steps of conducting an antigen-antibody reaction between an antibody of the invention, or a fragment thereof, or a combination thereof, and SARS-CoV-2-derived NPs. In one embodiment, the antibodies of the invention or fragments thereof, or combinations thereof, differ in epitope each time through the steps. In one aspect, an antibody or fragment thereof, or combination thereof, of the present invention is labeled with a labeling substance. For example, the method of the present invention includes a step of performing an antigen-antibody reaction between an unlabeled antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NPs, and labeled with a labeling substance. Antigen-antibody reaction between the antibody of the present invention or a fragment thereof, or a combination thereof (the epitope is different from the unlabeled antibody of the present invention, a fragment thereof, or a combination thereof) and SARS-CoV-2-derived NPs including the step of performing
一態様において、本発明の方法は、本発明の抗体もしくはその断片、またはそれらの組み合わせとＳＡＲＳ－ＣｏＶ－２由来ＮＰとの間で抗原抗体反応を行う工程、および／または、本発明の抗体もしくはその断片、またはそれらの組み合わせと該抗体もしくはその断片、またはそれらの組み合わせを認識する抗体もしくはその断片、またはそれらの組み合わせとの間で抗原抗体反応を行う工程をさらに含む。一態様において、本発明の抗体もしくはその断片、またはそれらの組み合わせ、および／または該抗体もしくはその断片、またはそれらの組み合わせを認識する抗体もしくはその断片、またはそれらの組み合わせは、標識物質により標識されている。
In one aspect, the method of the present invention includes the step of conducting an antigen-antibody reaction between the antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NPs, and/or the antibody of the present invention or It further comprises the step of conducting an antigen-antibody reaction between a fragment thereof, or a combination thereof, and an antibody or a fragment thereof, or a combination thereof, which recognizes the antibody, a fragment thereof, or a combination thereof. In one aspect, the antibody of the present invention or a fragment thereof, or a combination thereof, and/or an antibody or a fragment thereof, or a combination thereof that recognizes the antibody or a fragment thereof, or a combination thereof, is labeled with a labeling substance. there is
本発明において、「試料」は、ＳＡＲＳ－ＣｏＶ－２由来ＮＰが含まれ得る任意の試料であり、特に限定されないが、例えば生体試料が挙げられる。本発明において、「試料」は「検体」と互換的に用いられる。
In the present invention, a "sample" is any sample that can contain SARS-CoV-2-derived NPs, and is not particularly limited, but includes, for example, biological samples. In the present invention, "sample" is used interchangeably with "specimen."
本発明において、「生体試料」は、ヒトまたは動物の血液、血漿、血清、尿、***、髄液、唾液、口腔粘膜、鼻水、汗、涙、腹水、羊水、糞便、血管もしくは肝臓等の臓器、組織、細胞、またはそれらの抽出物等、ＳＡＲＳ－ＣｏＶ－２由来ＮＰが含まれ得る任意の試料であり、好ましくは、採取が容易である、口腔、扁桃腺、鼻腔、咽頭、喉頭、気管、気管支および肺等からの細胞および分泌液や、鼻腔ぬぐい液、鼻咽頭ぬぐい液、唾液、うがい液、喀痰、気管吸引液、気管支肺胞洗浄液等である。より好ましくは鼻咽頭拭い液、鼻腔拭い液、咽頭拭い液、唾液検体、もっとも好ましくは唾液検体が挙げられる。これらの検体を採取する方法は特に限定されず、公知の方法を採用することができる。具体的には、綿棒を用いた方法が多用される。動物は、ＳＡＲＳ－ＣｏＶ－２に感染し得るものであれば特に限定されず、例えば、イヌ、ネコ等が挙げられる。
生体試料は常法に従い採取することができる。 In the present invention, "biological sample" refers to human or animal blood, plasma, serum, urine, semen, cerebrospinal fluid, saliva, oral mucosa, nasal discharge, sweat, tears, ascites, amniotic fluid, feces, organs such as blood vessels or liver Any sample that can contain SARS-CoV-2-derived NPs, such as , tissues, cells, or extracts thereof, preferably from oral cavity, tonsil, nasal cavity, pharynx, larynx, trachea that is easy to collect. , cells and secretions from the bronchi and lungs, nasal swabs, nasopharyngeal swabs, saliva, gargles, sputum, tracheal aspirate, bronchoalveolar lavage, and the like. Nasopharyngeal swabs, nasal swabs, pharyngeal swabs, and saliva samples are more preferred, and saliva samples are most preferred. Methods for collecting these specimens are not particularly limited, and known methods can be adopted. Specifically, a method using a cotton swab is often used. Animals are not particularly limited as long as they can be infected with SARS-CoV-2, and examples thereof include dogs and cats.
A biological sample can be collected according to a conventional method.
生体試料は常法に従い採取することができる。 In the present invention, "biological sample" refers to human or animal blood, plasma, serum, urine, semen, cerebrospinal fluid, saliva, oral mucosa, nasal discharge, sweat, tears, ascites, amniotic fluid, feces, organs such as blood vessels or liver Any sample that can contain SARS-CoV-2-derived NPs, such as , tissues, cells, or extracts thereof, preferably from oral cavity, tonsil, nasal cavity, pharynx, larynx, trachea that is easy to collect. , cells and secretions from the bronchi and lungs, nasal swabs, nasopharyngeal swabs, saliva, gargles, sputum, tracheal aspirate, bronchoalveolar lavage, and the like. Nasopharyngeal swabs, nasal swabs, pharyngeal swabs, and saliva samples are more preferred, and saliva samples are most preferred. Methods for collecting these specimens are not particularly limited, and known methods can be adopted. Specifically, a method using a cotton swab is often used. Animals are not particularly limited as long as they can be infected with SARS-CoV-2, and examples thereof include dogs and cats.
A biological sample can be collected according to a conventional method.
例えば、血清は、非特許文献６に記載のとおり、分離後の血清を密栓できるプラスティックチューブに１～２ｍｌ入れ、蓋をした後、パラフィルムでシールする。凝固剤が入っていてもよく、血清分離剤入りの採血管を用いた場合は、遠心後の血清１～２ｍｌをプラスチックチューブ（滅菌チューブが望ましい）に移し蓋をした後、パラフィルムでシールする。
For example, as described in Non-Patent Document 6, put 1 to 2 ml of serum in a plastic tube that can be sealed after separation, cap it, and seal it with Parafilm. It may contain a coagulant, and if a blood collection tube containing a serum separator is used, transfer 1 to 2 ml of the serum after centrifugation into a plastic tube (preferably a sterilized tube), cap it, and seal it with Parafilm. .
例えば、全血は、非特許文献６に記載のとおり、血液凝固阻止剤（ＥＤＴＡ－ＮａまたＫ）入りの採血管に採取し、１～２ｍｌを密栓できるプラスティックチューブに分注し、蓋をした後、パラフィルムでシールする。可能であれば、血球分離し、末梢血単核球を細胞保存液に懸濁して凍結保存する。末梢血単核球の分離はＢＤバキュテイナ（登録商標）ＣＰＴ（商標）単核球分離用採血管を使うと簡便である。また、採血後の分注や血球分離ができない場合は、ＰＡＸｇｅｎｅ（登録商標）ＲＮＡ採血管を用いて採血し、そのまま凍結保存しておいてもよい。
For example, as described in Non-Patent Document 6, whole blood was collected in a blood collection tube containing an anticoagulant (EDTA-Na or K), and 1 to 2 ml was dispensed into a plastic tube that can be sealed and capped. Then seal with Parafilm. If possible, blood cells are separated and peripheral blood mononuclear cells are suspended in a cell preservation medium and cryopreserved. Isolation of peripheral blood mononuclear cells is facilitated using BD Vacutainer® CPT™ mononuclear cell isolation blood collection tubes. In addition, when it is not possible to perform dispensing or blood cell separation after blood collection, blood may be collected using a PAXgene (registered trademark) RNA collection tube and stored frozen as it is.
例えば、下気道由来液は、非特許文献６に記載のとおり、喀痰が出る場合喀痰を採取する。人工呼吸器管理下にある場合には、無菌的な操作のもとに、滅菌されたカテーテルを使って気管吸引液を採取する。臨床的に禁忌とならない場合は気管支肺胞洗浄液の採取も検討する。採取した喀痰または吸引液はスクリューキャップ付きプラスティックチューブに入れ蓋をし、パラフィルムでシールする。
For example, as for the liquid derived from the lower respiratory tract, as described in Non-Patent Document 6, sputum is collected when sputum is produced. If on mechanical ventilation, tracheal aspirate should be obtained using a sterile catheter under aseptic technique. Consider collecting bronchoalveolar lavage if not clinically contraindicated. The collected sputum or aspirate is placed in a plastic tube with a screw cap, capped and sealed with Parafilm.
例えば、鼻咽頭ぬぐい液は、非特許文献６に記載のとおり、滅菌綿棒（フロックスワブや材質にレーヨンやポリエステルを含む綿棒等、鼻腔用の細いもの）を鼻孔から挿入し、上咽頭を十分にぬぐい、綿棒を１～３ｍｌのウイルス輸送液（ＶＴＭ／ＵＴＭ）が入った滅菌スピッツ管に入れ蓋をし、パラフィルムでシールする。ウイルス輸送液が無い場合はＰＢＳや生理食塩水等を用いる。
For example, as described in Non-Patent Document 6, a nasopharyngeal swab is prepared by inserting a sterile cotton swab (thin swab for nasal cavity, such as a floss swab or a cotton swab containing rayon or polyester in the material) from the nostril, and thoroughly cleaning the nasopharynx. Place wipes and swabs into sterile spitz tubes containing 1-3 ml of viral transport fluid (VTM/UTM), cap and seal with Parafilm. If no virus transport solution is available, use PBS, physiological saline, or the like.
例えば、唾液は、非特許文献６に記載のとおり、滅菌されたプラスティックチューブに１～２ｍｌを入れ、蓋をした後、パラフィルムでシールする。また、サリベット（登録商標）等の唾液採取用キットを用いて、コットンに唾液を吸い込ませた後、遠心処理によって分離採取したものも用いられる。
For example, as described in Non-Patent Document 6, put 1 to 2 ml of saliva in a sterilized plastic tube, cap it, and seal it with Parafilm. In addition, a saliva collection kit such as Salivet (registered trademark) is used to suck saliva into cotton, and then the saliva is separated and collected by centrifugation.
例えば、尿は、非特許文献６に記載のとおり、１～２ｍｌを試験管（ファルコンチューブ等）に入れ、蓋をした後、パラフィルムでシールする。
例えば、便は、０．１ｇ程度（小豆大）を密栓できるプラスティックチューブに採取して蓋をした後、パラフィルムでシールする。 For example, as described in Non-Patent Document 6, 1 to 2 ml of urine is placed in a test tube (Falcon tube, etc.), capped, and sealed with Parafilm.
For example, about 0.1 g (adzuki bean size) of stool is collected in a hermetically sealed plastic tube, capped, and then sealed with Parafilm.
例えば、便は、０．１ｇ程度（小豆大）を密栓できるプラスティックチューブに採取して蓋をした後、パラフィルムでシールする。 For example, as described in Non-Patent Document 6, 1 to 2 ml of urine is placed in a test tube (Falcon tube, etc.), capped, and sealed with Parafilm.
For example, about 0.1 g (adzuki bean size) of stool is collected in a hermetically sealed plastic tube, capped, and then sealed with Parafilm.
本発明において、「標識物質」は、本発明の抗体もしくはその断片、またはそれらの組み合わせ、および／または該抗体もしくはその断片、またはそれらの組み合わせを認識する抗体もしくはその断片、またはそれらの組み合わせを標識する任意の物質を指し、抗原抗体反応を検出できるものであれば特に限定されない。正確、簡便、かつ迅速にＳＡＲＳ－ＣｏＶ－２由来ＮＰを検出する観点から、標識物質は、酵素、色素、金属コロイド粒子、ラテックス粒子、セルロース粒子等から選択される１以上のものであることが好ましい。
In the present invention, the “labeling substance” refers to the antibody of the present invention or a fragment thereof, or a combination thereof, and/or an antibody or a fragment thereof, or a combination thereof that recognizes the antibody or a fragment thereof, or a combination thereof. Any substance capable of detecting an antigen-antibody reaction is not particularly limited. From the viewpoint of accurate, convenient, and rapid detection of SARS-CoV-2-derived NPs, the labeling substance is one or more selected from enzymes, dyes, colloidal metal particles, latex particles, cellulose particles, and the like. preferable.
本発明において、「本発明の抗体もしくはその断片、またはそれらの組み合わせを認識する抗体もしくはその断片、またはそれらの組み合わせ」は、本発明の抗体もしくはその断片、またはそれらの組み合わせに特異的に結合し得る抗体もしくはその断片、またはそれらの組み合わせを指す。正確、簡便、かつ迅速にＳＡＲＳ－ＣｏＶ－２由来ＮＰを検出する観点から、本発明の抗体もしくはその断片、またはそれらの組み合わせを認識する抗体は、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、およびＩｇＥからなる群から選択される１以上のものであることが好ましく、ＩｇＧおよび／またはＩｇＭであることが特に好ましく、ＩｇＧであることがさらに好ましい。また、本発明の抗体もしくはその断片、またはそれらの組み合わせを認識する抗体は、キメラのおよびヒト化した抗体等の、修飾および／または変化した抗体で有り得る。また、本発明の抗体もしくはその断片、またはそれらの組み合わせを認識する抗体は、モノクローナルまたはポリクローナル抗体、修飾または変化したモノクローナルまたはポリクローナル抗体、または組換えもしくは合成的に産生したまたは合成した抗体であり得る。
In the present invention, "an antibody or fragment thereof of the present invention, or a combination thereof, or an antibody or fragment thereof, or a combination thereof that recognizes the antibody of the present invention or a fragment thereof, or a combination thereof" specifically binds to the antibody of the present invention, a fragment thereof, or a combination thereof. obtained antibodies or fragments thereof, or combinations thereof. From the viewpoint of accurate, convenient, and rapid detection of SARS-CoV-2-derived NPs, the antibodies of the present invention, fragments thereof, or antibodies that recognize a combination thereof consist of IgG, IgM, IgA, IgD, and IgE. It is preferably one or more selected from the group, particularly preferably IgG and/or IgM, more preferably IgG. Antibodies that recognize the antibodies of the invention or fragments thereof, or combinations thereof, can also be modified and/or altered antibodies, such as chimeric and humanized antibodies. Antibodies that recognize the antibodies of the present invention or fragments thereof, or combinations thereof, may also be monoclonal or polyclonal antibodies, modified or altered monoclonal or polyclonal antibodies, or recombinantly or synthetically produced or synthesized antibodies. .
本発明の抗体もしくはその断片、またはそれらの組み合わせを認識する抗体の断片は、ＦａｂフラグメントまたはＶＬ－、ＶＨ－またはＣＤＲ－領域等の、かかる免疫グロブリン分子の部分であり得るが、本発明の抗体もしくはその断片、またはそれらの組み合わせを認識する抗体の断片も、本発明の抗体もしくはその断片、またはそれらの組み合わせを認識する抗体と同程度に本発明の抗体もしくはその断片、またはそれらの組み合わせを特異的に認識し結合し得るものである。本発明の抗体もしくはその断片、またはそれらの組み合わせを認識する抗体の断片は、抗体フラグメント、ならびに、分離した軽鎖および重鎖、Ｆａｂ、Ｆａｂ／ｃ、Ｆｖ、Ｆａｂ’、Ｆ（ａｂ’）２等の、それらの部分であり得る。一態様において、本発明の抗体もしくはその断片、またはそれらの組み合わせを認識する抗体の断片は、Ｆａｂフラグメント、Ｆａｂ／ｃフラグメント、Ｆｖフラグメント、Ｆａｂ’フラグメントまたはＦ（ａｂ’）２フラグメントである。
An antibody of the invention or a fragment thereof, or a fragment of an antibody that recognizes a combination thereof, can be a Fab fragment or part of such immunoglobulin molecules, such as the VL-, VH- or CDR-regions, although the antibody of the invention Or a fragment thereof, or a fragment of an antibody that recognizes a combination thereof, is also as specific for an antibody of the invention, or a fragment thereof, or a combination thereof, as an antibody that recognizes an antibody of the invention, or a fragment thereof, or a combination thereof. can be recognized and bound to each other. Fragments of antibodies that recognize antibodies or fragments thereof, or combinations thereof, of the present invention include antibody fragments and separate light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab') 2 and so on. In one aspect, a fragment of an antibody that recognizes an antibody of the invention or a fragment thereof, or a combination thereof, is a Fab, Fab/c, Fv, Fab' or F(ab') 2 fragment.
また、本発明の抗体もしくはその断片、またはそれらの組み合わせを認識する抗体もしくはその断片、またはそれらの組み合わせは、二機能性抗体等の抗体派生物、または、単鎖Ｆｖ（ｓｃＦｖ）、二重特異性ｓｃＦｖまたは抗体融合タンパク質等の抗体構築物であり得る。
In addition, the antibody or fragment thereof of the present invention, or an antibody or fragment thereof, or a combination thereof that recognizes a combination thereof may be an antibody derivative such as a bifunctional antibody, or a single-chain Fv (scFv), bispecific It may be an antibody construct such as a sex scFv or an antibody fusion protein.
本発明において、ＳＡＲＳ－ＣｏＶ－２由来ＮＰは、本発明の抗体もしくはその断片、またはそれらの組み合わせとＳＡＲＳ－ＣｏＶ－２由来ＮＰとの抗原抗体反応を検出できる任意の免疫学検査によって検出される。免疫学検査は、例えば、酵素免疫測定法（ＥＬＩＳＡ、ＥＩＡ）、化学発光酵素免疫測定法（ＣＬＥＩＡ）、化学発光免疫測定法（ＣＬＩＡ）、電気化学発光免疫測定法（ＥＣＬＩＡ）、蛍光免疫測定法（ＦＩＡ）、放射免疫測定法（ＲＩＡ）、発光免疫測定法（ＬＩＡ）、酵素抗体法、蛍光抗体法、イムノクロマトグラフィー法（イムノクロマト法）、フィルター抗原アッセイ法、免疫比濁法、ラテックス比濁法、ラテックス凝集反応測定法、赤血球凝集反応法、または粒子凝集反応法等を含む。正確、簡便、かつ迅速にＳＡＲＳ－ＣｏＶ－２由来ＮＰを検出する観点から、ＳＡＲＳ－ＣｏＶ－２由来ＮＰは、ＥＬＩＳＡ法、ＣＬＥＩＡ法および／またはイムノクロマト法によって検出されることが好ましい。また、所定量の本発明の抗体もしくはその断片、またはそれらの組み合わせとＳＡＲＳ－ＣｏＶ－２由来ＮＰとの抗原抗体反応から検量線を作成し、測定値を検量線に内挿することによって試料中に含まれるＳＡＲＳ－ＣｏＶ－２由来ＮＰを定量することもできる。
In the present invention, SARS-CoV-2-derived NPs are detected by any immunological test that can detect an antigen-antibody reaction between the antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NPs. . Immunological tests, for example, enzyme immunoassay (ELISA, EIA), chemiluminescence enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescence immunoassay (LIA), enzyme antibody method, fluorescent antibody method, immunochromatographic method (immunochromatographic method), filter antigen assay method, immunoturbidimetric method, latex turbidimetric method , latex agglutination assays, hemagglutination assays, or particle agglutination assays. From the viewpoint of accurate, convenient, and rapid detection of SARS-CoV-2-derived NPs, SARS-CoV-2-derived NPs are preferably detected by ELISA, CLEIA and/or immunochromatography. In addition, a standard curve is prepared from the antigen-antibody reaction between a predetermined amount of the antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NP, and the measured values are interpolated into the standard curve. SARS-CoV-2-derived NPs contained in can also be quantified.
［ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出するためのキット］
本発明の別の側面は、本発明の抗体もしくはその断片、またはそれらの組み合わせを含む、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出するためのキット（以下、「本発明のキット」と記す場合がある）に関する。正確、簡便、かつ迅速にＳＡＲＳ－ＣｏＶ－２由来ＮＰを検出する観点、医療従事者が感染現場、防疫現場および臨床現場等の設備が十分とはいえない場所でＳＡＲＳ－ＣｏＶ－２由来ＮＰを検出する観点、疫学調査をする観点等から、本発明のキットは、イムノクロマトストリップの形態であることが好ましい。 [Kit for detecting proteins constituting SARS-CoV-2-derived nucleocapsid]
Another aspect of the present invention is a kit for detecting a protein that constitutes a SARS-CoV-2-derived nucleocapsid, comprising the antibody of the present invention, a fragment thereof, or a combination thereof (hereinafter referred to as "the kit of the present invention") may be noted). From the viewpoint of accurate, simple, and rapid detection of SARS-CoV-2-derived NPs, medical personnel can detect SARS-CoV-2-derived NPs in places where facilities such as infection sites, epidemic prevention sites, and clinical sites are not sufficient. From the viewpoint of detection, epidemiological investigation, etc., the kit of the present invention is preferably in the form of an immunochromatographic strip.
本発明の別の側面は、本発明の抗体もしくはその断片、またはそれらの組み合わせを含む、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出するためのキット（以下、「本発明のキット」と記す場合がある）に関する。正確、簡便、かつ迅速にＳＡＲＳ－ＣｏＶ－２由来ＮＰを検出する観点、医療従事者が感染現場、防疫現場および臨床現場等の設備が十分とはいえない場所でＳＡＲＳ－ＣｏＶ－２由来ＮＰを検出する観点、疫学調査をする観点等から、本発明のキットは、イムノクロマトストリップの形態であることが好ましい。 [Kit for detecting proteins constituting SARS-CoV-2-derived nucleocapsid]
Another aspect of the present invention is a kit for detecting a protein that constitutes a SARS-CoV-2-derived nucleocapsid, comprising the antibody of the present invention, a fragment thereof, or a combination thereof (hereinafter referred to as "the kit of the present invention") may be noted). From the viewpoint of accurate, simple, and rapid detection of SARS-CoV-2-derived NPs, medical personnel can detect SARS-CoV-2-derived NPs in places where facilities such as infection sites, epidemic prevention sites, and clinical sites are not sufficient. From the viewpoint of detection, epidemiological investigation, etc., the kit of the present invention is preferably in the form of an immunochromatographic strip.
例えば、イムノクロマトストリップは、本発明の抗体もしくはその断片、またはそれらの組み合わせおよび抗マウス免疫グロブリン抗体をそれぞれ別の部位、すなわちテストラインおよびコントロールラインに固定化したものである。イムノクロマトストリップのサンプルパットには、金コロイド粒子、ラテックス粒子、蛍光粒子、磁気粒子等で標識された本発明の抗体もしくはその断片、またはそれらの組み合わせを予め含浸させておく。サンプルパットに滴下された試料は、毛細管現象により展開され、テストラインに到達すると試料中のＳＡＲＳ－ＣｏＶ－２由来ＮＰのみが反応し、コントロールラインに到達すると金コロイド等で標識された本発明の抗体もしくはその断片、またはそれらの組み合わせのみが反応する。その他の試料中の成分は反応せずに吸水パッドまで移動する。テストラインおよびコントロールラインの色の濃さ、反射強度、吸光度、蛍光強度、または磁気強度等をイムノクロマトリーダーで測定することにより、試料中のＳＡＲＳ－ＣｏＶ－２由来ＮＰを検出できるだけでなく、試料中のＳＡＲＳ－ＣｏＶ－２由来ＮＰを定量できる。
For example, an immunochromatographic strip is obtained by immobilizing the antibody of the present invention or a fragment thereof, or a combination thereof, and an anti-mouse immunoglobulin antibody at separate sites, that is, a test line and a control line. A sample pad of an immunochromatographic strip is pre-impregnated with the antibody of the present invention or a fragment thereof labeled with colloidal gold particles, latex particles, fluorescent particles, magnetic particles, or the like, or a combination thereof. The sample dropped onto the sample pad is developed by capillary action, and when it reaches the test line, only the SARS-CoV-2-derived NPs in the sample react, and when it reaches the control line, it is labeled with colloidal gold or the like. Only antibodies or fragments thereof, or combinations thereof react. Other components in the sample migrate to the absorbent pad without reacting. By measuring the color depth, reflection intensity, absorbance, fluorescence intensity, or magnetic intensity of the test line and control line with an immunochromatographic reader, not only can SARS-CoV-2-derived NPs in the sample be detected, but also of SARS-CoV-2-derived NPs can be quantified.
［ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出するための組成物］
本発明の別の側面は、本発明の抗体もしくはその断片、またはそれらの組み合わせを含む、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出するための組成物（以下、「本発明の組成物」と記す場合がある）に関する。 [Composition for detecting proteins constituting SARS-CoV-2-derived nucleocapsid]
Another aspect of the present invention is a composition for detecting a protein that constitutes a SARS-CoV-2-derived nucleocapsid, comprising the antibody of the present invention, a fragment thereof, or a combination thereof (hereinafter referred to as "composition of the present invention ”).
本発明の別の側面は、本発明の抗体もしくはその断片、またはそれらの組み合わせを含む、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出するための組成物（以下、「本発明の組成物」と記す場合がある）に関する。 [Composition for detecting proteins constituting SARS-CoV-2-derived nucleocapsid]
Another aspect of the present invention is a composition for detecting a protein that constitutes a SARS-CoV-2-derived nucleocapsid, comprising the antibody of the present invention, a fragment thereof, or a combination thereof (hereinafter referred to as "composition of the present invention ”).
［ＳＡＲＳ－ＣｏＶ－２感染の診断方法］
本発明の別の側面は、試料を本発明の抗体もしくはその断片、またはそれらの組み合わせと接触させる工程を含む、ＳＡＲＳ－ＣｏＶ－２感染の診断方法（以下、「本発明の診断方法」と記す場合がある）に関する。一態様において、本発明の診断方法は、ＥＬＩＳＡ法、ＣＬＥＩＡ法、イムノクロマト法およびフィルター抗原アッセイ法からなる群から選択される少なくとも１つの免疫学検査によりＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出する工程をさらに含む。 [Method for diagnosing SARS-CoV-2 infection]
Another aspect of the present invention is a method for diagnosing SARS-CoV-2 infection comprising the step of contacting a sample with an antibody or fragment thereof of the present invention, or a combination thereof (hereinafter referred to as "diagnostic method of the present invention"). (as may be the case). In one aspect, the diagnostic method of the present invention identifies proteins that make up the SARS-CoV-2-derived nucleocapsid by at least one immunological test selected from the group consisting of ELISA, CLEIA, immunochromatography, and filter antigen assay. Further comprising the step of detecting.
本発明の別の側面は、試料を本発明の抗体もしくはその断片、またはそれらの組み合わせと接触させる工程を含む、ＳＡＲＳ－ＣｏＶ－２感染の診断方法（以下、「本発明の診断方法」と記す場合がある）に関する。一態様において、本発明の診断方法は、ＥＬＩＳＡ法、ＣＬＥＩＡ法、イムノクロマト法およびフィルター抗原アッセイ法からなる群から選択される少なくとも１つの免疫学検査によりＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出する工程をさらに含む。 [Method for diagnosing SARS-CoV-2 infection]
Another aspect of the present invention is a method for diagnosing SARS-CoV-2 infection comprising the step of contacting a sample with an antibody or fragment thereof of the present invention, or a combination thereof (hereinafter referred to as "diagnostic method of the present invention"). (as may be the case). In one aspect, the diagnostic method of the present invention identifies proteins that make up the SARS-CoV-2-derived nucleocapsid by at least one immunological test selected from the group consisting of ELISA, CLEIA, immunochromatography, and filter antigen assay. Further comprising the step of detecting.
一態様において、本発明の診断方法は、本発明の抗体もしくはその断片、またはそれらの組み合わせとＳＡＲＳ－ＣｏＶ－２由来ＮＰとの間で抗原抗体反応を行う工程を含む。一態様において、本発明の診断方法は、本発明の抗体もしくはその断片、またはそれらの組み合わせとＳＡＲＳ－ＣｏＶ－２由来ＮＰとの間で抗原抗体反応を行う工程を複数回含む。一態様において、該工程の各回毎に本発明の抗体もしくはその断片、またはそれらの組み合わせはエピトープが異なる。一態様において、本発明の抗体もしくはその断片、またはそれらの組み合わせは、標識物質により標識されている。例えば、本発明の診断方法は、無標識の本発明の抗体もしくはその断片、またはそれらの組み合わせとＳＡＲＳ－ＣｏＶ－２由来ＮＰとの間で抗原抗体反応を行う工程、および標識物質により標識されている本発明の抗体もしくはその断片、またはそれらの組み合わせ（前記無標識の本発明の抗体もしくはその断片、またはそれらの組み合わせとはエピトープが異なる）とＳＡＲＳ－ＣｏＶ－２由来ＮＰとの間で抗原抗体反応を行う工程を含む。
In one aspect, the diagnostic method of the present invention comprises the step of conducting an antigen-antibody reaction between an antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NPs. In one aspect, the diagnostic method of the present invention comprises multiple steps of conducting an antigen-antibody reaction between an antibody of the present invention or a fragment thereof, or a combination thereof, and SARS-CoV-2-derived NPs. In one embodiment, the antibodies of the invention or fragments thereof, or combinations thereof, differ in epitope each time through the steps. In one aspect, an antibody or fragment thereof, or combination thereof, of the present invention is labeled with a labeling substance. For example, the diagnostic method of the present invention includes a step of performing an antigen-antibody reaction between the unlabeled antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NPs, and labeled with a labeling substance. Antigen antibody between the antibody of the present invention or a fragment thereof, or a combination thereof (the epitope is different from the unlabeled antibody of the present invention or a fragment thereof, or a combination thereof) and the SARS-CoV-2-derived NP including the step of conducting the reaction.
一態様において、本発明の診断方法は、本発明の抗体もしくはその断片、またはそれらの組み合わせとＳＡＲＳ－ＣｏＶ－２由来ＮＰとの間で抗原抗体反応を行う工程、および／または、本発明の抗体もしくはその断片、またはそれらの組み合わせと該抗体もしくはその断片、またはそれらの組み合わせを認識する抗体もしくはその断片、またはそれらの組み合わせとの間で抗原抗体反応を行う工程をさらに含む。一態様において、本発明の抗体もしくはその断片、またはそれらの組み合わせ、および／または該抗体もしくはその断片、またはそれらの組み合わせを認識する抗体もしくはその断片、またはそれらの組み合わせは、標識物質により標識されている。
In one aspect, the diagnostic method of the present invention includes the step of conducting an antigen-antibody reaction between the antibody of the present invention or a fragment thereof, or a combination thereof and SARS-CoV-2-derived NPs, and/or the antibody of the present invention. or a fragment thereof, or a combination thereof, and an antibody or fragment thereof, or a combination thereof, which recognizes the antibody, a fragment thereof, or a combination thereof, and a step of conducting an antigen-antibody reaction. In one aspect, the antibody of the present invention or a fragment thereof, or a combination thereof, and/or an antibody or a fragment thereof, or a combination thereof that recognizes the antibody or a fragment thereof, or a combination thereof, is labeled with a labeling substance. there is
例１ ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片の調製
［免疫原の設計］
データベース上に公開された配列を元に、ＳＡＲＳ－ＣｏＶ－２のＮタンパク質のアミノ酸配列について、既存のヒトコロナウイルス（ＳＡＲＳ、ＭＥＲＳ、ＨＫＵ－１、ＯＣ４３、ＮＬ６３、２２９Ｅ）のＮタンパク質と相同性・類似性解析を行った（図１Ｂ）。その結果、ＳＡＲＳ－ＣｏＶ－２－ＮＰはＳＡＲＳと約９０％のアミノ酸配列が相同であり、ＭＥＲＳとは約４７％、その他の弱毒性ヒトコロナウイルスとは相同性が約３０％以下であることが分かった。そこで、ヒトコロナウイルス間で共通して保存されているモチーフ（ＦＹＹＬＧＴＧＰ）を除いた、Ｎタンパク質中のアミノ酸１２１～４１９の領域を特定した。
今回、その領域とは異なる部分を認識する抗体を得るためにＮ末端側の１～２０７の領域を免疫原として用いることとした（図１Ａ）。 Example 1 Preparation of fragment of NP derived from SARS-CoV-2 [design of immunogen]
Based on the sequence published on the database, the amino acid sequence of the N protein of SARS-CoV-2 is homologous to the N protein of existing human coronaviruses (SARS, MERS, HKU-1, OC43, NL63, 229E) • A similarity analysis was performed (Fig. 1B). As a result, SARS-CoV-2-NP has about 90% amino acid sequence homology with SARS, about 47% with MERS, and about 30% or less homology with other attenuated human coronaviruses. I found out. Therefore, the region of amino acids 121 to 419 in the N protein was identified, excluding the motif (FYYLGTGP) commonly conserved among human coronaviruses.
In this study, we decided to use the N-terminal 1-207 region as an immunogen in order to obtain an antibody that recognizes a region different from that region (Fig. 1A).
［免疫原の設計］
データベース上に公開された配列を元に、ＳＡＲＳ－ＣｏＶ－２のＮタンパク質のアミノ酸配列について、既存のヒトコロナウイルス（ＳＡＲＳ、ＭＥＲＳ、ＨＫＵ－１、ＯＣ４３、ＮＬ６３、２２９Ｅ）のＮタンパク質と相同性・類似性解析を行った（図１Ｂ）。その結果、ＳＡＲＳ－ＣｏＶ－２－ＮＰはＳＡＲＳと約９０％のアミノ酸配列が相同であり、ＭＥＲＳとは約４７％、その他の弱毒性ヒトコロナウイルスとは相同性が約３０％以下であることが分かった。そこで、ヒトコロナウイルス間で共通して保存されているモチーフ（ＦＹＹＬＧＴＧＰ）を除いた、Ｎタンパク質中のアミノ酸１２１～４１９の領域を特定した。
今回、その領域とは異なる部分を認識する抗体を得るためにＮ末端側の１～２０７の領域を免疫原として用いることとした（図１Ａ）。 Example 1 Preparation of fragment of NP derived from SARS-CoV-2 [design of immunogen]
Based on the sequence published on the database, the amino acid sequence of the N protein of SARS-CoV-2 is homologous to the N protein of existing human coronaviruses (SARS, MERS, HKU-1, OC43, NL63, 229E) • A similarity analysis was performed (Fig. 1B). As a result, SARS-CoV-2-NP has about 90% amino acid sequence homology with SARS, about 47% with MERS, and about 30% or less homology with other attenuated human coronaviruses. I found out. Therefore, the region of amino acids 121 to 419 in the N protein was identified, excluding the motif (FYYLGTGP) commonly conserved among human coronaviruses.
In this study, we decided to use the N-terminal 1-207 region as an immunogen in order to obtain an antibody that recognizes a region different from that region (Fig. 1A).
［ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片の合成］
配列番号１で表されるアミノ酸配列からなるＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片を以下の方法により調製した。なお、ＳＡＲＳ－ＣｏＶ－２由来ＮＰは配列番号２で表されるアミノ酸配列からなり、該アミノ酸配列に対応するＲＮＡ配列は配列番号３で表され、該ＲＮＡ配列に対応するＤＮＡ配列は配列番号４で表される。なお、該ＤＮＡ配列は、アミノ酸配列に基づき真核細胞でのタンパク質合成用にコドン最適化させている。 [Synthesis of fragment of NP derived from SARS-CoV-2]
A SARS-CoV-2-derived NP fragment consisting of the amino acid sequence represented by SEQ ID NO: 1 was prepared by the following method. The SARS-CoV-2-derived NP consists of the amino acid sequence represented by SEQ ID NO: 2, the RNA sequence corresponding to the amino acid sequence is represented by SEQ ID NO: 3, and the DNA sequence corresponding to the RNA sequence is SEQ ID NO: 4. is represented by The DNA sequence is codon-optimized for protein synthesis in eukaryotic cells based on the amino acid sequence.
配列番号１で表されるアミノ酸配列からなるＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片を以下の方法により調製した。なお、ＳＡＲＳ－ＣｏＶ－２由来ＮＰは配列番号２で表されるアミノ酸配列からなり、該アミノ酸配列に対応するＲＮＡ配列は配列番号３で表され、該ＲＮＡ配列に対応するＤＮＡ配列は配列番号４で表される。なお、該ＤＮＡ配列は、アミノ酸配列に基づき真核細胞でのタンパク質合成用にコドン最適化させている。 [Synthesis of fragment of NP derived from SARS-CoV-2]
A SARS-CoV-2-derived NP fragment consisting of the amino acid sequence represented by SEQ ID NO: 1 was prepared by the following method. The SARS-CoV-2-derived NP consists of the amino acid sequence represented by SEQ ID NO: 2, the RNA sequence corresponding to the amino acid sequence is represented by SEQ ID NO: 3, and the DNA sequence corresponding to the RNA sequence is SEQ ID NO: 4. is represented by The DNA sequence is codon-optimized for protein synthesis in eukaryotic cells based on the amino acid sequence.
まず、配列番号４で表されるＤＮＡ配列を人工合成し、該ＤＮＡ配列を鋳型とするＰＣＲ法により、配列番号１で表されるアミノ酸配列の１～２０７番目の領域（図１）に対応する配列番号４で表されるＤＮＡ配列の１～６２１番目の領域を増幅した。１μＭフォワードプライマー（配列番号５）５μＬ、１μＭリバースプライマー（配列番号６）５μＬ、配列番号４で表されるＤＮＡ配列が挿入された１０ｎｇ／μＬ ＤＮＡベクター（ｐＵＣ５７（ジーンウィズ社））１μＬ、ＰｒｉｍｅＳＴＡＲ（登録商標）Ｍａｘ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（タカラバイオ社）２５μＬ、および超純水１４μＬを混合して全量５０μＬの反応液を調製した。ＰＣＲ法を表１に示すサーマルサイクラーの設定で行った。これにより、配列番号４で表されるＤＮＡ配列の１～６２１番目の領域を含むＤＮＡ配列を得た。そして、ｐＥＵ－Ｅ０１―ＭＣＳ－ＴＥＶ－Ｈｉｓ－Ｃベクター（セルフリーサイエンス社）を鋳型とするＰＣＲ法により、配列番号７および８に示したプライマーを用いて同様な手法でＤＮＡベクターを増幅した。
First, the DNA sequence represented by SEQ ID NO: 4 was artificially synthesized, and by PCR using the DNA sequence as a template, the amino acid sequence represented by SEQ ID NO: 1 corresponded to the 1st to 207th regions (Fig. 1). A region from 1 to 621 of the DNA sequence represented by SEQ ID NO:4 was amplified. 5 μL of 1 μM forward primer (SEQ ID NO: 5), 5 μL of 1 μM reverse primer (SEQ ID NO: 6), 10 ng/μL DNA vector (pUC57 (Genewith)) into which the DNA sequence represented by SEQ ID NO: 4 was inserted, 1 μL, PrimeSTAR ( 25 μL of registered trademark Max DNA Polymerase (Takara Bio Inc.) and 14 μL of ultrapure water were mixed to prepare a reaction solution with a total volume of 50 μL. The PCR method was performed with the thermal cycler settings shown in Table 1. As a result, a DNA sequence containing the 1-621st region of the DNA sequence represented by SEQ ID NO:4 was obtained. Then, a DNA vector was amplified by PCR using pEU-E01-MCS-TEV-His-C vector (Cell Free Science) as a template, using the primers shown in SEQ ID NOS: 7 and 8 in the same manner.
次に、前記ＰＣＲ法から得られたＤＮＡ配列およびＤＮＡベクターをアガロースゲル電気泳動により精製した後、Ｉｎ－Ｆｕｓｉｏｎ（登録商標）ＨＤ Ｃｌｏｎｉｎｇ Ｋｉｔ（タカラバイオ社）を用いることで、ＤＮＡを連結させてＤＮＡベクターを調製した。得られたＤＮＡベクターを大腸菌ＪＭ１０９株に形質転換し、形質転換後の大腸菌をアンピシリン含有ＬＢ培地に播種した。増殖後の大腸菌からプラスミド精製キット（プロメガ社）を使用してＤＮＡベクターを調製した。
Next, after purifying the DNA sequence and DNA vector obtained from the PCR method by agarose gel electrophoresis, the DNA is ligated by using the In-Fusion (registered trademark) HD Cloning Kit (Takara Bio Inc.). A DNA vector was prepared. E. coli JM109 strain was transformed with the obtained DNA vector, and the transformed E. coli was seeded on ampicillin-containing LB medium. A DNA vector was prepared from the grown E. coli using a plasmid purification kit (Promega).
次に、得られたＤＮＡベクターからＳＰ６ＲＮＡポリメラーゼを用いてｍＲＮＡを転写し、セルフリーサイエンス社のＷＥＰＲＯ７２４０キットの使用説明書に従って、Ｈｉｓタグが付加されたＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片（配列番号１で表されるアミノ酸配列の１～２０７番目の領域（図１Ａ））を小麦胚芽抽出液により無細胞タンパク質合成した。
［ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片の精製］ Next, mRNA is transcribed from the resulting DNA vector using SP6 RNA polymerase, and a His-tagged SARS-CoV-2-derived NP fragment (SEQ ID NO: The 1-207th region of the amino acid sequence represented by 1 (FIG. 1A)) was subjected to cell-free protein synthesis using wheat germ extract.
[Purification of SARS-CoV-2-derived NP fragment]
［ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片の精製］ Next, mRNA is transcribed from the resulting DNA vector using SP6 RNA polymerase, and a His-tagged SARS-CoV-2-derived NP fragment (SEQ ID NO: The 1-207th region of the amino acid sequence represented by 1 (FIG. 1A)) was subjected to cell-free protein synthesis using wheat germ extract.
[Purification of SARS-CoV-2-derived NP fragment]
無細胞タンパク質合成の反応液からＮｉカラムを用いてＨｉｓタグが付加されたＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片を精製した。転写、無細胞タンパク質合成、および精製の一連の工程は、自動合成装置（セルフリーサイエンス社）を用いて行った。Ｈｉｓタグが付加されたＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片を３回にわたって精製し、１～３回目の精製画分をそれぞれＥ１～Ｅ３とした。Ｅ１～Ｅ３を２×ＳＤＳサンプル緩衝液（１２５ｍＭ Ｔｒｉｓ－ＨＣｌ、４％ＳＤＳ、２０％グリセロール、０．０１％ブロモフェノールブルー、および１０％２－メルカプトエタノール）と混合して熱処理し、ポリアクリルアミドゲルに供して電気泳動し、Ｒａｐｉｄ ＣＢＢ ＫＡＮＴＯ ３Ｓ（関東化学株式会社）によって染色した。
A His-tagged SARS-CoV-2-derived NP fragment was purified from the cell-free protein synthesis reaction solution using a Ni column. A series of steps of transcription, cell-free protein synthesis, and purification were performed using an automated synthesizer (Cell Free Science). The His-tagged SARS-CoV-2-derived NP fragment was purified three times, and the first to third purified fractions were labeled E1 to E3, respectively. E1-E3 were mixed with 2×SDS sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 0.01% bromophenol blue, and 10% 2-mercaptoethanol), heat-treated and run on a polyacrylamide gel. and subjected to electrophoresis and stained with Rapid CBB Kanto 3S (Kanto Kagaku Co., Ltd.).
その結果、精製前の溶液（Ｓ：可溶性画分）と比較してＥ１～Ｅ３では夾雑タンパク質の存在量が低減されたことから、ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片が精製されたことを確認することができた（図２）。
As a result, compared to the solution before purification (S: soluble fraction), the abundance of contaminating proteins was reduced in E1 to E3, confirming that the SARS-CoV-2-derived NP fragment was purified. (Fig. 2).
例２ ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片に対する抗体の作製
［マウスの免疫、ハイブリドーマ細胞の作製］
精製した３００μｇのＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片をフロイントアジュバントと混合して乳化させ、ＢＡＬＢ／ｃマウスに複数回皮下注射して免疫した。免疫したマウスから１か月後にＢ細胞を回収し、ポリエチレングリコール溶液を用いてＳＰ２／０ミエローマ細胞と融合した。融合したハイブリドーマ細胞を選択培地と共に９６ウェルマイクロプレートに播種した。その結果、Ｗ１～２４、Ｗ’１～５９、Ｅ１～３５のクローンを得た。 Example 2 Production of antibodies against fragments of SARS-CoV-2-derived NP [immunization of mice, production of hybridoma cells]
300 μg of purified SARS-CoV-2-derived NP fragments were emulsified by mixing with Freund's adjuvant and immunized by multiple subcutaneous injections into BALB/c mice. B cells were harvested from immunized mice one month later and fused with SP2/0 myeloma cells using a polyethylene glycol solution. Fused hybridoma cells were seeded in 96-well microplates with selective medium. As a result, clones W1-24, W'1-59 and E1-35 were obtained.
［マウスの免疫、ハイブリドーマ細胞の作製］
精製した３００μｇのＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片をフロイントアジュバントと混合して乳化させ、ＢＡＬＢ／ｃマウスに複数回皮下注射して免疫した。免疫したマウスから１か月後にＢ細胞を回収し、ポリエチレングリコール溶液を用いてＳＰ２／０ミエローマ細胞と融合した。融合したハイブリドーマ細胞を選択培地と共に９６ウェルマイクロプレートに播種した。その結果、Ｗ１～２４、Ｗ’１～５９、Ｅ１～３５のクローンを得た。 Example 2 Production of antibodies against fragments of SARS-CoV-2-derived NP [immunization of mice, production of hybridoma cells]
300 μg of purified SARS-CoV-2-derived NP fragments were emulsified by mixing with Freund's adjuvant and immunized by multiple subcutaneous injections into BALB/c mice. B cells were harvested from immunized mice one month later and fused with SP2/0 myeloma cells using a polyethylene glycol solution. Fused hybridoma cells were seeded in 96-well microplates with selective medium. As a result, clones W1-24, W'1-59 and E1-35 were obtained.
［ハイブリドーマ細胞のスクリーニングとクローニング］
次に、各ハイブリドーマが産生するモノクローナル抗体のＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片に対する反応性を、ＥＬＩＳＡ法、ＡｌｐｈａＳｃｒｅｅｎ法、染色法、および結合定数により調べ、ＮＰと反応する抗体を産生するハイブリドーマ細胞を選別した（図３、図４）。 [Screening and Cloning of Hybridoma Cells]
Next, the reactivity of monoclonal antibodies produced by each hybridoma to fragments of SARS-CoV-2-derived NP is examined by ELISA, AlphaScreen, staining, and binding constants, and hybridoma cells that produce antibodies that react with NP. were selected (Figs. 3 and 4).
次に、各ハイブリドーマが産生するモノクローナル抗体のＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片に対する反応性を、ＥＬＩＳＡ法、ＡｌｐｈａＳｃｒｅｅｎ法、染色法、および結合定数により調べ、ＮＰと反応する抗体を産生するハイブリドーマ細胞を選別した（図３、図４）。 [Screening and Cloning of Hybridoma Cells]
Next, the reactivity of monoclonal antibodies produced by each hybridoma to fragments of SARS-CoV-2-derived NP is examined by ELISA, AlphaScreen, staining, and binding constants, and hybridoma cells that produce antibodies that react with NP. were selected (Figs. 3 and 4).
ＥＬＩＳＡ法では、まず、ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片をＥＬＩＳＡプレート上に固定化した。プレートをブロッキングした後、プレートにハイブリドーマの培養上清を加えて反応させた後、ペルオキシダーゼ標識したヤギ抗マウス免疫グロブリン抗体を加えて反応させた。その後、ペルオキシダーゼ基質溶液を加えて発色させ、その吸光度を測定した。
In the ELISA method, a SARS-CoV-2-derived NP fragment was first immobilized on an ELISA plate. After blocking the plate, the hybridoma culture supernatant was added to the plate for reaction, and then peroxidase-labeled goat anti-mouse immunoglobulin antibody was added for reaction. Then, a peroxidase substrate solution was added to develop color, and the absorbance was measured.
ＡｌｐｈａＳｃｒｅｅｎ法はメーカー推奨法に従って実施した。まずＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片について、ビオチン標識したタンパク質を例１に記載の方法と同様な手順で無細胞タンパク質合成した。その後、合成したビオチン標識タンパク質とハイブリドーマの培養上清をプレートに添加して混合した後、室温で３０分間インキュベートした。そして、アクセプタービーズ（Ｐｒｏｔｅｉｎ Ｇコーティング）１０μｌおよびドナービーズ（ストレプトアビジンコーティング）を加えた。さらに３０分間、室温でインキュベートした後、ＥｎＶｉｓｉｏｎ（ＰｅｒｋｉｎＥｌｍｅｒ）にて測定を行った。
The AlphaScreen method was performed according to the manufacturer's recommended method. First, for the SARS-CoV-2-derived NP fragment, a biotin-labeled protein was synthesized in a cell-free manner in the same manner as described in Example 1. Thereafter, the synthesized biotin-labeled protein and the hybridoma culture supernatant were added to the plate, mixed, and then incubated at room temperature for 30 minutes. Then, 10 μl of acceptor beads (Protein G coating) and donor beads (streptavidin coating) were added. After an additional 30 min incubation at room temperature, measurements were taken on an EnVision (PerkinElmer).
染色法は、メーカー推奨法で実施した。具体的には、アフリカミドリザル腎由来細胞ＶｅｒｏＥ６／ＴＭＰＲＳＳ２（ＪＣＲＢ１８１９）に対し、国立感染症研究所から供与されたＳＡＲＳ－ＣｏＶ－２を、２４時間、３７℃で感染させた。次にハイブリドーマの培養上清を感染細胞の培養液に加えて反応させた後、蛍光標識したヤギ抗マウス免疫グロブリン抗体を加えて細胞を免疫染色した。その後、蛍光顕微鏡を用いて免疫染色した細胞をイメージングした。なお、非感染細胞をネガティブコントロールとした。
The staining method was carried out according to the manufacturer's recommended method. Specifically, African green monkey kidney-derived cells VeroE6/TMPRSS2 (JCRB1819) were infected with SARS-CoV-2 provided by the National Institute of Infectious Diseases for 24 hours at 37°C. Next, the culture supernatant of the hybridoma was added to the culture solution of the infected cells for reaction, and then the cells were immunostained by adding a fluorescently labeled goat anti-mouse immunoglobulin antibody. The immunostained cells were then imaged using a fluorescence microscope. Non-infected cells were used as a negative control.
結合定数の測定は、ＯｃｔｅｔＲＥＤ９６（ザルスタット）を用いたＢｉｏ－Ｌａｙｅｒ Ｉｎｔｅｒｆｅｒｏｍｅｔｒｙ（ＢＬＩ法）により、９６ウェル黒色マイクロプレート（Ｇｒｅｉｎｅｒ Ｂｉｏ－Ｏｎｅ）を用いて１，０００ｒｐｍで撹拌しながら３０℃の条件でメーカー推奨法で実施した。具体的にはＡｎｔｉ－ｍｏｕｓｅ ＩｇＧ Ｃａｐｔｕｒｅバイオセンサーチップ（ＡＭＣ）に０．１％ＢＳＡと０．０２％Ｔｗｅｅｎ２０を含むＰＢＳで５倍希釈したハイブリドーマの培養上清と５分間反応させた。そして、３００ｎＭのＮＰを用いて、結合時間３分間、解離時間５分間にて測定を行った。すべての測定値は、コントロールのウェルの値を差し引くことで、ベースラインドリフトを補正した。また、データは、ＦｏｒｔｅＢｉｏデータ解析ソフトウェアのローカルフィッティングアルゴリズムを用いた１：１結合モデルで解析した。
The binding constant was measured by Bio-Layer Interferometry (BLI method) using OctetRED96 (Sarstedt) using a 96-well black microplate (Greiner Bio-One) under the conditions of 30°C while stirring at 1,000 rpm. The recommended method was performed. Specifically, an anti-mouse IgG Capture biosensor chip (AMC) was reacted for 5 minutes with hybridoma culture supernatant diluted 5-fold with PBS containing 0.1% BSA and 0.02% Tween20. Measurement was then performed using 300 nM NP with a binding time of 3 minutes and a dissociation time of 5 minutes. All measurements were corrected for baseline drift by subtracting control well values. Data were also analyzed with a 1:1 binding model using the local fitting algorithm of the ForteBio data analysis software.
以上のスクリーニングで特定したハイブリドーマをクローニングすることにより、ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片に高い反応性を示すモノクローナル抗体を産生するハイブリドーマ細胞３７種を選択した。
By cloning the hybridomas identified by the above screening, 37 types of hybridoma cells that produce monoclonal antibodies that are highly reactive to the SARS-CoV-2-derived NP fragment were selected.
例３ 各モノクローナル抗体が認識するエピトープの解析
ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片のアミノ酸配列の各領域をそれぞれ欠損させた４種の組換えタンパク質（欠損変異体）を作製し、それらとの反応性を比較することで、各モノクローナル抗体が認識するエピトープを調べた。 Example 3 Analysis of Epitopes Recognized by Each Monoclonal Antibody Four types of recombinant proteins (deletion mutants) were prepared by deleting each region of the amino acid sequence of the SARS-CoV-2-derived NP fragment, and reacted with them. The epitopes recognized by each monoclonal antibody were examined by comparing the sexes.
ＳＡＲＳ－ＣｏＶ－２由来ＮＰの断片のアミノ酸配列の各領域をそれぞれ欠損させた４種の組換えタンパク質（欠損変異体）を作製し、それらとの反応性を比較することで、各モノクローナル抗体が認識するエピトープを調べた。 Example 3 Analysis of Epitopes Recognized by Each Monoclonal Antibody Four types of recombinant proteins (deletion mutants) were prepared by deleting each region of the amino acid sequence of the SARS-CoV-2-derived NP fragment, and reacted with them. The epitopes recognized by each monoclonal antibody were examined by comparing the sexes.
各組換えタンパク質を調製するため、まず例１で作製した配列番号１で表されるアミノ酸配列の１～２０７番目のアミノ酸配列をコードするコムギ無細胞系発現用ベクターｐＥＵ－Ｅ０１－ＴＥＶ－Ｈｉｓ－ＳＡＲＳ－ＣｏＶ－２－ＮＰ（１－２０７）を鋳型として、配列番号１で表されるアミノ酸配列の１～５０番目（Δ１－５０）、５１～１０５番目（Δ５１－１０５）、１０６～１６０番目（Δ１０６－１６０）、および１６１～２０７番目（Δ１６１－２０７）のアミノ酸配列領域をそれぞれコードするｃＤＮＡ断片を欠損させた発現ベクターをそれぞれＰｒｉｍｅＳＴＡＲ（登録商標）Ｍｕｔａｇｅｎｅｓｉｓ Ｂａｓａｌ Ｋｉｔ（タカラバイオ社）の方法に従って作製した。作製したＤＮＡのベクターからＳＰ６ＲＮＡポリメラーゼを用いてｍＲＮＡを合成し、セルフリーサイエンス社のＷＥＰＲＯ７２４０キットの使用方法に従って、各組換えタンパク質を例１に記載の方法と同様な手順で無細胞タンパク質合成した。
In order to prepare each recombinant protein, first, the wheat cell-free expression vector pEU-E01-TEV-His- encoding the 1st to 207th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 1 prepared in Example 1 was used. Using SARS-CoV-2-NP (1-207) as a template, 1st to 50th (Δ1-50), 51st to 105th (Δ51-105), 106th to 160th of the amino acid sequence represented by SEQ ID NO: 1 (Δ106-160) and 161st to 207th (Δ161-207) amino acid sequence regions, respectively, were deleted using expression vectors lacking cDNA fragments according to the method of PrimeSTAR (registered trademark) Mutagenesis Basal Kit (Takara Bio Inc.). made. mRNA was synthesized from the prepared DNA vector using SP6 RNA polymerase, and each recombinant protein was subjected to cell-free protein synthesis in the same manner as described in Example 1 according to the method for using the WEPRO7240 kit from Cell Free Science.
得られた各タンパク質を用いて、ＥＬＩＳＡ法によって各モノクローナル抗体のエピトープを解析した。まず、得られた各タンパク質をＥＬＩＳＡプレート上に固定化した。プレートをブロッキングした後、プレートにハイブリドーマの培養上清を加えて反応させた後、ペルオキシダーゼ標識したヤギ抗マウス免疫グロブリン抗体を加えて反応させた。その後、ペルオキシダーゼ基質溶液を加えて発色させ、その吸光度を測定した（図５）。
その結果、取得抗体は１－５０、５１付近、５１－１０５、１０６付近、１６１－２０７、５１－２０７の６種類に分類された（表２）。 Using each obtained protein, the epitope of each monoclonal antibody was analyzed by ELISA. First, each protein obtained was immobilized on an ELISA plate. After blocking the plate, the hybridoma culture supernatant was added to the plate for reaction, and then peroxidase-labeled goat anti-mouse immunoglobulin antibody was added for reaction. Then, a peroxidase substrate solution was added to develop color, and the absorbance was measured (Fig. 5).
As a result, the obtained antibodies were classified into six types: 1-50, around 51, 51-105, around 106, 161-207, and 51-207 (Table 2).
その結果、取得抗体は１－５０、５１付近、５１－１０５、１０６付近、１６１－２０７、５１－２０７の６種類に分類された（表２）。 Using each obtained protein, the epitope of each monoclonal antibody was analyzed by ELISA. First, each protein obtained was immobilized on an ELISA plate. After blocking the plate, the hybridoma culture supernatant was added to the plate for reaction, and then peroxidase-labeled goat anti-mouse immunoglobulin antibody was added for reaction. Then, a peroxidase substrate solution was added to develop color, and the absorbance was measured (Fig. 5).
As a result, the obtained antibodies were classified into six types: 1-50, around 51, 51-105, around 106, 161-207, and 51-207 (Table 2).
例４ 各モノクローナル抗体の、他のヒトコロナウイルス由来ＮＰに対する特異性の解析
ＳＡＲＳ－ＣｏＶ－２およびＳＡＲＳ、ＭＥＲＳのＮＰを合成し、それらに対する抗体の反応性をＥＬＩＳＡ法にて測定した。
まず、他のヒトコロナウイルス（ＳＡＲＳ、ＭＥＲＳ）に由来する各ＮＰをコードする各ＤＮＡ配列を合成した（ジーンウィズ社に委託）（それぞれ配列番号９および１０で表される）。なお、各ＤＮＡ配列は、アミノ酸配列に基づき真核細胞でのタンパク質合成用にコドン最適化させている。
次に、他のヒトコロナウイルス（ＳＡＲＳ、ＭＥＲＳ）に由来する各ＮＰを、それぞれ例１に記載の方法と同様な手順で無細胞タンパク質合成した。
得られた各タンパク質を用いて、例３に記載したＥＬＩＳＡ法と同様に各モノクローナル抗体の特異性を解析した。 Example 4 Analysis of Specificity of Each Monoclonal Antibody to Other Human Coronavirus-Derived NPs NPs of SARS-CoV-2, SARS, and MERS were synthesized, and the reactivity of the antibodies to them was measured by ELISA.
First, each DNA sequence encoding each NP derived from other human coronaviruses (SARS, MERS) was synthesized (entrusted to Genewiz) (represented by SEQ ID NOs: 9 and 10, respectively). Each DNA sequence is codon-optimized for protein synthesis in eukaryotic cells based on the amino acid sequence.
Next, each NP derived from other human coronaviruses (SARS, MERS) was subjected to cell-free protein synthesis by the same procedure as described in Example 1.
Using each protein obtained, the specificity of each monoclonal antibody was analyzed in the same manner as the ELISA method described in Example 3.
ＳＡＲＳ－ＣｏＶ－２およびＳＡＲＳ、ＭＥＲＳのＮＰを合成し、それらに対する抗体の反応性をＥＬＩＳＡ法にて測定した。
まず、他のヒトコロナウイルス（ＳＡＲＳ、ＭＥＲＳ）に由来する各ＮＰをコードする各ＤＮＡ配列を合成した（ジーンウィズ社に委託）（それぞれ配列番号９および１０で表される）。なお、各ＤＮＡ配列は、アミノ酸配列に基づき真核細胞でのタンパク質合成用にコドン最適化させている。
次に、他のヒトコロナウイルス（ＳＡＲＳ、ＭＥＲＳ）に由来する各ＮＰを、それぞれ例１に記載の方法と同様な手順で無細胞タンパク質合成した。
得られた各タンパク質を用いて、例３に記載したＥＬＩＳＡ法と同様に各モノクローナル抗体の特異性を解析した。 Example 4 Analysis of Specificity of Each Monoclonal Antibody to Other Human Coronavirus-Derived NPs NPs of SARS-CoV-2, SARS, and MERS were synthesized, and the reactivity of the antibodies to them was measured by ELISA.
First, each DNA sequence encoding each NP derived from other human coronaviruses (SARS, MERS) was synthesized (entrusted to Genewiz) (represented by SEQ ID NOs: 9 and 10, respectively). Each DNA sequence is codon-optimized for protein synthesis in eukaryotic cells based on the amino acid sequence.
Next, each NP derived from other human coronaviruses (SARS, MERS) was subjected to cell-free protein synthesis by the same procedure as described in Example 1.
Using each protein obtained, the specificity of each monoclonal antibody was analyzed in the same manner as the ELISA method described in Example 3.
その結果ＳＡＲＳおよびＭＥＲＳと反応せず、ＳＡＲＳ－ＣｏＶ－２を特異的に認識する抗体が計１２種類得られた（図６）。また、ＳＡＲＳとは反応するものの、ＭＥＲＳとは反応しない抗体が計２２種類得られた。
As a result, a total of 12 antibodies that specifically recognize SARS-CoV-2 without reacting with SARS and MERS were obtained (Fig. 6). In addition, a total of 22 types of antibodies that reacted with SARS but did not react with MERS were obtained.
例５ 各モノクローナル抗体またはそれらの組み合わせの反応性解析
本発明の抗体の反応性について調査するために、サンドイッチＥＬＩＳＡ法を用いた検討を行った。初めにハイブリドーマ培養上清から精製した抗体をプレートに固相化し、洗浄後、２％スキムミルクにてブロッキングを行った。次に洗浄後、コムギ無細胞系を用いて合成したＳＡＲＳ－ＣｏＶ－２のＮＰを２ｎｇ／ｍＬの濃度に調製し、プレートに添加した。洗浄後、ＨＲＰ標識を行った本発明の抗体を反応させた。その結果を図７に示す。その結果、これらの抗体のうち、Ｅ１５およびＥ１６では既存の抗体との組み合わせによって高い反応性を示した。 Example 5 Analysis of Reactivity of Each Monoclonal Antibody or Combination of Monoclonal Antibodies In order to examine the reactivity of the antibody of the present invention, a sandwich ELISA method was used. First, an antibody purified from a hybridoma culture supernatant was immobilized on a plate, washed, and then blocked with 2% skimmed milk. Next, after washing, SARS-CoV-2 NPs synthesized using a wheat cell-free system were adjusted to a concentration of 2 ng/mL and added to the plate. After washing, the HRP-labeled antibody of the present invention was allowed to react. The results are shown in FIG. As a result, among these antibodies, E15 and E16 showed high reactivity in combination with existing antibodies.
本発明の抗体の反応性について調査するために、サンドイッチＥＬＩＳＡ法を用いた検討を行った。初めにハイブリドーマ培養上清から精製した抗体をプレートに固相化し、洗浄後、２％スキムミルクにてブロッキングを行った。次に洗浄後、コムギ無細胞系を用いて合成したＳＡＲＳ－ＣｏＶ－２のＮＰを２ｎｇ／ｍＬの濃度に調製し、プレートに添加した。洗浄後、ＨＲＰ標識を行った本発明の抗体を反応させた。その結果を図７に示す。その結果、これらの抗体のうち、Ｅ１５およびＥ１６では既存の抗体との組み合わせによって高い反応性を示した。 Example 5 Analysis of Reactivity of Each Monoclonal Antibody or Combination of Monoclonal Antibodies In order to examine the reactivity of the antibody of the present invention, a sandwich ELISA method was used. First, an antibody purified from a hybridoma culture supernatant was immobilized on a plate, washed, and then blocked with 2% skimmed milk. Next, after washing, SARS-CoV-2 NPs synthesized using a wheat cell-free system were adjusted to a concentration of 2 ng/mL and added to the plate. After washing, the HRP-labeled antibody of the present invention was allowed to react. The results are shown in FIG. As a result, among these antibodies, E15 and E16 showed high reactivity in combination with existing antibodies.
次に反応性上位２０の組み合わせで検出限界について調査した。抗原の濃度を２ｎｇ／ｍＬから１２５ｐｇ／ｍＬまで２倍ずつ希釈し、サンドイッチＥＬＩＳＡ法を行った。その結果、抗原濃度１２５ｐｇ／ｍＬにおいても既存の抗体同士の組み合わせよりも高い反応性を示した。本発明の抗体同士の組み合わせでは固相化にＥ５を用い、ＨＲＰ標識したＥ１５を用いた場合に高い反応性を示した（表３）。
Next, we investigated the detection limit for the top 20 combinations of reactivity. Sandwich ELISA was performed by diluting the antigen concentration from 2 ng/mL to 125 pg/mL in 2-fold steps. As a result, even at an antigen concentration of 125 pg/mL, it showed higher reactivity than the combination of existing antibodies. A combination of the antibodies of the present invention showed high reactivity when E5 was used for immobilization and HRP-labeled E15 was used (Table 3).
例６ サンドイッチＥＬＩＳＡによる各変異株の培養ウイルス由来のＮＰの検出
各ＮＰ抗体を組み合わせたサンドイッチＥＬＩＳＡについて、Ｎ末端側を認識する抗体を用いることで、各種変異株ウイルス由来のＮＰの検出に差が生じるかどうか調べた。既存抗体として、（＃９×＃９８）の組み合わせをコントロールとして用いた。常法に従って抗体を固相化、洗浄および２％スキムミルクによりブロッキングした。そして、各種ウイルス培養液を１％ＮＰ４０／ＰＢＳを等量混合することで不活化し、その後、１０倍希釈したものを原液とし、５倍希釈系列を作製し、６０分間反応させた。その後、ＨＲＰ標識抗体と反応させて検出した。その結果、既存の抗体（＃９×＃９８）と比べて、Ｎ末端側に対する抗体を用いた場合では、各種変異株由来のＮＰに対して同等レベルの反応性を維持する組み合わせが確認された（図８）。 Example 6 Detection of NPs derived from cultured virus of each mutant strain by sandwich ELISA By using an antibody that recognizes the N-terminal side in sandwich ELISA using a combination of each NP antibody, NPs derived from various mutant viruses can be detected. We investigated whether there was a difference in detection. As an existing antibody, a combination of (#9×#98) was used as a control. Antibodies were immobilized, washed and blocked with 2% skimmed milk according to a conventional method. Then, each virus culture solution was inactivated by mixing equal amounts of 1% NP40/PBS, and then diluted 10-fold to prepare a 5-fold dilution series as a stock solution, which was allowed to react for 60 minutes. Then, it was reacted with an HRP-labeled antibody and detected. As a result, compared to the existing antibody (#9 × #98), when using an antibody against the N-terminal side, a combination that maintains an equivalent level of reactivity to NPs derived from various mutant strains was confirmed. (Fig. 8).
各ＮＰ抗体を組み合わせたサンドイッチＥＬＩＳＡについて、Ｎ末端側を認識する抗体を用いることで、各種変異株ウイルス由来のＮＰの検出に差が生じるかどうか調べた。既存抗体として、（＃９×＃９８）の組み合わせをコントロールとして用いた。常法に従って抗体を固相化、洗浄および２％スキムミルクによりブロッキングした。そして、各種ウイルス培養液を１％ＮＰ４０／ＰＢＳを等量混合することで不活化し、その後、１０倍希釈したものを原液とし、５倍希釈系列を作製し、６０分間反応させた。その後、ＨＲＰ標識抗体と反応させて検出した。その結果、既存の抗体（＃９×＃９８）と比べて、Ｎ末端側に対する抗体を用いた場合では、各種変異株由来のＮＰに対して同等レベルの反応性を維持する組み合わせが確認された（図８）。 Example 6 Detection of NPs derived from cultured virus of each mutant strain by sandwich ELISA By using an antibody that recognizes the N-terminal side in sandwich ELISA using a combination of each NP antibody, NPs derived from various mutant viruses can be detected. We investigated whether there was a difference in detection. As an existing antibody, a combination of (#9×#98) was used as a control. Antibodies were immobilized, washed and blocked with 2% skimmed milk according to a conventional method. Then, each virus culture solution was inactivated by mixing equal amounts of 1% NP40/PBS, and then diluted 10-fold to prepare a 5-fold dilution series as a stock solution, which was allowed to react for 60 minutes. Then, it was reacted with an HRP-labeled antibody and detected. As a result, compared to the existing antibody (#9 × #98), when using an antibody against the N-terminal side, a combination that maintains an equivalent level of reactivity to NPs derived from various mutant strains was confirmed. (Fig. 8).
例７ 唾液検体中におけるＮＰの分析
段階希釈した唾液検体に対して、全長のＮＰを混合して３７℃で３０分インキュベーションした後、ＳＤＳ－ＰＡＧＥとＣＢＢ染色により分析することで、ＮＰが分解されるかどうかを調べた（図９）。その結果、高濃度の唾液検体と混合したサンプルでは、ＮＰのバンドが消失しており唾液検体中においてＮＰは分解されると考えられた。また、唾液を１／５００、１／２５００倍に希釈した検体と混合したサンプルでは、全長のＮＰ（約５０ｋＤａ付近）と、分解途中産物と推定される、やや短い約４０ｋＤａ程度のバンドも認められた。同時に、唾液を９５℃で５分間加熱した後に使用したものでも試験したところ、加熱処理したサンプルでは、全長のＮＰのバンドは消失しなかった。次に、本サンプルについて各抗体を用いたウエスタンブロットで分析したところ、Ｃ末端側を認識する既存抗体＃６０や＃９８では、唾液の添加量依存的にＮＰが分解されるとバンドはほとんど検出できなかったのに対して、それ以外の抗体では、分解途中産物と推定される約４０ｋＤａ付近のバンドについても検出された（図１０）。したがって、ＮＰは唾液検体中においてＣ末端側から分解を受けると考えられた。 Example 7 Analysis of NPs in Saliva Samples Serially diluted saliva samples were mixed with full-length NPs, incubated at 37° C. for 30 minutes, and then analyzed by SDS-PAGE and CBB staining to degrade NPs. (Fig. 9). As a result, the NP band disappeared in the sample mixed with the high-concentration saliva sample, suggesting that NP was degraded in the saliva sample. In addition, in samples mixed with specimens obtained by diluting saliva to 1/500 and 1/2500, full-length NP (approximately 50 kDa) and a slightly shorter band of approximately 40 kDa, presumed to be intermediate decomposition products, were also observed. Ta. At the same time, when saliva used after heating at 95°C for 5 minutes was also tested, the full-length NP band did not disappear in the heat-treated sample. Next, when this sample was analyzed by Western blot using each antibody, with the existing antibodies #60 and #98 that recognize the C-terminal side, almost no band was detected when NP was degraded depending on the amount of saliva added. A band around 40 kDa, which is presumed to be a degradation product, was also detected with the other antibodies (FIG. 10). Therefore, NP was considered to be degraded from the C-terminal side in saliva samples.
段階希釈した唾液検体に対して、全長のＮＰを混合して３７℃で３０分インキュベーションした後、ＳＤＳ－ＰＡＧＥとＣＢＢ染色により分析することで、ＮＰが分解されるかどうかを調べた（図９）。その結果、高濃度の唾液検体と混合したサンプルでは、ＮＰのバンドが消失しており唾液検体中においてＮＰは分解されると考えられた。また、唾液を１／５００、１／２５００倍に希釈した検体と混合したサンプルでは、全長のＮＰ（約５０ｋＤａ付近）と、分解途中産物と推定される、やや短い約４０ｋＤａ程度のバンドも認められた。同時に、唾液を９５℃で５分間加熱した後に使用したものでも試験したところ、加熱処理したサンプルでは、全長のＮＰのバンドは消失しなかった。次に、本サンプルについて各抗体を用いたウエスタンブロットで分析したところ、Ｃ末端側を認識する既存抗体＃６０や＃９８では、唾液の添加量依存的にＮＰが分解されるとバンドはほとんど検出できなかったのに対して、それ以外の抗体では、分解途中産物と推定される約４０ｋＤａ付近のバンドについても検出された（図１０）。したがって、ＮＰは唾液検体中においてＣ末端側から分解を受けると考えられた。 Example 7 Analysis of NPs in Saliva Samples Serially diluted saliva samples were mixed with full-length NPs, incubated at 37° C. for 30 minutes, and then analyzed by SDS-PAGE and CBB staining to degrade NPs. (Fig. 9). As a result, the NP band disappeared in the sample mixed with the high-concentration saliva sample, suggesting that NP was degraded in the saliva sample. In addition, in samples mixed with specimens obtained by diluting saliva to 1/500 and 1/2500, full-length NP (approximately 50 kDa) and a slightly shorter band of approximately 40 kDa, presumed to be intermediate decomposition products, were also observed. Ta. At the same time, when saliva used after heating at 95°C for 5 minutes was also tested, the full-length NP band did not disappear in the heat-treated sample. Next, when this sample was analyzed by Western blot using each antibody, with the existing antibodies #60 and #98 that recognize the C-terminal side, almost no band was detected when NP was degraded depending on the amount of saliva added. A band around 40 kDa, which is presumed to be a degradation product, was also detected with the other antibodies (FIG. 10). Therefore, NP was considered to be degraded from the C-terminal side in saliva samples.
例８ 各組み合わせのサンドイッチＥＬＩＳＡによる唾液検体中のＮＰの検出
各ＮＰ抗体を組み合わせたサンドイッチＥＬＩＳＡについて、Ｎ末端側を認識する抗体を用いることで、唾液検体中での検出率に差が生じるかを調べた。既存抗体として、（＃９×＃９８）の組み合わせをコントロールとして用いた。常法に従って抗体を固相化、洗浄および２％スキムミルクによりブロッキングした。そして、唾液検体６種類をＰＢＳで１／５０、１／２５０、１／１２５０倍に希釈し、０．１％ＮＰ－４０を含むＰＢＳで１０ｎｇ／ｍＬとなるように希釈したＮＰと等量混合後、各ウェルに添加して３７℃で３０分間反応させた。その後、ＨＲＰ標識抗体と反応させて検出した。その結果、既存の抗体（＃９×＃９８）と比べて、Ｎ末端側に対する抗体を用いた場合では、唾液検体の添加による反応性の低下が改善された。カットオフをＯＤ４５０＝０．１とし、１／５０倍に希釈した唾液検体での検出率を比較したところ、既存の抗体の組み合わせの場合では、６検体中２検体のみが検出可能であった。一方で、既存抗体と新規抗体を組み合わせた場合（＃９×Ｅ１５）、反応性が向上し４検体の検出が可能となった。さらに新規抗体同士のＥ５×Ｅ１５では５検体、Ｗ１１×Ｅ５の組み合わせではすべての検体が検出可能であった（図１１）。 Example 8 Detection of NP in Saliva Specimens by Sandwich ELISA of Each Combination For sandwich ELISA in which each NP antibody is combined, whether or not there is a difference in the detection rate in saliva specimens by using an antibody that recognizes the N-terminal side. Examined. As an existing antibody, a combination of (#9×#98) was used as a control. Antibodies were immobilized, washed and blocked with 2% skimmed milk according to a conventional method. Then, six types of saliva specimens were diluted 1/50, 1/250, and 1/1250 times with PBS, and mixed with equal amounts of NP diluted to 10 ng/mL with PBS containing 0.1% NP-40. Then, it was added to each well and reacted at 37°C for 30 minutes. Then, it was reacted with an HRP-labeled antibody and detected. As a result, compared with the existing antibody (#9×#98), when the antibody against the N-terminal side was used, the decrease in reactivity due to the addition of the saliva sample was improved. A cutoff of OD450 = 0.1 was used to compare the detection rates of 1/50-fold diluted saliva specimens. In the case of the combination of existing antibodies, only 2 specimens out of 6 specimens were detectable. On the other hand, when the existing antibody and the new antibody were combined (#9×E15), the reactivity was improved and four specimens could be detected. Furthermore, 5 specimens were detectable with E5×E15 between the novel antibodies, and all specimens were detectable with the combination of W11×E5 (FIG. 11).
各ＮＰ抗体を組み合わせたサンドイッチＥＬＩＳＡについて、Ｎ末端側を認識する抗体を用いることで、唾液検体中での検出率に差が生じるかを調べた。既存抗体として、（＃９×＃９８）の組み合わせをコントロールとして用いた。常法に従って抗体を固相化、洗浄および２％スキムミルクによりブロッキングした。そして、唾液検体６種類をＰＢＳで１／５０、１／２５０、１／１２５０倍に希釈し、０．１％ＮＰ－４０を含むＰＢＳで１０ｎｇ／ｍＬとなるように希釈したＮＰと等量混合後、各ウェルに添加して３７℃で３０分間反応させた。その後、ＨＲＰ標識抗体と反応させて検出した。その結果、既存の抗体（＃９×＃９８）と比べて、Ｎ末端側に対する抗体を用いた場合では、唾液検体の添加による反応性の低下が改善された。カットオフをＯＤ４５０＝０．１とし、１／５０倍に希釈した唾液検体での検出率を比較したところ、既存の抗体の組み合わせの場合では、６検体中２検体のみが検出可能であった。一方で、既存抗体と新規抗体を組み合わせた場合（＃９×Ｅ１５）、反応性が向上し４検体の検出が可能となった。さらに新規抗体同士のＥ５×Ｅ１５では５検体、Ｗ１１×Ｅ５の組み合わせではすべての検体が検出可能であった（図１１）。 Example 8 Detection of NP in Saliva Specimens by Sandwich ELISA of Each Combination For sandwich ELISA in which each NP antibody is combined, whether or not there is a difference in the detection rate in saliva specimens by using an antibody that recognizes the N-terminal side. Examined. As an existing antibody, a combination of (#9×#98) was used as a control. Antibodies were immobilized, washed and blocked with 2% skimmed milk according to a conventional method. Then, six types of saliva specimens were diluted 1/50, 1/250, and 1/1250 times with PBS, and mixed with equal amounts of NP diluted to 10 ng/mL with PBS containing 0.1% NP-40. Then, it was added to each well and reacted at 37°C for 30 minutes. Then, it was reacted with an HRP-labeled antibody and detected. As a result, compared with the existing antibody (#9×#98), when the antibody against the N-terminal side was used, the decrease in reactivity due to the addition of the saliva sample was improved. A cutoff of OD450 = 0.1 was used to compare the detection rates of 1/50-fold diluted saliva specimens. In the case of the combination of existing antibodies, only 2 specimens out of 6 specimens were detectable. On the other hand, when the existing antibody and the new antibody were combined (#9×E15), the reactivity was improved and four specimens could be detected. Furthermore, 5 specimens were detectable with E5×E15 between the novel antibodies, and all specimens were detectable with the combination of W11×E5 (FIG. 11).
Claims (12)
- ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質に結合する抗体もしくはその断片、またはそれらの組み合わせであって、該抗体もしくはその断片、またはそれらの組み合わせの認識するエピトープが、配列番号１で表されるアミノ酸配列の１～２０７番目にある、前記抗体もしくはその断片、またはそれらの組み合わせ。 An antibody, a fragment thereof, or a combination thereof that binds to a protein that constitutes a SARS-CoV-2-derived nucleocapsid, wherein the epitope recognized by the antibody, a fragment thereof, or a combination thereof is represented by SEQ ID NO: 1 The antibody or fragment thereof, or a combination thereof, which is located at positions 1-207 of the amino acid sequence.
- エピトープが、配列番号１で表されるアミノ酸配列の１～５０番目、５１～１０５番目、１０６～１６０番目、または１６１～２０７番目にある、請求項１に記載の抗体もしくはその断片、またはそれらの組み合わせ。 The antibody or fragment thereof according to claim 1, wherein the epitope is located at positions 1 to 50, 51 to 105, 106 to 160, or 161 to 207 of the amino acid sequence represented by SEQ ID NO: 1, or combination.
- エピトープが、配列番号１で表されるアミノ酸配列の５１～１０５番目にある、請求項１に記載の抗体またはその断片と、１０６～１６０番目にある、請求項１または２に記載の抗体またはその断片の組み合わせ。 The antibody or fragment thereof according to claim 1, wherein the epitope is located at positions 51-105 of the amino acid sequence represented by SEQ ID NO: 1, and the antibody according to claim 1 or 2, which is located at positions 106-160, or its combination of fragments.
- ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質に特異的に結合する、請求項１～３のいずれか一項に記載の抗体もしくはその断片、またはそれらの組み合わせ。 The antibody or fragment thereof according to any one of claims 1 to 3, or a combination thereof, which specifically binds to a protein that constitutes a SARS-CoV-2-derived nucleocapsid.
- 抗体が、モノクローナル抗体である、請求項１～４のいずれか一項に記載の抗体もしくはその断片、またはそれらの組み合わせ。 The antibody or fragment thereof according to any one of claims 1 to 4, or a combination thereof, wherein the antibody is a monoclonal antibody.
- その断片が、Ｆａｂフラグメント、Ｆａｂ／ｃフラグメント、Ｆｖフラグメント、Ｆａｂ’フラグメントまたはＦ（ａｂ’）２フラグメントである、請求項１～５のいずれか一項に記載の抗体もしくはその断片、またはそれらの組み合わせ。 6. The antibody or fragment thereof of any one of claims 1 to 5, or a combination.
- ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を試料から検出する方法であって、試料を、請求項１～６のいずれか一項に記載の抗体もしくはその断片、またはそれらの組み合わせと接触させる工程を含む、前記方法。 A method for detecting a protein that constitutes a SARS-CoV-2-derived nucleocapsid from a sample, the step of contacting the sample with the antibody or fragment thereof according to any one of claims 1 to 6, or a combination thereof. The above method, comprising
- 試料が、生体試料である、請求項７に記載の方法。 The method according to claim 7, wherein the sample is a biological sample.
- 試料が、体液中の試料である、請求項７または８に記載の方法。 The method according to claim 7 or 8, wherein the sample is a sample in body fluid.
- ＥＬＩＳＡ法、ＣＬＥＩＡ法、イムノクロマト法およびフィルター抗原アッセイ法からなる群から選択される少なくとも１つの免疫学検査によりＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出する工程をさらに含む、請求項７～９のいずれか一項に記載の方法。 Further comprising the step of detecting proteins that make up the SARS-CoV-2-derived nucleocapsid by at least one immunological test selected from the group consisting of ELISA, CLEIA, immunochromatography and filter antigen assay, claims 7- 10. The method of any one of 9.
- 請求項１～６のいずれか一項に記載の抗体もしくはその断片、またはそれらの組み合わせを含む、ＳＡＲＳ－ＣｏＶ－２由来ヌクレオカプシドを構成するタンパク質を検出するためのキット。 A kit for detecting proteins constituting a SARS-CoV-2-derived nucleocapsid, comprising the antibody or fragment thereof according to any one of claims 1 to 6, or a combination thereof.
- イムノクロマトストリップの形態である、請求項１１に記載のキット。 The kit according to claim 11, which is in the form of an immunochromatographic strip.
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| JP2022033238A JP2023128698A (en) | 2022-03-04 | 2022-03-04 | Anti-sars-cov-2 antibody against sars-cov-2 antigen in body fluid, comprising mutant strain, method for detecting sars-cov-2 using the antibody, and kit comprising the antibody |
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