CN114790239B - Antibody for resisting coronavirus N protein and application thereof - Google Patents

Abstract

The invention discloses an antibody for resisting coronavirus N protein and application thereof, wherein the antibody comprises SEQ ID NO:2, HCDR2 of SEQ ID No. 3 and HCDR3 of SEQ ID No. 4, with LCDR1 of SEQ ID No. 10, LCDR2 of SEQ ID No. 11 and LCDR3 of SEQ ID No. 12; the antibodies have a higher affinity.

Description

Antibody for resisting coronavirus N protein and application thereof

Technical Field

The invention belongs to the fields of cell biotechnology and immunology, and relates to an antibody for resisting coronavirus N protein and application thereof.

Background

The pathogen laboratory detection comprises real-time fluorescence RT-PCR, virus gene sequencing and serum immunological detection, although RT-PCR and virus gene sequencing are the gold standard for diagnosis, the detection time is long, the laboratory biological safety requirement standard is high, and the like, and the serum immunological detection has certain clinical significance as an index for early disease screening due to short detection time and simple operation.

Disclosure of Invention

In order to remedy the deficiencies of the prior art, it is an object of the present invention to provide an antibody against the N protein of coronavirus which has a high activity of binding to the N protein.

In a first aspect, the present invention provides a monoclonal antibody against the N protein, the monoclonal antibody comprising:

SEQ ID NO:2, HCDR1 of SEQ id no:3 and HCDR2 of SEQ id no:4, and SEQ id no:10, LCDR1 of SEQ id no:11 and LCDR2 of SEQ id no: LCDR3 of 12.

Further, the monoclonal antibody further comprises a heavy chain variable region having a heavy chain variable region identical to SEQ id no: 5. 6, 7, 8, and HFR1, HFR2, HFR3, and HFR4, wherein the amino acid sequences are at least 90% identical; and has a sequence identical to SEQ id no: 13. 14, 15, 16, and light chain variable region framework regions LFR1, LFR2, LFR3, and LFR4 having at least 90% sequence identity to the amino acid sequences set forth in seq id no.

Further, the heavy chain variable region of the monoclonal antibody has a sequence identical to that of SEQ ID NO:9, and preferably 95%, and the light chain variable region has a VH with at least 90% sequence identity, preferably 95% sequence identity, to the amino acid sequence set forth in SEQ id no:17, and preferably at least 90% sequence identity, and preferably at least 95% sequence identity, to the VL.

Further, the VH of the monoclonal antibody has the amino acid sequence of SEQ id no: 9; the VL of the monoclonal antibody has SEQ id no: 17.

Further, the monoclonal antibody further comprises a heavy chain constant region and a light chain constant region.

A second aspect of the invention provides a substance according to any one of the following:

1) A nucleic acid molecule encoding the monoclonal antibody according to the first aspect of the invention or a functional fragment thereof;

2) A recombinant expression vector comprising the nucleic acid molecule of 1);

3) A host cell comprising the recombinant expression vector of 2);

4) A drug conjugate comprising a monoclonal antibody according to the first aspect of the invention or a functional fragment thereof;

5) A product for detecting N protein of coronavirus, said product comprising a monoclonal antibody according to the first aspect of the invention or a functional fragment thereof; or a nucleic acid molecule, recombinant expression vector, host cell, drug conjugate encoding the monoclonal antibody or functional fragment thereof according to the first aspect of the invention;

6) A composition comprising a monoclonal antibody according to the first aspect of the invention or a functional fragment thereof, or a nucleic acid molecule encoding a monoclonal antibody according to the first aspect of the invention or a functional fragment thereof, a recombinant expression vector, a host cell, a drug conjugate.

Further, the recombinant expression vector has a signal peptide operably linked to an antibody.

Further, the recombinant expression vector further comprises a transcription regulatory element.

Further, the product may further comprise a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction.

Further, the reagent for performing the antigen-antibody reaction includes a buffer, a salt, and the like.

Further, the drug conjugate further comprises a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, or enzyme.

A third aspect of the invention provides a pharmaceutical composition comprising a monoclonal antibody according to the first aspect of the invention or a substance according to the second aspect of the invention; and/or a pharmaceutically acceptable carrier.

A fourth aspect of the invention provides a method as defined in any one of the following:

1) A method of preparing a monoclonal antibody according to the first aspect of the invention, the method comprising: culturing the host cell of the second aspect of the invention, optionally isolating the antibody from the host cell and/or the medium in which the host cell is grown;

2) A method of detecting N protein in a sample, the method comprising: contacting a test sample with a monoclonal antibody according to the first aspect of the invention, and determining the presence or level of N protein in the test sample.

Further, the method of 1) further comprises purifying the monoclonal antibody.

Further, the host cell is selected from mammalian cells.

Further, the cell is selected from HEK293 cells.

A fourth aspect of the invention provides a use as claimed in any one of:

1) Use of a monoclonal antibody according to the first aspect of the invention, a substance according to the second aspect of the invention for detecting N protein;

2) Use of a monoclonal antibody according to the first aspect of the invention, or a substance according to the second aspect of the invention, in the manufacture of a product for detecting coronavirus infection;

3) Use of a monoclonal antibody according to the first aspect of the invention, or a substance according to the second aspect of the invention, in the preparation of a product for detecting a disease associated with a coronavirus infection;

4) Use of a monoclonal antibody according to the first aspect of the invention, a substance according to the second aspect of the invention or a pharmaceutical composition according to the third aspect of the invention for the manufacture of a medicament for the prevention and/or treatment of a disease associated with a coronavirus infection.

Further, the product comprises a kit.

Further, the kit comprises: colloidal gold immunoassay kit, chemiluminescence kit, radioimmunoassay kit, enzyme linked immunosorbent assay (ELISA), fluorescence immunoassay kit and microfluid chip.

Further, the coronavirus is selected from SARS-CoV-2, MERS-CoV, and SARS-CoV.

Further, the coronavirus is SARS-CoV-2.

Further, the disease related to coronavirus infection is COVID.

Further, the disease related to coronavirus infection is COVID-19.

Drawings

FIG. 1 is an electrophoretogram for detection of monoclonal antibody 8D 7;

FIG. 2 is an HPLC chart for detecting monoclonal antibody 8D 7;

FIG. 3 is a graph showing the binding activity of monoclonal antibody 8D7 detected by ELISA.

Detailed Description

Before the present methods are described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety.

The term "coronavirus" or "CoV" refers to any virus of the coronavirus family, including but not limited to SARS-CoV-2, MERS-CoV, and SARS-CoV. SARS-CoV-2 refers to a coronavirus that was identified as emerging. SARS-CoV-2 is also known as 2019-nCoV. It binds to the human host cell receptor angiotensin converting enzyme 2 (ACE 2) via the viral spike protein.

As used herein, the term "coronavirus infection" or "CoV infection" refers to infection of a coronavirus, such as SARS-CoV-2, MERS-CoV, or SARS-CoV. The term encompasses coronavirus respiratory infections, typically in the lower respiratory tract. Symptoms may include high fever, dry cough, shortness of breath, pneumonia, gastrointestinal symptoms (such as diarrhea), organ failure (renal failure and dysfunction), septic shock and death in severe cases.

As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a particular antigen. Natural intact antibodies comprise two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ and μ, each heavy chain consisting of a variable region (VH) and first, second, third and optionally fourth constant regions (CH 1, CH2, CH3, CH4, respectively); mammalian light chains are classified as λ or κ, whereas each light chain consists of a variable region (VL) and a constant region. The antibody is "Y" shaped, wherein the stem of Y consists of the second and third constant regions of two heavy chains that are joined together by disulfide bonds. Each arm of Y comprises the variable and first constant regions of a single heavy chain in combination with the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically contain three highly variable loops, called Complementarity Determining Regions (CDRs) (light chain CDRs include LCDR1, LCDR2, and LCDR3, and heavy chain CDRs include HCDR1, HCDR2, and HCDR 3). The CDR boundaries of the antibodies and antigen binding fragments disclosed in the present invention may be defined or identified by the convention of Kabat, IMGT, chothia or Al-Lazikani, with three CDRs inserted between flanking segments (stretch) called Framework Regions (FR) (light chain FR including LFR1, LFR2, LFR3 and LFR4, heavy chain FR including HFR1, HFR2, HFR3 and HFR 4) that are more highly conserved than CDRs and form a scaffold that supports highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of the constant region of the heavy chain of the antibody. The five major classes or isotypes of antibodies are large immunoglobulin a (IgA), igD, igE, igG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ 1 heavy chain), igG2 (γ 2 heavy chain), igG3 (γ 3 heavy chain), igG4 (γ 4 heavy chain), igA1 (α 1 heavy chain), or IgA2 (α 2 heavy chain).

In certain embodiments, the antibodies provided herein encompass any antigen binding fragment thereof. As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody that comprises one or more CDRs or any other antibody fragment that binds an antigen but does not comprise the entire native antibody structure. Examples of antigen binding fragments include, but are not limited to, diabodies, fabs, fab ', F (ab ') 2, fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv) 2, bispecific dsFv (dsFv-dsFv '), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (diabodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. The antigen binding fragment is capable of binding to the same antigen as the parent antibody.

The term "operably linked" refers to the joining of two or more biological sequences of interest, with or without a spacer or linker, such that they are in a relationship that allows them to function in the intended manner. When used with respect to a polypeptide, it means that the polypeptide sequences are linked in a manner that allows the linked product to have the intended biological function. The term may also be used with respect to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it means that the polynucleotide sequences are linked in a manner that allows for the regulation of expression of the polypeptide by the polynucleotide. In one embodiment, the operably linked nucleotide sequences are contiguous (e.g., in the case of a signal sequence). Alternatively, operably linked nucleotide sequences may not be contiguous (e.g., in the case of an enhancer).

Antibodies against coronavirus N protein

The invention provides a monoclonal antibody directed against the N protein, said antibody comprising SEQ id no:2, HCDR1 of SEQ id no:3 and HCDR2 of SEQ id no:4, and SEQ id no:10, LCDR1 of SEQ id no:11 and LCDR2 of SEQ id no: LCDR3 of 12.

In one embodiment, the monoclonal antibody further comprises a heavy chain variable region having a sequence identical to SEQ id no: 5. 6, 7, 8, and at least 90% sequence identity to the amino acid sequence of the heavy chain variable region framework regions HFR1, HFR2, HFR3, and HFR4; and has a sequence identical to SEQ id no: 13. 14, 15, 16, and LFR1, LFR2, LFR3, and LFR4, respectively, which are light chain variable region framework regions having at least 90% sequence identity to each other.

In yet another embodiment, the monoclonal antibody heavy chain variable region has a heavy chain variable region identical to SEQ id no:9, and preferably 95%, and the light chain variable region has a VH with at least 90% sequence identity, preferably 95% sequence identity, to the amino acid sequence set forth in SEQ id no:17, and preferably at least 90% sequence identity, preferably 95% sequence identity, to the amino acid sequence set forth in VL.

As a preferred embodiment, the VH of the monoclonal antibody has the amino acid sequence of SEQ id no: 9; the VL of the monoclonal antibody has an amino acid sequence shown in SEQ ID NO. 17.

In some embodiments, the antibodies and antigen binding fragments thereof provided herein comprise all or a portion of a heavy chain variable domain and/or all or a portion of a light chain variable domain. In one embodiment, the antibodies and antigen binding fragments thereof provided herein are single domain antibodies consisting of all or a portion of a heavy chain variable domain provided herein.

In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein further comprise an immunoglobulin (Ig) constant region, which optionally further comprises a heavy chain and/or light chain constant region. In certain embodiments, the heavy chain constant region comprises a CH1, hinge, and/or CH2-CH3 region (or optionally a CH2-CH3-CH4 region). In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain constant region of human IgG1, igG2, igG3, igG4, igA1, igA2, or IgM. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain constant region of human IgG 1. In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain constant region of human IgG 4. In certain embodiments, the light chain constant region comprises Ckappa (C κ) or Clambda (C λ). The constant regions of the antibodies and antigen-binding fragments thereof provided herein can be identical in sequence to, or differ from, the wild-type constant region by one or more mutations.

The antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein.

In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise one or more amino acid residue substitutions in one or more of the CDR sequences and/or in one or more of the FR sequences. In certain embodiments, the affinity variant comprises no more than 20, 15, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 substitutions in total in the CDR sequence and/or FR sequence.

In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise 1, 2, or 3 CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to a CDR sequence (or those) listed herein, but still retain specific binding to the coronavirus N protein at a level similar to or even higher than that of its parent antibody.

In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise one or more variable region sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to a variable region sequence (or sequences) listed herein, but still retain a level of specific binding affinity for the coronavirus N protein that is similar to, or even higher than, that of its parent antibody. In some embodiments, the mutation occurs in a region outside of the CDR (e.g., in the FR).

Recombinant expression vector

As used herein, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to cause expression of the protein. The vector may be used to transform, transduce or transfect a host cell so that it expresses the carried genetic element in the host cell. Examples of vectors include plasmids; phagemid; cosmids and artificial chromosomes, such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda or M13 bacteriophages; and animal viruses. Classes of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papovaviruses (e.g., SV 40). The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain an origin of replication. The term "origin of replication" refers to a sequence that, when present in a vector, initiates replication. The origin of replication may be recognized by the replication initiating factor or alternatively by a DNA helicase. The vector may also include materials that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein envelopes.

The vector may be a recombinant expression vector or a cloning vector. The invention provides vectors (e.g., expression vectors) comprising a nucleic acid sequence provided herein encoding an anti-N protein antibody, at least one promoter operably linked to the nucleic acid sequence, and/or at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papovaviruses (e.g., SV 40), lambda and M13 phages, plasmids, such as pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELET, pUNO, pUO, psg5L, pBABE, pWPXL, pBI, p15 TV-8978 zx8978, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, DNAPC 1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pORT 3, BOORF-VSP, etc.

A "recombinant expression vector" is a nucleic acid molecule encoding a gene that is expressed in a host cell and that, in addition, contains the necessary elements to control the expression of the gene. Typically, expression vectors comprise a transcription promoter, a gene of interest, and a transcription terminator.

In certain embodiments, the recombinant expression vector is a viral-based vector. In certain embodiments, the recombinant expression vector is a lentiviral vector. In certain embodiments, the recombinant expression vector is an adeno-associated virus (AAV) vector.

In certain embodiments, the nucleic acid sequence encoding the anti-N protein antibody or antigen-binding fragment thereof provided herein is codon optimized. As used herein, the term "codon-optimized" refers to the alteration of a nucleic acid sequence to enhance expression in a vertebrate (e.g., a human) of interest by replacing at least one, more than one, or a substantial number of codons of the native sequence with codons that are more frequently or most frequently used in the gene of the vertebrate, but which do not alter the amino acid sequence of the original translated protein. Various species exhibit specific preferences for certain codons for particular amino acids.

In certain embodiments, the nucleic acid sequence encoding the anti-N protein antibody is codon optimized. In certain embodiments, the nucleic acid sequence encoding the heavy chain variable region of the anti-N protein antibody is codon optimized. In certain embodiments, the nucleic acid sequence encoding the light chain variable region of the anti-N protein antibody is codon optimized. In certain embodiments, the nucleic acid sequence encoding the heavy chain constant region of the anti-N protein antibody is codon optimized. In certain embodiments, the nucleic acid sequence encoding the light chain constant region of the anti-N protein antibody is codon optimized.

Host cell

Vectors comprising polynucleotide sequences encoding the antibodies may be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors of the invention are prokaryotes, yeast or higher eukaryote cells. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example of the family Enterobacteriaceae (Enterobacteriaceae), such as the genus Escherichia (Escherichia), for example Escherichia coli; enterobacter (Enterobacter); erwinia (Erwinia); klebsiella (Klebsiella); proteus (Proteus); salmonella (Salmonella), such as Salmonella typhimurium (Salmonella typhimurium); serratia species (Serratia), such as Serratia marcescens (Serratia marcescens); and Shigella (Shigella), and bacillus (bacillus), such as bacillus subtilis and bacillus licheniformis (b.licheniformis); pseudomonas (Pseudomonas), such as Pseudomonas aeruginosa (p. Aeruginosa); and Streptomyces (Streptomyces).

In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for the expression of anti-coronavirus N proteins. Saccharomyces cerevisiae (Saccharomyces cerevisiae) or common baker's yeast is the most commonly used among lower eukaryotic host microorganisms. However, a variety of other genera, species and strains are generally available and suitable for use in the present invention, such as Schizosaccharomyces pombe (Schizosaccharomyces pombe); kluyveromyces (Kluyveromyces) hosts, such as Kluyveromyces lactis (K.lactis), kluyveromyces fragilis (K.fragilis) (ATCC 12,424), kluyveromyces bulgaricus (K.bulgaricus) (ATCC 16,045), kluyveromyces williamsii (K.wickeramii) (ATCC 24,178), kluyveromyces androgens (K.wallei) (ATCC 56,500), kluyveromyces drosophilus (K.drosophilarum) (ATCC 36,906), kluyveromyces thermotolerans (K.the yeast) and Kluyveromyces marxianus (K.marxianus); yarrowia (yarrowia) (EP 402,226); pichia pastoris (EP 183,070); candida genus (Candida); trichoderma reesei (Trichoderma reesei) (EP 244,234); neurospora crassa (Neurosporacrassa); schwanniomyces (Schwanniomyces), such as Schwanniomyces occidentalis (Schwanniomyces occidentalis); and filamentous fungi, such as Neurospora (Neurospora), penicillium (Penicillium), torticollis (Tolypocladium), and Aspergillus (Aspergillus) hosts, such as Aspergillus nidulans (a. Nidulans) and Aspergillus niger (a. Niger).

Host cells suitable for expression of the antibodies or antigen fragments provided by the invention are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains and variants have been identified as well as corresponding permissive insect host cells from: spodoptera frugiperda (Spodoptera frugiperda) (caterpillars), aedes aegypti (mosquitoes), aedes albopictus (mosquitoes), drosophila melanogaster (Drosophila melanogaster), and Bombyx mori (Bombyx mori). A variety of viral strains for transfection are publicly available, such as L-1 variants of Autographa californica (NPV) and Bm-5 viral strains of Bombyx mori NPV, and the viruses can be used according to the invention as viruses in the present invention, in particular for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

However, vertebrate cells have also attracted considerable attention, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable mammalian host cell lines are monkey kidney CV1 strain transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary cells/-DHFR; mouse support cells (TM 4); monkey kidney cells (CV 1 ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat (Buffalo rat) liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI MRC 5 cells; FS4 cells; and human liver tumor lines (Hep G2).

Host cells are transformed with the expression or cloning vectors described above to produce anti-N protein antibodies and cultured in modified conventional nutrient media, as appropriate, to induce promoters, select transformants, or amplify genes encoding the desired sequences. In another embodiment, the antibody can be made by homologous recombination as known in the art.

Pharmaceutical compositions and methods of administration

The invention further provides pharmaceutical compositions comprising a recombinant expression vector expressing an antibody against coronavirus N protein, or an antigen-binding fragment thereof, provided by the invention, and one or more pharmaceutically acceptable carriers.

The term "pharmaceutically acceptable" indicates that the indicated carrier is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

Pharmaceutically acceptable carriers for use in the compositions of the present invention may include, but are not limited to, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles (e.g., sodium chloride injection, ringer's injection, isotonic glucose injection, sterile water injection, or ringer's glucose and lactate injection), non-aqueous vehicles (e.g., non-volatile oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial agents, isotonic agents (e.g., sodium chloride or dextrose), buffers (e.g., phosphate or citrate buffers), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending/dispersing agents (e.g., sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifiers (e.g., polysorbate 80 (Tween-80)), diluents, adjuvants, excipients, or nontoxic auxiliary substances, other components known in the art, or various combinations thereof. Suitable components may include, for example, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, or emulsifying agents.

The present invention provides an agent for treating or preventing a disease associated with an antigen (e.g., N protein) positive cell of interest, which comprises the anti-N protein antibody or an antigen-binding fragment thereof of the present invention as an active ingredient, and a therapeutically effective amount or a prophylactically effective amount of the agent can be administered to a subject in need thereof to treat or prevent a coronavirus positive disease. The anti-N protein antibody or antigen binding fragment thereof can inhibit coronavirus-induced disease-associated activity or eliminate or reduce the number of coronaviruses.

In addition, the present invention relates to a method for immunodetection or measurement of a target antigen (e.g., N protein), a reagent for immunodetection or measurement of a target antigen (e.g., N protein), a method for immunodetection or measurement of a coronavirus expressing a target antigen (e.g., N protein), and a diagnostic agent for diagnosis of a disease associated with infection with a target antigen (e.g., N protein) coronavirus, which comprises the antibody or antibody fragment specifically recognizing binding of a target antigen (e.g., human N protein) of the present invention as an active ingredient.

In the present invention, the method for detecting or determining the amount of the target antigen (e.g., N protein) may be any known method. For example, it includes immunodetection or assay methods.

The immunoassay or measurement method is a method of detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunological detection or measurement method include a radioactive substance-labeled immune antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a Fluorescence Immunoassay (FIA), a luminescence immunoassay, a western immunoblotting method, a physicochemical method, and the like.

The above-mentioned diseases associated with coronavirus infection can be diagnosed by detecting or measuring the expression N protein with the antibody or antibody fragment of the present invention.

For detecting cells expressing the polypeptide, a known immunoassay method can be used, and immunoprecipitation, fluorescent cell staining, immunohistological staining, or the like is preferably used. In addition, a fluorescent antibody staining method using FMAT8100HTS (Applied biosystems) and the like can be used.

In the present invention, the sample for detecting or measuring the target antigen (e.g., N protein) is not particularly limited as long as it has a possibility of containing the expressed target antigen (e.g., N protein), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, interstitial fluid, or culture fluid.

The diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present invention may further contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction, depending on the desired diagnostic method. Reagents for performing antigen-antibody reactions include buffers, salts, and the like. The reagent for detection includes reagents generally used in immunodetection or assay methods, such as a labeled secondary antibody recognizing the monoclonal antibody, an antibody fragment thereof or a binding substance thereof, a substrate corresponding to the label, and the like.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.

EXAMPLE 1 screening of monoclonal antibodies

1. Synthesis of recombinant novel crown N protein

Synthesizing an N protein sequence of SARS-CoV-2 virus, and constructing into a pEM5.1 vector; extracting plasmids for transfection; transfecting to HEK293 cells, and culturing the cells for 7 days; and (3) harvesting the supernatant, purifying by using a Ni column, and concentrating and replacing a buffer solution to obtain the recombinant new crown N protein, wherein the sequence of the recombinant new crown N protein is derived from Uniprot P0DTC9. The sequence is shown as SEQ ID NO:1 is shown.

2. Immunization

The first time, 100 mug of Freund's complete adjuvant is used, the injection is carried out in the abdominal cavity, the total dose is 0.5 ml/piece, and the second immunization is carried out at the interval of 3 weeks; the second time of using Freund's incomplete adjuvant with the dose of 50 mug/0.5 ml/piece, and carrying out the third immunization at the interval of 2 weeks; cell fusion was prepared 10 days after the third injection;

taking feeder cells, and pressing 10 ⁵ Perforamen, plating 10 the day before fusion ⁵ Each well is 100 mu l; mice immunized spleen cells were fused with prepared myeloma cells using the fusion agent PEG, and plated on 96-cell culture plates to which feeder cells had been added at 100. Mu.l/well.

3. Screening and cloning of hybridoma cells

Screening positive holes by an ELISA detection method, and laying recombinant expression N protein overnight; washing the plate, adding skimmed milk powder, sealing, and standing at 37 deg.C for 1h; washing the plate, adding 100 mul of culture solution supernatant of 96 holes, and incubating for 1h at 37 ℃; washing the plate, adding a goat anti-mouse secondary antibody marked by HRP, and incubating for 30min at 37 ℃; washing the plate, adding a developing solution, developing for 10min, adding a stop solution, and reading the numerical value of OD 450; screening high expression cell strain for subcloning culture.

4. Sequencing

The cells were collected, total RNA was extracted using Trizol, and cDNA was generated by reverse transcription using oligo (dT) 20 as a primer. Then, specific primers are used for PCR amplification of the heavy chain variable region gene and the light chain variable region gene respectively. And (3) after the PCR product is subjected to electrophoretic purification, inserting the PCR product into a vector through TA cloning, converting, and selecting positive clones for sequencing.

5. Results

The sequence of the monoclonal antibody 8D7 against SARS-CoV-2 was selected and shown in Table 1.

TABLE 1 sequence of monoclonal antibody 8D7

Example 2 functional study of monoclonal antibody 8D7

1. Expression and purification of monoclonal antibodies

1) The selected sequence is chemically synthesized and cloned into a eukaryotic expression vector.

2) And amplifying and extracting the plasmid.

3) Plasmids encoding the antibodies were transiently transfected into mammalian cells HEK293.

4) Collecting the supernatant, and purifying by affinity chromatography to obtain the monoclonal antibody.

5) As a result, the expression amount of the purified antibody was 225mg/L.

2. Detection of physicochemical Properties of monoclonal antibodies

2.1 gel electrophoresis detection of purity of monoclonal antibodies

1) Instrumentation and equipment

Name (R)	Manufacturer of the product	Type number
			Chemiluminescence imager	Tanon	Tanon-5200
Electrophoresis apparatus	BIO-RAD	poweerpac basic
			Electrophoresis tank	BIO-RAD	DYC-Mini4

2) Primary reagent

3) Sample preparation

Mixing 20 μ l sample with 5 μ l5 × reducing buffer, heating at 95 deg.C for 5min, and cooling;

mu.l of the sample was mixed well with 5. Mu.l of 5 Xnon-reducing buffer.

4) Electrophoresis

Preparing gel, adding a proper amount of electrophoresis buffer solution, adding sample, and performing electrophoresis.

5) Dyeing and bleaching

After electrophoresis, putting the gel into a proper amount of Coomassie brilliant blue staining solution, and staining for 1 hour or more at room temperature; pouring out the staining solution, adding appropriate amount of Coomassie brilliant blue staining decolorization solution, and decolorizing at room temperature for 4-24h. After completion of decolorization, ddH was used ₂ O soaking, comparing with the undyed gel according to Marker protein, cutting off the gel of the needed protein component, and collecting. The protein to be purified is then separated from the gel.

6) Results

The results are shown in FIG. 1, and the bands from left to right are marker, reduced band and non-reduced band; the detection purity of the monoclonal antibody is more than 95 percent.

2.2HPLC detection of purity of monoclonal antibodies

1) Instrumentation and equipment

2) Primary reagent

Name (R)	Manufacturer of the product	Specification of	Goods number
				Dipotassium hydrogen phosphate trihydrate	Sinopharm Group Chemical Reagent Co., Ltd.	500 g/bottle	10017592
Potassium dihydrogen phosphate	Sinopharm Group Chemical Reagent Co., Ltd.	500 g/bottle	10017692
				Potassium chloride	Sinopharm Group Chemical Reagent Co., Ltd.	500 g/bottle	10016392

3) Fluid phase system

Dipotassium phosphate trihydrate, potassium dihydrogen phosphate and potassium chloride are added into about 900ml of purified water, stirred and dissolved, the volume is adjusted to 1L, and the pH value is determined to be between 6.2 +/-0.1 by measuring with a pH meter. Filtering with 0.22 μm filter membrane, and storing at room temperature.

4) Sample preparation

System applicability sample: MIL62 Standard substance diluted to 2mg/ml with mobile phase

And (3) testing the sample: the sample to be tested was diluted to 2mg/ml with mobile phase.

5) Chromatographic conditions

6) Results

As shown in FIG. 2, the purity of monoclonal antibody was greater than 95%.

3. Detection of binding Activity of monoclonal antibodies

1) Coating: diluting antigen N protein to 2 μ g/ml with coating solution, mixing well, adding 96-well coated plate, 100 μ l/well, sealing with membrane, and standing at 4 deg.C overnight.

2) The plate washer washes 3 times, no liquid can remain on the plate in the last time, and the liquid on the surface of the plate is patted dry by using absorbent paper.

3) And (3) sealing: add 5% milk powder (0.5 g milk powder in 10ml DPBS), 300. Mu.l/well, incubate 1h at 37 ℃ and wash plate 3 times as per step 2).

4) The antibody was diluted in a gradient of 100. Mu.l/well, reacted at 37 ℃ for 1h, and the plate was washed 3 times according to step 2).

5) Adding a secondary antibody: diluted with DPBS according to 1.

6) Color development: TMB was added at 100. Mu.l/well and developed in the dark at room temperature for 10min.

7) And (4) terminating: 2N H was added ₂ SO ₄ 100. Mu.l/well.

8) Detecting OD450 with enzyme labeling instrument within 10min

9) Results

As a result, as shown in FIG. 3, monoclonal antibody 8D7 can specifically bind to antigen N protein and exhibit concentration dependence with an EC50 of 0.01505. Mu.g/ml.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> eastern Biotechnology Ltd of Suzhou

<120> antibody against coronavirus N protein and application thereof

<141> 2022-03-29

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 425

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr

1 5 10 15

Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg

20 25 30

Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn

35 40 45

Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu

50 55 60

Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro

65 70 75 80

Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly

85 90 95

Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr

100 105 110

Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp

115 120 125

Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp

130 135 140

His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln

145 150 155 160

Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser

165 170 175

Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn

180 185 190

Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala

195 200 205

Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu

210 215 220

Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln

225 230 235 240

Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys

245 250 255

Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln

260 265 270

Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp

275 280 285

Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile

290 295 300

Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile

305 310 315 320

Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala

325 330 335

Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu

340 345 350

Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro

355 360 365

Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln

370 375 380

Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu

385 390 395 400

Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser

405 410 415

Thr Gln Ala His His His His His His

420 425

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gly Tyr Ser Phe Ile Asn Tyr Gly

1 5

<210> 3

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ile Asn Thr Tyr Thr Gly Asp Pro

1 5

<210> 4

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ala Arg Ser Pro Gly Ser Gly Ala Trp Phe Ala Tyr

1 5 10

<210> 5

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Asn Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser

20 25

<210> 6

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Asn Trp Val Lys Gln Ala Pro Gly Lys Ala Leu Lys Trp Met Gly

1 5 10 15

Trp

<210> 7

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Thr Tyr Val Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr

1 5 10 15

Ser Ala Ser Thr Ala Tyr Leu Gln Ile Thr Asn Leu Lys Asn Glu Asp

20 25 30

Thr Ala Thr Tyr Phe Cys

35

<210> 8

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

1 5 10

<210> 9

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Asn Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ile Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Ala Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Asp Pro Thr Tyr Val Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Thr Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Ser Pro Gly Ser Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 10

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gln Ser Ile Ser Glu Tyr

1 5

<210> 11

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Tyr Ala Ser

1

<210> 12

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gln Gln Gly His Ser Phe Pro Tyr Thr

1 5

<210> 13

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser

20 25

<210> 14

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

1 5 10 15

Lys

<210> 15

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Ile Gly

20 25 30

Val Tyr Tyr Cys

35

<210> 16

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 17

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Glu Tyr

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Ile Gly Val Tyr Tyr Cys Gln Gln Gly His Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

Claims (15)

1. A monoclonal antibody against the N protein of SARS-CoV-2 virus, comprising:

SEQ ID NO:2, HCDR1 of SEQ ID NO:3 and HCDR2 of SEQ ID NO:4, and HCDR3 of SEQ ID NO:10, LCDR1 of SEQ ID NO:11 and LCDR2 of SEQ ID NO: LCDR3 of 12.

2. The monoclonal antibody of claim 1, further comprising a heavy chain variable region having an amino acid sequence substantially identical to SEQ ID NO: 5. 6, 7, 8, and HFR1, HFR2, HFR3, and HFR4, wherein the amino acid sequences are at least 90% identical; and a polypeptide having a sequence identical to SEQ ID NO: 13. 14, 15, 16, and light chain variable region framework regions LFR1, LFR2, LFR3, and LFR4 having at least 90% sequence identity to the amino acid sequences set forth in seq id no.

3. The monoclonal antibody of claim 1 or 2, wherein the heavy chain variable region of the monoclonal antibody has an amino acid sequence identical to SEQ ID NO:9 and the light chain variable region has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:17 has at least 90% sequence identity.

4. The monoclonal antibody of claim 3, wherein the heavy chain variable region of the monoclonal antibody has an amino acid sequence identical to SEQ ID NO:9, VH having at least 95% sequence identity to the amino acid sequence set forth in seq id no; the light chain variable region has a sequence identical to SEQ ID NO:17, and VL having at least 95% sequence identity to the amino acid sequence set forth in seq id no.

5. The monoclonal antibody of claim 1 or 2, further comprising a heavy chain constant region and a light chain constant region.

6. A substance according to any one of the following:

1) A nucleic acid molecule encoding the monoclonal antibody or functional fragment thereof according to any one of claims 1-5;

2) A recombinant expression vector comprising the nucleic acid molecule of 1);

3) A host cell comprising the recombinant expression vector of 2);

4) A drug conjugate comprising the monoclonal antibody or functional fragment thereof according to any one of claims 1-5;

5) A product for detecting the protein SARS-CoV-2N, which comprises the monoclonal antibody or functional fragment thereof of any one of claims 1-5, the nucleic acid molecule of 1), the recombinant expression vector of 2), the host cell of 3), or the drug conjugate of 4);

6) A product for detecting infection by coronavirus SARS-CoV-2 virus, said product comprising the monoclonal antibody or functional fragment thereof of any one of claims 1-5, the nucleic acid molecule of 1), the recombinant expression vector of 2), the host cell of 3), or the drug conjugate of 4);

7) A composition comprising the monoclonal antibody or functional fragment thereof according to any one of claims 1-5, the nucleic acid molecule described in 1), the recombinant expression vector described in 2), and the host cell described in 3).

7. The agent according to claim 6, wherein the recombinant expression vector has a signal peptide operably linked to an antibody.

8. The agent according to claim 7, wherein said recombinant expression vector further comprises a transcriptional regulatory element.

9. The agent according to claim 6, wherein the drug conjugate further comprises a coupling moiety selected from the group consisting of: a detectable label, drug, toxin, cytokine, or enzyme.

10. A pharmaceutical composition comprising a monoclonal antibody according to any one of claims 1 to 5 or a substance according to any one of claims 6 to 9; and/or a pharmaceutically acceptable carrier.

11. The method of any one of:

1) A method of making the monoclonal antibody of any one of claims 1-5, the method comprising: culturing the host cell of claim 6, optionally isolating the antibody from the host cell and/or the medium in which the host cell is grown;

2) A method for detecting SARS-CoV-2 virus N protein in a sample for non-diagnostic purposes, the method comprising: contacting the monoclonal antibody of claim 1 with a test sample and determining the presence or level of N protein in the test sample.

12. The method of claim 11, wherein the method of 1) further comprises purifying the monoclonal antibody.

13. The method of claim 11, wherein the host cell is selected from mammalian cells.

14. The method of claim 13, wherein the cell is selected from HEK293 cells.

15. Use according to any one of the following:

1) Use of the monoclonal antibody according to any one of claims 1 to 5, the substance according to any one of claims 6 to 9 for the detection of the N protein of the SARS-CoV-2 virus for non-diagnostic purposes;

2) Use of the monoclonal antibody of any one of claims 1-5, the substance of any one of claims 6-9 for the preparation of a product for the detection of infection by coronavirus SARS-CoV-2;

3) Use of the monoclonal antibody of any one of claims 1-5 and the substance of any one of claims 6-9 for the preparation of a product for the diagnosis of a disease associated with infection by the coronavirus SARS-CoV-2.

Images