KR20230039786A - An antibody specific to C-terminal region of coronavirus nucleocapsid protein and uses thereof - Google Patents

Abstract

The present invention relates to an antibody specific to a C-terminus region of a coronavirus nucleocapsid (NP) protein and uses thereof. An objective of the present invention is to provide the antibody that specifically binds to the C-terminus of the NP protein or an antigen-binding fragment that specifically binds to the C-terminus.

Description

An antibody specific to C-terminal region of coronavirus nucleocapsid protein and uses thereof {An antibody specific to C-terminal region of coronavirus nucleocapsid protein and uses thereof}

The present invention relates to antibodies specific for the C-terminal region of a coronavirus nucleocapsid protein and uses thereof.

Coronavirus disease 2019 (COVID-19) is a severe disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a human coronavirus first reported in December 2019. It is an acute respiratory disease. Coronavirus was first discovered in chickens in the late 1920s, then in animals such as dogs, pigs and birds, and in 1965 in humans. Human coronaviruses are known to be transmitted when droplets from an infected person penetrate the respiratory tract or the mucous membranes of the eyes, nose, or mouth. However, SARS, which occurred in 2003 and Middle East Respiratory Syndrome (MERS), which occurred in 2012, which caused fatal respiratory diseases, are also known as infectious diseases caused by human coronaviruses. In particular, COVID-19 caused by SARS-CoV-2 infection spread around the world due to its high fatality rate and rapid spread, and was declared a pandemic by the World Health Organization (WHO) in March 2020, and some The country is making all-out efforts to prevent the spread by controlling entry from countries with a large number of confirmed cases. In addition, as the SARS-CoV-2 mutant virus emerges in many parts of the world, diagnosing and controlling COVID-19 is more difficult.

The most essential method for controlling infectious diseases is virus infection diagnosis, and the most important factors in diagnosis are accuracy and speed. Accordingly, it is necessary to develop a method capable of quickly and accurately diagnosing whether an object to be analyzed is infected with the coronavirus.

Accordingly, the present inventors developed an antibody that specifically binds to the protein C-terminal region of nucleocapsid (NP), which is a region with low variation in gene sequence among antigens expressed by coronaviruses, to be used for diagnosis of coronavirus. The present invention was completed by confirming that it could be.

KR 10-2245849 B1 KR 10-2021-0076854 A

An object of the present invention is to provide an antibody that specifically binds to the C-terminus of a coronavirus nucleocapsid (NP) protein or an antigen-binding fragment that specifically binds to the C-terminus.

Another object of the present invention is to provide a polynucleotide encoding the antibody or antigen-binding fragment, an expression vector containing the same, and a host cell containing the same.

Another object of the present invention is to provide a composition for diagnosing coronavirus infection comprising the antibody or the antigen-binding fragment, and a kit including the same.

Another object of the present invention is to provide an information providing method for diagnosing coronavirus infection, comprising the step of detecting coronavirus present in a biological sample using the antibody or the antigen-binding fragment.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below. In addition, a number of papers and patent documents are referenced throughout this specification and their citations are indicated. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention belongs and the contents of the present invention.

One aspect of the present invention provides an antibody that specifically binds to the C-terminus of a coronavirus nucleocapsid (NP) protein or an antigen-binding fragment that specifically binds to the C-terminus.

In the present invention, the term "coronavirus" refers to a positive-sense single-stranded RNA virus (ssRNA) belonging to the family Coronaviridae and having a spherical outer membrane and having a size of about 80-220 nm. . Coronaviruses are the outermost spike (S) protein, hemagglutinin-esterase (HE) protein, membrane (M) protein, envelope (E) protein and nucleosomes. It is known to contain five structural proteins, including the capsid (nucleocapsid, NP) protein (Lai and Homes, 2001. Fields Virology).

In one embodiment, the coronavirus may be SARS-CoV-2, but the coronavirus detectable by the antibody or antigen-binding fragment thereof of the present invention is included without limitation.

The term of the present invention, "SARS-CoV-2 (SARS-coronavirus-2)" is a virus that causes coronavirus infection-19 (COVID-19), a respiratory disease.

SARS-CoV-2 is an ssRNA virus belonging to the genus betacoronavirus with an envelope. SARS-CoV-2 is taxonomically considered to be a strain of SARS-CoV, has zoonotic origins, and has close genetic similarities to bat coronaviruses. The SARS-CoV-2 is mainly transmitted through close contact between people or through droplets generated during coughing or sneezing, and mainly binds to the receptor angiotensin converting enzyme 2 (ACE2) to enter human cells. It is known. According to classification based on the phylogenetic relationship of coronaviruses, SARS-CoV-2 has about 70% or more identical genetic sequence with SARS-CoV-1, which caused SARS in 2003, and in particular, NP's The gene sequences are 91% identical.

The major structural proteins of SARS-CoV-2 include nucleocapsid (NP), spike (S), membrane (M), envelope (E), proteins and/or genes encoding the proteins, like SARS-CoV-1. can do. Among the structural proteins of SARS-CoV-2, the nucleocapsid (NP) protein surrounds the genetic material ssRNA and is involved in RNA replication. Spike (S) protein exists on the surface of virus particles and is involved in host cell invasion, and is divided into S1 and S2. The S1 protein interacts with the host cell receptor, and the receptor-binding domain (RBD) present in the S1 protein is known as a key site that binds to the ACE2 receptor present on the host cell surface. The S2 protein is involved in fusion with the cell membrane of the host cell. The structure is described in Cascella, M., Rajnik, M., Cuomo, A., Dulebohn, S. C., & Di Napoli, R. (2020). Features, Evaluation and Treatment of Coronavirus (COVID-19). In StatPearls. It is detailed in Treasure Island (FL).

The SARS-CoV-2 of the present invention includes all mutant forms that can be classified as SARS-CoV-2 without limitation, and examples thereof include alpha (B.1.1.7 strain) and beta (B.1.351 strain: B. 1.351.2, B.1.351.3), delta (B.1.617.2 and AY strains: AY.1, AY.2, AY.3), gamma (P.1 strain: P.1.1, P.1.2) It may include a mutation, referred to as a mutation. Mutant SARS-CoV-2 may include specific substitutions or substitution combinations in the spike protein, examples of which include L452R, E484K, K417N/T, E484K, and N501Y.

In one embodiment, the antibody or fragment thereof provided in the present invention may specifically bind to the nucleocapsid (NP) protein (also referred to as "N protein") of the coronavirus.

The antibody or fragment thereof provided in the present invention may bind to the C-terminus of the nucleocapsid (NP) protein of the coronavirus. Specifically, the binding site of the antibody or antigen-binding fragment thereof provided in the present invention is amino acids 248 to 419 from the N-terminus of the nucleocapsid (NP) protein of the coronavirus shown in FIG. 12 (SEQ ID NO: 19) , Specifically, it may be included in the sequence corresponding to positions 364 to 419. The antibody or antigen-binding fragment thereof provided in the present invention in any one of the above embodiments may have an epitope included in the amino acid region at positions 364 to 419 from the N-terminus of SEQ ID NO: 19. there is.

In one embodiment of the present invention, it was confirmed that the antibody 1G6 of the present invention has a binding site within the sequence corresponding to amino acids 248 to 419 from the N-terminus of the nucleocapsid (NP) protein of coronavirus, and as a result of further analysis, the above It was confirmed that the binding site was included in the sequence corresponding to amino acids 364 to 419.

On the other hand, the sequence of FIG. 12 (SEQ ID NO: 19) is a representative example of the nucleocapsid protein of coronavirus, and is used as a reference sequence to indicate the binding position of the antibody or binding fragment thereof provided in the present invention. , Those skilled in the art can align any sequence with the sequence of SEQ ID NO: 19 shown in FIG. 12 to identify a position corresponding to a specific residue number from the N-terminus of FIG. 12 or a corresponding sequence region in any sequence. .

In the present invention, the term "antibody" refers to a protein molecule that serves as a ligand that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, Both whole antibodies and antibody fragments are included. The term also includes chimeric antibodies (eg humanized murine antibodies) and bivalent or bispecific molecules (eg bispecific antibodies), diabodies, triabodies and tetrabodies. The term further includes single-chain antibodies, scabs, derivatives of antibody constant regions and artificial antibodies based on protein scaffolds that have a binding function to FcRn (neonatal Fc receptor). A whole antibody has a structure having two full-length light chains (LC) and two full-length heavy chains (HC), and each light chain is connected to the heavy chain by a disulfide bond. The total antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG includes IgG1, IgG2, IgG3, and IgG4 as subtypes. In one embodiment, the antibody provided in the present invention may be an IgG antibody.

The terms "fragment", "binding fragment of a polypeptide" and "antibody fragment" are used interchangeably to refer to any fragment of an antibody of the invention that retains the antigen-binding activity of the antibody. Exemplary antibody fragments include, but are not limited to, single chain antibodies, Fd, Fab, Fab', F(ab')2, dsFv or scFv. The Fd refers to the heavy chain part included in the Fab fragment. The Fab has a structure having light and heavy chain variable regions, a light chain constant region (framework region, FR), and a heavy chain first constant region (CH1 domain), and has one antigen binding site. Fab' is different from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. An F(ab')2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'. Fv (variable fragment) means a minimum antibody fragment having only the heavy chain variable region and the light chain variable region. In double disulfide Fv (dsFv), the heavy chain variable region and light chain variable region are linked by a disulfide bond, and in single chain Fv (scFv), the heavy chain variable region and light chain variable region are generally covalently linked through a peptide linker. . Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), For example, it can be produced through genetic recombination technology.

In one embodiment, the antibody provided in the present invention may be a monoclonal antibody.

As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and such a monoclonal antibody has a single binding specificity and affinity for a specific epitope. indicates the figure.

Typically, an immunoglobulin has a heavy chain and a light chain, each comprising a constant region and a variable region (the regions are also known as domains). The variable regions of the light chain and the heavy chain include three variable regions and four structural regions called complementarity-determining regions (CDRs).

The CDR mainly serves to bind to an epitope of an antigen. The CDRs of each chain are typically called CDR1, CDR2, CDR3 sequentially starting from the C-terminus, and are also identified by the chain in which a particular CDR is located. The complementarity-determining regions are located between regions that are relatively conserved, called constant regions (FR). Each heavy chain variable region (VH) and light chain variable region (VL) consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3 , CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigen. The CDRs of the heavy chain variable region are HCDR1, HCDR2, and HCDR3, the CDRs of the light chain variable region are LCDR1, LCDR2, and LCDR3, the FRs of the heavy chain variable region are HFR1, HFR2, HFR3, and HFR4, and the FRs of the light chain variable region are LFR1 and LFR2 , LFR3, LFR4.

In one embodiment, the antibody specifically binding to the C-terminus of the coronavirus nucleocapsid (NP) protein of the present invention or the antigen-binding fragment specifically binding to the C-terminus is HCDR1 of SEQ ID NO: 2, a heavy chain variable region comprising HCDR2 of SEQ ID NO: 4 and HCDR3 of SEQ ID NO: 6; and a light chain variable region including LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 12, and LCDR3 of SEQ ID NO: 14.

In any one of the embodiments described above, the antibody or antigen-binding fragment thereof comprises HFR (heavy chain FR)1 of SEQ ID NO: 1, HFR2 of SEQ ID NO: 3, HFR3 of SEQ ID NO: 5, and HFR4 of SEQ ID NO: 7. Heavy chain variable region containing; and a light chain variable region comprising LFR (light chain FR)1 of SEQ ID NO: 9, LFR2 of SEQ ID NO: 11, LFR3 of SEQ ID NO: 13, and LFR4 of SEQ ID NO: 15.

In any one of the embodiments described above, the antibody or antigen-binding fragment thereof may further include a heavy chain tail domain of SEQ ID NO: 8.

In any one of the embodiments described above, the antibody or antigen-binding fragment thereof may further include a light chain tail domain of SEQ ID NO: 16.

In any one of the above embodiments, the antibody or antigen-binding fragment thereof may include a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 18.

In one embodiment of the present invention, an antibody that specifically binds to the C-terminus of the coronavirus nucleocapsid (NP) protein was developed, and it was confirmed that the developed antibody had CDR and FR sequences as shown in FIG. The invention was completed.

In the present invention, the CDRs of the variable regions were determined by a conventional method according to the system devised by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest (5th), National Institutes of Health, Bethesda, MD. (1991)) CDR numbering used in the present invention was performed using the Kabat method, but antibodies containing CDRs determined according to other methods such as the IMGT method, the Chothia method, and the AbM method are also included in the scope of the present invention.

Another aspect of the present invention provides a polynucleotide encoding the antibody or antigen-binding fragment thereof provided in the present invention.

Polynucleotides encoding the antibodies of the present invention can be readily isolated and sequenced using conventional procedures.

For example, oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions from the phage template DNA can be used. Once the polynucleotide has been isolated, it can be placed into an expression vector, which can then be introduced into a suitable host cell to produce the desired monoclonal antibody from the transformed host cell (i.e., transformant).

Another aspect of the present invention provides an expression vector containing the antibody or antigen-binding fragment thereof provided in the present invention and a host cell into which the vector is introduced.

The antibody is as described above.

The expression vector containing the polynucleotide encoding the antibody provided in the present invention is not particularly limited thereto, but is mammalian cell (eg, human, monkey, rabbit, rat, hamster, mouse cell, etc.), plant cell, yeast It can be a vector capable of replicating and / or expressing the polynucleotide in eukaryotic or prokaryotic cells, including cells, insect cells, or bacterial cells (eg, E. coli, etc.), specifically, the nucleotide in the host cell It can be a vector operably linked to an appropriate promoter for expression and containing at least one selectable marker. For example, the polynucleotide may be introduced into a phage, plasmid, cosmid, mini-chromosome, virus or retroviral vector.

The expression vector including the polynucleotide encoding the antibody may be an expression vector each including a polynucleotide encoding the heavy chain or light chain of the antibody, or an expression vector including both polynucleotides encoding the heavy chain or light chain of the antibody.

Host cells into which the expression vector provided in the present invention is introduced are not particularly limited thereto, but bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium transformed by the introduction of the expression vector; yeast cells; fungal cells such as Pichia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells, chinese hamster ovary cells), SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), Bowes melanoma cells, HT-1080 , animal cells such as BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), and PER.C6 (human retinal cells); or plant cells.

As used herein, the term "transduction" refers to a method of delivering a vector containing a polynucleotide encoding the antibody to a host cell. Such introduction is carried out by calcium phosphate-DNA co-precipitation method, DEAE-dextran-mediated transfection method, polybrene-mediated transfection method, electroporation method, microinjection method, liposome fusion method, lipofectamine and protoplast fusion method, etc. It can be performed by several methods known in the art. In addition, transduction means the transfer of a target into a cell using virus particles as a means of infection. In addition, vectors can be introduced into host cells by gene bombardment or the like. In the present invention, introduction may be used in combination with transfection and transformation.

Another aspect of the present invention provides a composition for diagnosing coronavirus infection, comprising the antibody or antigen-binding fragment thereof provided in the present invention.

Another aspect of the present invention provides a kit for diagnosing coronavirus infection, including the antibody or antigen-binding fragment thereof provided in the present invention.

Another aspect of the present invention is an antibody or antigen-binding fragment thereof provided by the present invention; Or a composition for diagnosing infection comprising the same; Alternatively, a method for diagnosing a coronavirus infection or providing information for diagnosis, including detecting a coronavirus present in a biological sample isolated from a subject suspected of having a coronavirus infection, using a kit containing the composition is provided.

As used herein, the term "diagnosis" refers to determining an individual's susceptibility to a specific disease or disorder, determining whether an individual currently has a specific disease or disorder, or providing information on treatment efficacy. This includes monitoring the health of the object for

For the purpose of the present invention, the diagnosis may be to determine whether or not a coronavirus infectious disease has occurred. The "coronavirus infectious disease" refers to a disease caused by an infection in which a coronavirus invades the body of an organism that is a host. Specifically, the coronavirus is SARS-CoV-2 (SARS-coronavirus-2 ) can be For example, the coronavirus infectious disease may be COVID-19, but is not limited thereto.

The composition for diagnosing coronavirus infection of the present invention may include other antibodies or antigen-binding fragments thereof other than the antibodies or antigen-binding fragments thereof of the present invention. In addition, materials necessary for the binding reaction between the antibody or antigen-binding fragment thereof of the present invention and the antigen, for example, reagents may be further included. In addition, a substance necessary for detecting this binding, a substance for stabilizing the antibody or antigen-binding fragment thereof of the present invention, and the like may be further included.

In one embodiment, the diagnostic composition of the present invention may further include an antibody or antigen-binding fragment thereof that specifically binds to the N-terminus of the coronavirus nucleocapsid protein. The antibody or antigen-binding fragment thereof specifically binding to the N-terminus may have an epitope included in the region of amino acids 1 to 101 from the N-terminus of SEQ ID NO: 19. For example, it may have an epitope included in any one region selected from the following:

i) amino acids 1 to 47 from the N-terminus of SEQ ID NO: 19; and

ii) amino acids 47 to 101 from the N-terminus of SEQ ID NO: 19.

In any one of the above embodiments, the diagnostic composition of the present invention

(i) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 28, HCDR2 of SEQ ID NO: 30, and HCDR3 of SEQ ID NO: 32; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising LCDR1 of SEQ ID NO: 36, LCDR2 of SEQ ID NO: 38, and LCDR3 of SEQ ID NO: 40;

(ii) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 46, HCDR2 of SEQ ID NO: 48, and HCDR3 of SEQ ID NO: 50; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising LCDR1 of SEQ ID NO: 54, LCDR2 of SEQ ID NO: 56, and LCDR3 of SEQ ID NO: 58; and

(iii) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 64, HCDR2 of SEQ ID NO: 66, and HCDR3 of SEQ ID NO: 68; And it may further include any one or more selected from an antibody comprising a light chain variable region including LCDR1 of SEQ ID NO: 72, LCDR2 of SEQ ID NO: 74, and LCDR3 of SEQ ID NO: 76, or an antigen-binding fragment thereof.

For example, the antibody or antigen-binding fragment thereof included in the diagnostic composition of the present invention may be labeled for detection. A variety of methods available for molecular labeling are well known to those skilled in the art and are contemplated within the scope of the present invention.

Examples of types of labels that can be used in the present invention include enzymes, radioactive isotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds. Commonly used labels include fluorescent substances (eg, flurescin, rhodamine, Texas red, etc.), enzymes (eg, horseradish peroxidase, beta-galactosidase, alkaline phosphatase), radioactive isotopes (eg, ³² P or ¹²⁵ I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds such as dioxetane, luminol or acridinium. Labeling methods such as covalent bonding of enzymes or biotinyl groups, iodination, phosphorylation, and biotinylation are also well known.

The kit of the present invention may further include reagents, instruments, etc. necessary for detecting the binding of the antibody or antigen-binding fragment thereof of the present invention and an antigen.

For example, a solid carrier may be included in the kit container of the present invention. An antibody of the present invention may be attached to a solid carrier, and such a solid carrier may be porous or non-porous, planar or non-planar.

For example, the kit may include a solid carrier; a sample pad accommodating a sample to be analyzed and having a buffer input unit and a sample input unit; a conjugate pad containing an antibody that specifically binds to the coronavirus contained in the sample introduced from the sample pad; a signal detection pad including a signal detection unit for detecting whether or not coronavirus is present in the sample and a control unit for determining whether the sample has moved to the absorbent pad regardless of the presence or absence of an analyte; and an absorbent pad for absorbing the specimen after the signal detection reaction is completed, but is not limited thereto.

For example, the kit of the present invention may be in the form of an enzyme-linked immunosorbent assay (ELISA) kit. The term "ELISA" of the present invention, also referred to as an enzyme immunoassay method, is a method of quantification using absorbance through a reaction between an enzyme and a substrate by binding an enzyme to an antibody to form an antigen-antibody complex. The ELISA includes direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, and Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of attached antibody and antigen, followed by reaction with another antibody that recognizes an antigen in a complex of antibody and antigen attached to a solid support and then recognizing this antibody Indirect sandwich ELISA using labeled secondary antibodies and the like.

In one embodiment, the kit of the present invention may be a sandwich ELISA kit. In any one of the embodiments described above, the sandwich ELISA kit of the present invention includes an antibody that binds to the C-terminus of the coronavirus nucleocapsid protein or an antibody that recognizes a site different from an antigen-binding fragment thereof or an antigen-binding fragment thereof. Fragments may be further included.

In any one of the above embodiments, the sandwich ELISA kit of the present invention may further include an antibody or antigen-binding fragment thereof that binds to the N-terminus of the coronavirus nucleocapsid protein. The antibody or antigen-binding fragment thereof that binds to the N-terminus may have an epitope included in any one region selected from the following:

i) amino acids 1 to 47 from the N-terminus of SEQ ID NO: 19; and

ii) amino acids 47 to 101 from the N-terminus of SEQ ID NO: 19.

For example, the antibody or antigen-binding fragment thereof that binds to the N-terminus may be any one or more selected from the following:

(i) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 28, HCDR2 of SEQ ID NO: 30, and HCDR3 of SEQ ID NO: 32; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising LCDR1 of SEQ ID NO: 36, LCDR2 of SEQ ID NO: 38, and LCDR3 of SEQ ID NO: 40;

(ii) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 46, HCDR2 of SEQ ID NO: 48, and HCDR3 of SEQ ID NO: 50; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising LCDR1 of SEQ ID NO: 54, LCDR2 of SEQ ID NO: 56, and LCDR3 of SEQ ID NO: 58;

(iii) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 64, HCDR2 of SEQ ID NO: 66, and HCDR3 of SEQ ID NO: 68; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising LCDR1 of SEQ ID NO: 72, LCDR2 of SEQ ID NO: 74, and LCDR3 of SEQ ID NO: 76.

The antibody or fragment thereof of the present invention can be used to detect the presence or absence of a coronavirus in a biological sample by contacting it with a biological sample and confirming the reaction.

The biological sample may be any one selected from the group consisting of sputum, saliva, blood, sweat, lung cells, mucus of lung tissue, respiratory tissue and saliva of the subject, but is not limited thereto, and is prepared by a conventional method known to those skilled in the art possible.

As an example, the information providing method for diagnosing coronavirus infection of the present invention includes (a) contacting the antibody or antigen-binding fragment thereof provided in the present invention with a biological sample; and (b) detecting a complex of the antibody or antigen-binding fragment thereof of the present invention and the coronavirus NP protein formed by the contact.

As used herein, the term "antigen-antibody complex" refers to a combination of a corresponding protein antigen in a sample and an antibody recognizing it. Detection of the antigen-antibody complex can be performed using methods known in the art, for example, spectroscopic, photochemical, biochemical, immunochemical, electrical, spectroscopic, chemical and other methods. by colormetric method, electrochemical method, fluorescence method, luminometry, particle counting method, visual assessment and scintillation counting method It can be detected by any method selected from the group consisting of western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion method, rocket Methods such as immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complete fixation assay, fluorescence-activated cell sorting (FACS), and protein chip can be used. Not limited to this.

By using the antibody of the present invention, a much smaller amount of coronavirus can be detected compared to conventional coronavirus detection antibodies, so the antibody of the present invention can be usefully used for detecting and diagnosing coronavirus.

1 shows the results of confirming the reactivity of antibodies produced by mouse-derived B cell hybridomas immunized through inoculation with inactivated SARS-CoV-2 to the SARS-CoV-2 NP antigen by ELISA. The spleen of the immunized mouse was collected to collect B lymphocytes, and a B cell hybridoma cell population was constructed by fusing with mouse myeloma cells. Anti-NP antibody-secreting B cell hybridomas were selected by ELISA to confirm reactivity to NP protein for the antibodies secreted by each hybridoma, and subcloning by sequentially diluting the hybridoma parent cells. was performed several times to finally select the 1G6 anti-NP antibody.
2 is a result confirming the NP antigen specificity of the discovered 1G6 anti-NP antibody. Nucleocapsid (NP) commercially used by ELISA method, S1 and S2 proteins of SARS-CoV-2 spike protein, and recombinant proteins for receptor binding domain (RBD) antigen present in S1 protein (Sino Biological), respectively An anti-NP antibody purified with Protein G resin was added to the coated well, and the bound antibody was detected with an anti-mouse IgG-HRP (CST) antibody.
3 is an ELISA result comparing SARS-CoV-2 NP antigen reactivity using the novel antibody 1G6 and two commercially available anti-SARS-CoV-2 NP antibodies 4B21 and B46F. Anti-NP antibodies were serially diluted and added to wells coated with SARS-CoV-2 NP antigen, and bound antibodies were detected with an anti-mouse IgG-HRP antibody.
Figure 4 shows SARS-CoV NP antigen (91% identical in amino acid sequence to SARS-CoV-2 NP) reactivity using novel antibody 1G6 and two commercially available anti-SARS-CoV-2 NP antibodies 4B21 and B46F. It is an ELISA result comparing the . Anti-NP antibodies were serially diluted and added to wells coated with SARS-CoV NP antigen protein, and the bound antibodies were detected with an anti-mouse IgG-HRP antibody.
FIG. 5 shows ELISA results comparing reactivity to inactivated SARS-CoV-2 lysates using commercially available anti-SARS-CoV-2 NP antibodies 4B21 and B46F and purified novel antibody 1G6. Anti-NP antibodies were serially diluted and added to wells coated with the inactivated SARS-CoV-2 lysate, and bound antibodies were detected with an anti-mouse IgG-HRP antibody.
Figure 6 is a Western blot (WB) result performed to investigate whether the novel antibody 1G6 antibody recognizes a linear or conformational epitope of the SARS-CoV-2 NP antigen protein. The SARS-CoV-2 NP recombinant antigen protein was developed on reducing 12% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane, and then 1G6 antibody was used as the primary antibody and anti-mouse-IgG-HRP as the secondary antibody. Antibodies were treated. NP developed on SDS-PAGE was confirmed to be slightly above the predicted molecular weight of 47.33 kDa, and it was confirmed that a protein band was detected at the same position in Western blotting.
Figure 7 is a Western blot result confirming that the anti-NP antibody specifically binds to the intracellularly expressed SARS-CoV-2 NP antigen protein. The commercially available SARS-CoV-2 NP-10xHis/pCMV3 plasmid was transduced into human embryonic kidney cells (HEK293T), and the cell homogenate was developed on reducing 12% SDS-PAGE, transferred to a PVDF membrane, and stained with 1G6 antibody. This is a Western blot result of detecting a specific protein. By treating the anti-His antibody as a positive control antibody, it was confirmed that the NP-His protein was expressed at the predicted molecular weight of 47.33 kDa through post-translational modification in cells, and the 1G6 antibody was the anti-His antibody. It was confirmed that it binds to the protein at the same position as
Figure 8 is a commercially used SARS-CoV-2 NP-His using the specific reactivity of the 1G6 antibody to the intracellular antigen of Figure 7 to confirm whether the new anti-NP antibody is also applicable to immunoprecipitation. /pCMV3 plasmid (Sino Biological) was transduced into human embryonic kidney cells (HEK293T), the cell homogenate was mixed with the new antibody, and the antibody was captured with Protein G resin, and the antigen bound to the antibody was also detected. This is the result of confirming that The 1G6 antibody was mixed with the same subtype (IgG2a) antibody as a negative control antibody with the cell homogenate and compared. As a result, the immunoprecipitation ability of the 1G6 antibody was confirmed.
9 is a schematic diagram showing the constituent parts of recombinant proteins cloned with NP fragment variants in which a specific amino acid sequence is missing in order to determine the precise epitope of an anti-SARS-CoV-2 NP antibody. A total of two types of partially defective recombinant proteins were prepared, NP (1-174 AA)-p1-His (sequence 1-174 amino acid partial defect protein), NP (1-248 AA) -p2-His (SEQ ID NO: 1 partially deleted protein with ~248 amino acids).
10 shows the results of expressing the SARS-CoV-2 NP partially defective recombinant protein and determining the epitope by performing Western blotting with an anti-NP antibody. NP (1-174 AA)-p1-HisNP (SEQ ID NO: 1-174 amino acids) containing the N-terminal region and NP (1-248 AA) -p2-His (NP containing the N-terminal region and SR-rich portion) Sequence 1 to 247 amino acid) protein was induced in E. coli, and the cell lysate and commercially used total NP (sequence 1 to 419 amino acid) protein (Sino Biological) were developed on reducing 12% SDS-PAGE, followed by PVDF It was transferred to the membrane and it was confirmed whether or not a specific NP partially defective protein containing an epitope was detected with an anti-NP antibody. Based on this result, it was confirmed that the C-terminal region of the 1G6 antibody, rather than the 1st to 248th region of the NP protein, was the epitope of these antibodies. Correct expression of the recombinant protein was confirmed using an anti-His antibody as a positive control.
11 is a result of analyzing the complementarity determining regions (CDR) of the heavy chain variable region (VH) and the light chain variable region (VL) of the novel 1G6 anti-NP C-terminal recognizing antibody. Complementary DNA (cDNA) is synthesized for total RNA (total RNA) extracted from antibody-producing B cell hybridomas, and a polymerase chain reaction is performed using heavy chain variable region complementarity determining region primers and light chain variable region complementarity determining region primers. reaction, PCR), and then ligated to a T-vector to prepare a recombinant plasmid. The recombinant plasmid was amplified in host cells, extracted, and the corresponding region of the complementarity determining region was sequenced. The protein sequence was confirmed from the analyzed nucleotide sequence, and the CDR sequence was determined based on the KaBat CDR definition.
12 shows sequence information of the SARS-CoV-2 NP protein.
13 to 16 are results confirming the characteristics of 3E10, 3F10, 5B6, and 5B6.8 anti-NP antibodies.
17 is a schematic diagram of the SARS-CoV-2 NP antigen protein and fragment protein and the results of additional analysis to more precisely determine the epitope. 17A is a schematic diagram of the structure of SARS-CoV-2 NPs, full-length NPs numbered 1-419; NP-NTD numbers 47-174; NP-CTD numbers 248-364; NP-p1 is 1-174; NP-p2 is numbered 1-248; NP-p3 is numbered 1-305; 101-419 for NP-p4; NP-p5 prepared a partially defective recombinant protein having amino acids 248-419. FIG. 17B shows Western Blot results. Recombinant proteins were developed on reducing 12% SDS-PAGE and transferred to a PVDF membrane, and it was confirmed whether a specific NP partially defective protein containing an epitope was detected with an anti-NP antibody. Based on these results, it was confirmed that the epitope of the 3F10 antibody was the 1st to 47th region of the NP protein, and the 3E10 and 5B6 were confirmed to be the 47th to 101st region of the NP protein. It was confirmed that the 1G6 antibody was the epitope of these antibodies at positions 364 to 419 of the NP protein.
18 is a result of comparative analysis of SARS-CoV-2 NP and SARS-CoV-2 viral lysate recognition in a sandwich ELISA method loaded with a novel anti-NP antibody pair. 1G6 was coated on the bottom of the ELISA plate with capture antibody and then washed. SARS-CoV-2 NP recombinant protein was added, washed, and then detected with an HRP-conjugated anti-NP antibody. 18A shows that the most effective capture and detection antibody pairs in the sandwich ELISA method for detecting SARS-CoV-2 NP antigen protein are 1G6 and 3E10. 18B shows that the most effective capture and detection antibody pairs in the sandwich ELISA method for detecting SARS-CoV-2 virus lysates are 1G6 and 3F10, and 1G6 and 3E10.
19 is a result of comparing the anti-NP antibody binding ability to NP of alpha, beta, and gamma mutant SARS-CoV-2 viruses. The left side is a schematic diagram showing the positions of amino acid mutations in the NP protein of SARS-CoV-2 virus variants, and mutations are indicated in red. The right side is the result of ELISA using the antibody of the present invention. Anti-NP antibodies were serially diluted and added to wells coated with SARS-CoV-2 mutant virus NP antigen, and the bound antibody was anti-mouse IgG-HRP antibody was detected with It was confirmed that 1G6 had binding ability to NP of alpha, beta, and gamma mutant SARS-CoV-2 viruses.
20 is a result of confirming the NP recognition ability of SARS-CoV-2 mutant virus in a sandwich ELISA loaded with a 3E10, 1G6 antibody pair. SARS-CoV-2 variant antigen NP recombinant protein was added to an ELISA plate coated with 3E10 capture antibody, and then detected with HRP-conjugated 1G6 antibody. It was confirmed that all NPs of alpha, beta, and gamma mutant SARS-CoV-2 viruses were detected in a sandwich ELISA loaded with 3E10 and 1G6 antibody pairs. It was confirmed that it was not detected when using the influenza NP antibody bound to HRP, a negative control.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

Example 1: Preparation of mouse-derived anti-SARS-CoV-2 NP monoclonal antibody

To generate hybridomas secreting anti-SARS-CoV-2 NP antibodies, we used an inactivated SARS-CoV-2 virus as an antigen. Six-week-old female BALB/c mice were purchased from Orient Bio (Gyeonggi-do, Korea) and reared in sterile facilities. TiterMax Gold adjuvant (Sigma -Aldrich) and immunized with 10 μg virus a total of three times. Two weeks after the final immunization, immune cells were isolated from lymph nodes and used to prepare B cell hybridomas producing anti-SARS-CoV-2 NP antibodies. Fusion with follicular (F0) cells, which are mouse myeloma cells, was performed according to a general B cell hybridoma preparation method and HAT (hypoxanthine-aminopterin-thymidine medium) media selection method.

Reactivity to SARS-CoV-2 NP (SEQ ID NO: 1 to 419 amino acids) recombinant protein antigen was confirmed through ELISA technique using the culture supernatant of 503 initial clones. Specifically, the antigen (antigen, Ag) to be attached to the bottom of a MaxiSorp 96-well plate (Thermo Scientific) is a SARS-CoV-2 NP (sequence 1 to 419 amino acids) recombinant protein (Sino Biological) in phosphate-buffered PBS (PBS). saline), treated with 100 ng per well, and attached for 16 hours at 4°C. In order to remove non-adhered substances, the plate was washed three times with PBS (PBST) containing 0.05% Tween-20, and then treated with PBS containing 1% bovine serum albumin (BSA) at 37 ° C for 1 hour to obtain non-specific Prepared so that the reaction does not occur. The culture supernatant of the primary clone was incubated at 37°C for 2 hours, washed three times with PBST to remove the residue, and then horse-radish peroxidase (HRP)-attached anti-mouse IgG (1:5000 dilution, CST g) and reacted at 37 ° C for 1 hour. After washing the plate 5 times with PBST, the reaction product was developed using a color-developing tetramethylbenzidine substrate (TMB, 3, 3', 5, 5'-Tetramethyl-benzidine; BD Biosciences) and reacted with 2N H ₂ SO ₄ has been terminated. Optical density (OD) was measured at 450 nm using a VICTOR Nivo plate reader (PerkinElmer). A monoclonal hybridoma selection process was performed by selecting clones having a high optical density (OD) value among a total of 503 initial clones (FIG. 1).

Example 2: Anti-SARS-CoV-2 NP monoclonal antibody subtyping

In order to confirm the isotype of the anti-SARS-CoV-2 NP monoclonal antibody secreted by the hybridomas selected in Example 1, it was determined using a mouse antibody isotyping kit (Pierce).

On an ELISA plate coated with mouse antibody subtype (IgG1, IgG2a, IgG2b, IgG3, IgA, IgM) specific anti-mouse capture antibody, the hybridoma culture medium was diluted 1/50 in TBS (Tris-buffered saline) to 50 per well. μl was treated, and anti-mouse-IgG+IgA+IgM-HRP antibody was simultaneously treated to induce a reaction for 1 hour, followed by a color reaction using TMB.

As a result of confirmation, 1G6 was found to be an IgG2a kappa-chain subtype.

Antibody Isotype light chain 1G6 IgG2a k

Example 3. Confirmation of reactivity of anti-SARS-CoV-2 NP antibody to antigen derived from SARS-CoV-2

In order to confirm the specificity of the anti-SARS-CoV-2 NP antibody discovered in Example 1, SARS-CoV-2 structural proteins, nucleocapsid (NP, amino acids SEQ ID NO: 1 to 419), spike ( S) S1 (spike sequence 1-685 amino acids) and S2 (spike sequence 686-1273 amino acids) constituting proteins, and recombinant protein (Sino Biological Co.) antigen against RBD (spike sequence 319-541 amino acids) antigen present in S1 100 ng was used per well, and the specific experimental method was the same as in Example 1.

As a result of the experiment, the antibody 1G6 obtained in Example 1 showed high absorbance only to the NP antigen, but showed no reactivity to the other antigens (S1, S2, RBD) (FIG. 2).

Accordingly, it can be confirmed that the 1G6 antibody specifically reacts to NP among SARS-CoV-2-derived antigens.

Example 4. Comparison of anti-SARS-CoV-2 NP monoclonal antibody sensitivity and specificity

In order to confirm the difference in binding ability to the SARS-CoV-2 NP antigen, ELISA was performed by preparing new purified anti-SARS-CoV-2 NP antibodies at various concentrations.

For comparative analysis of binding ability, commercially available anti-SARS-CoV-2 NP monoclonal antibodies B46F (Thermo Scientific) and 4B21 (Creative Diagnostics) were used together as controls.

First, using an ELISA plate to which SARS-CoV-2 NP antigen was attached at 100 ng per well, a solution in which the antibody was serially diluted from 3 μg was incubated with the primary antibody at 37 ° C. for 2 hours. After removing the residue by washing three times with PBST, the mixture was reacted with HRP-attached anti-mouse IgG (1:5000 dilution) at 37°C for 1 hour. After washing the plate 5 times with PBST, TMB solution was added to develop the reaction, 2N H ₂ SO ₄ was added to terminate the reaction, and absorbance was measured at 450 nm.

As a result, 1G6 showed the highest binding ability to SARS-CoV-2 NP, commercial antibody B46F showed the next highest binding ability, and 4B21 had little binding ability (FIG. 3).

In order to confirm the specificity of the developed antibody, the ability to recognize SARS-CoV-2 NP and gene sequence 91% similar to SARS-CoV NP was confirmed.

For the ELISA technique, SARS-CoV NP (Sino biological) antigen was attached at 100 ng per well, and the experiment was performed in the same manner as above. The binding ability to the SARS-CoV NP antigen was confirmed in the order of B46F, 4B21, and 1G6 (FIG. 4).

The EC50 (effective concentration 50%, effective concentration until the ability to recognize the antigen reaches 50%) values that can quantify the binding ability of an antibody are 1G6 (5 ng/mL) for SARS-CoV-2 NP, B46F (68 ng/mL) and 4B21 (12690 ng/mL) were identified in that order, and the EC50s for SARS-CoV NPs were B46F (43 ng/mL), 4B21 (60 ng/mL), and 1G6 (6661 ng/mL). It was confirmed in order (Table 2).

Antibody EC ₅₀ (ng/mL) to
SARS-CoV-2 NPs EC ₅₀ (ng/mL) to
SARS-CoV-NPs 1G6 5 6661 B46F (Commercial mAb) 68 43 4B21 (Commercial mAb) 12690 60

These results indicate that the novel antibody 1G6 is an antibody having specific binding only to SARS-CoV-2 NP.

Example 5: SARS-CoV-2 virus diagnosis using anti-SARS-CoV-2 NP monoclonal antibody

In order to confirm that the novel anti-SARS-CoV-2 NP antibody can be used to detect the actual SARS-CoV-2 virus, it was confirmed using an inactivated SARS-CoV-2 lysate.

After attaching the inactivated SARS-CoV-2 lysate (Native Antigen) at 500 ng per well at 4° C. for 16 hours, ELISA was performed in the same manner as above.

As a result of measuring the absorbance, it was confirmed that the novel antibody 1G6 binds to SARS-CoV-2 lysate higher than commercially available antibodies (4B21, B46F) (FIG. 5).

EC50 values were also confirmed in the order of 1G6 (6 ng/mL), B46F (1246 ng/mL), and 4B21 (20760 ng/mL) (Table 3).

Antibody EC ₅₀ (ng/mL) to
SARS-CoV-2 viral lysates 1G6 6 B46F (Commercial mAb) 1246 4B21 (Commercial mAb) 20760

Based on these results, it is judged that the novel antibody 1G6 can be used to diagnose SARS-CoV-2 lysates.

Example 6. Confirmation of epitope recognition specificity of anti-SARS-CoV-2 NP monoclonal antibody

To investigate whether the anti-SARS-CoV-2 NP antibody recognizes a linear or conformational epitope of the SARS-CoV-2 NP antigen protein, western blot (WB) was performed.

Specifically, SARS-CoV-2 NPs (1 μg) were developed in reducing 12% SDS-PAGE conditions and transferred to a PVDF membrane. To prevent non-specific adhesion, the membrane was blocked with PBST supplemented with 5% skim milk for 1 hour at room temperature, and then reacted with the developed 1G6 monoclonal antibody (2 μg/mL) at 4°C for 12 hours. After washing the membrane three times for 10 minutes each time with PBST, it was further reacted with HRP-conjugated anti-mouse IgG (1:5000 dilution). Immunoreactive bands were reacted with enhanced chemi-luminescence (ECL) and visualized using an Azure C300 Western blot imager (Azure Biosystems).

As a result of the confirmation, the 1G6 antibody detected the protein at the position of the predicted SARS-CoV-2 NP antigen protein having a molecular weight of 47.08 kDa, suggesting that these antibodies can recognize the linear epitope of the SARS-CoV-2 NP protein (Fig. 6).

Example 7. Confirmation of anti-NP antibody specificity for intracellularly expressed SARS-CoV-2 NP antigen

In order to confirm that the anti-SARS-CoV-2 antibody 1G6 derived from inactivated SARS-CoV-2 immunized mice can recognize the intracellularly expressed SARS-CoV2 NP protein, it was confirmed by Western blotting.

Specifically, the NP-10xHis (NP-His) expression vector was transduced into human embryonic kidney cells (HEK293T) using polyethylenimine (PEI). After 72 hours, the cells were collected and dissolved in RIPA buffer [PBS containing 1.0% (v/v) NP40, 0.1% Sodium Dodecyl Sulfate, 0.5% Sodium Deoxycholate, protease inhibitor cocktail (Roche)] and used as a sample for protein analysis. Protein quantification of the cell lysate was performed by the Bradford method. Prepared protein samples were developed in reducing 12% SDS-PAGE conditions by 10 μg, and transferred to a PVDF membrane. The membrane onto which the protein was transferred was blocked by soaking in a TBS solution containing 5% (w/v) skim milk, and then purified anti-NP antibodies, 1G6 antibody (0.5 μg/mL) or mouse anti-6xHis antibody (1: 5000, Qiagen) was diluted in a blocking solution and treated with a primary antibody. After treatment with the primary antibody, sufficient washing with TBST (TBS containing 0.1% (v/v) tween-20), treatment with anti-mouse IgG-HRP as the secondary antibody, and protein bands bound to the antibody were detected by enhanced chemiluminescence. Confirmed.

As a result, the 1G6 anti-NP antibody binding band was confirmed at the same position as the anti-6xHis antibody binding band of the NP-His protein having the predicted molecular weight of 47.33 kDa (FIG. 7).

Example 8. Confirmation of immunoprecipitation ability of anti-NP antibody against SARS-CoV-2 NP antigen expressed in cells

Based on the specific reactivity of the 1G6 anti-NP antibody to the SARS-CoV-2 NP antigen expressed in cells in Example 7, the immunoprecipitation ability of these antibodies against the NP antigen was verified.

Specifically, for antigen immunoprecipitation, the NP-10xHis expression vector was transduced into human embryonic kidney cells (HEK293T) using PEI. After 72 hours, cells were collected, dissolved in RIPA buffer, and used as samples for immunoprecipitation protein analysis. The cell lysate was quantified by the Bradford method, and 800 μg of the protein sample was mixed with 5 μg of 1G6 antibody or negative control antibody of the same subtype (IgG2a) and mixed well at 4° C. for 16 hours to induce antigen-antibody binding. . Antibodies bound to the antigen were captured with Protein G resin (Santacruz) at 4°C for 4 hours. After the capture reaction, the resin was sufficiently washed with RIPA buffer, 40 μl of SDS-PAGE sample buffer was added, and immunoprecipitated protein samples containing the antigen were prepared by heating at 100°C. Protein samples were developed under reducing 12% SDS-PAGE conditions, transferred to a PVDF membrane, and then Western blotted with anti-rabbit-IgG-HRP to detect non-specific protein bands. After confirming that it was not, rabbit anti-6xHis antibody (Rabbit anti-6xHis-mAb, Sigma-Aldrich) was treated as the primary antibody. After treatment with the primary antibody, sufficient washing with TBST, treatment with anti-rabbit IgG-HRP as the secondary antibody, and immunoprecipitation protein bands were confirmed by enhanced chemiluminescence.

As a result, it was confirmed that the NP-10xHis protein having a molecular weight of 47.33 kDa was detected by the rabbit anti-6xHis antibody only in the immunoprecipitation sample using the 1G6 antibody, confirming the immunoprecipitation ability of the anti-NP antibody 1G6 (FIG. 8). .

Example 9. Fine Epitope Mapping of Anti-NP 1G6 Antibody in SARS-CoV-2 NP Protein

NP, one of the structural proteins of SARS-CoV-2, is responsible for packaging the genome of SARS-CoV-2, has 419 amino acids, and has N-arm (sequence 1 to 46 amino acids) RNA binding site N-terminal domain (sequence 47-174 amino acids), a linker with multiple serine and arginine (SEQ ID NOs: 175-247 amino acids), a C-terminal domain that allows NP proteins to form dimers (SEQ ID NOs: 248-364 amino acids), a C-tail (SEQ ID NOs: 365 ~419 amino acids). As a result of Example 3, it was confirmed that the 1G6 antibody derived from inactivated SARS-CoV-2 immunized mice specifically binds to NP among structural proteins of SARS-CoV-2. In addition, a more elaborate epitope was determined.

Specifically, an NP recombinant protein (Sino Biological Co.) and two NP partially deleted proteins having the entire amino acid sequence were cloned. 9 shows a schematic diagram and amino acid sequence information of NP domain configuration and expressed NP recombinant proteins (NP-His, NP-p1-His, NP-p2-His). To this end, using each of the specific primers shown in Table 4 and pfu DNA polymerase (Biofact), SARS-CoV-2 Nucleocapsid Gene ORF cDNA clone expression plasmid, C-His tag (Sino Biological) 10 ng as a template A PCR reaction was performed.

primer order One NP F 5' agcAAGCTTgccaccATGAGTGACAATGGACCACAGA 3' 2 NP-p1R 5' agcGATATCCTCAGCATAGAAGCCCTTTG 3' 3 NP-p2R 5' agcGATATCGGTCACTGTTTGTCCCTGTTG 3' 4 IgG2a heavy chain F 5' cttccggaattcSARGTNMAGCTGSAGSAGTCWGG 3' 5 IgG2a heavy chain R 5' ggaagatctCTTGACCAGGCATCCTAGAGT 3' 6 kappa light chain F 5' gggagctcGAYATTTGTGMTSACMCARWCTMCA 3' 7 kappa light chain R 5' ggtgcatgcGGATACAGTTGGTGCAGCATC 3'

After treating the secured gene and pCMV3 vector (Sino Biological Co.) with restriction enzymes (HindIII/EcoRV) at 37°C for 15 hours, the cut gene and vector were mixed at a certain ratio and ligase (Roche Co.) was added. After that, a ligation reaction was performed at 16° C. for 16 hours. The conjugation reaction mixture was transformed into DH5α, spread on LB-restricted medium plates containing kanamycin antibiotic, and incubated at 37°C for 15 hours. The colonies grown on the plate were selected and cultured in LB-limited liquid medium to isolate the amplified recombinant plasmid. The pCMV3 and NPp2-10xHis/pCMV3 expression vectors were transduced into human embryonic kidney cells (HEK293T) using PEI. After 72 hours, cells were collected, dissolved in RIPA buffer, and used as protein analysis samples.

Protein quantification of the cell lysate was performed by the Bradford method. Prepared protein samples were developed in reducing 12% SDS-PAGE conditions by 20 μg, and transferred to a PVDF membrane. The membrane onto which the protein was transferred was blocked by soaking in a TBS solution containing 5% (w/v) skim milk, and then purified anti-NP antibody (0.5 μg/mL) and mouse anti-6xHis antibody (1 :5000, Qiagen) was diluted in a blocking solution and treated with a primary antibody. After treatment with the primary antibody, it was thoroughly washed with TBST, treated with anti-mouse IgG-HRP as the secondary antibody, and the antibody-reactive protein band was confirmed by enhanced chemiluminescence.

As a result, the 1G6 antibody bound only to NP-His (47.33 kDa, amino acid sequence 1 to 419), and based on this, the C-terminus (sequence 248 to 419 amino acid sequence) of SARS-CoV-2 NP was determined as the epitope. (FIG. 10).

Example 10. Analysis of complementarity determining regions (CDRs) of anti-NP antibodies

After confirming that the novel 1G6 antibody specifically recognizes the C-terminal part of the SARS-CoV-2 NP protein, the complementarity determining region (CDR) sequence of the antibody was analyzed to obtain information on the antigen recognition specificity of the 1G6 antibody. .

Specifically, about 10 ⁶ cells producing the 1G6 antibody were collected and total RNA was extracted using an RNA extraction kit (Nanohelix). cDNA was synthesized from 5 μg of total RNA using a complementary DNA synthesis kit (Promega), and polymerase chain using 1 μg of synthesized cDNA and the mouse heavy chain region primers and mouse light chain region primers in Table 4. The CDR-containing regions of the mouse heavy and light chains were amplified by reaction (PCR). The amplified DNA was ligated with pTOP Blunt V2 vector using TOPcloner Blunt kit (Enzynomics). The recombinant plasmid was transformed into E. coli DH5α, plated on an LB-restricted medium plate containing ampicillin, and incubated at 37°C for 15 hours. Each colony formed on the plate was cultured in LB liquid medium for 12 hours or more, and the plasmid was extracted and the corresponding nucleotide sequence was analyzed. The protein sequence was determined from the analyzed nucleotide sequence, and the CDR of the 1G6 antibody was determined by analyzing it based on the Kabat CDR definition (FIG. 11).

Preparation Example: Preparation of monoclonal antibody binding to N-terminus of coronavirus NP protein and confirmation of construction

Among the 503 initial clones obtained in Example 1, clones having a high optical density (OD) value were additionally selected, and 3E10, 3F10, 5B6, and 5B6.8 anti-NP antibodies were selected by performing a monoclonal hybridoma selection process (Fig. 13a). Their properties were confirmed in the same manner as in the previous example.

As a result of confirming the monoclonal antibody subtypes, 3E10 was an IgG2b kappa-chain subtype, 3F10 and 5B6 were an IgG2a kappa-chain subtype, and 5B6.8 was an IgG3 kappa-chain subtype. 3E10, 3F10, 5B6, and 5B6.8 showed high absorbance only to the NP antigen, but showed no reactivity to the other antigens (S1, S2, RBD) (FIG. 13b). The results of confirming the response to SARS-CoV-2 NPs and SARS-CoV NPs are shown in FIGS. 14a and b and the table below. It was confirmed that all four new antibodies recognizing the SARS-CoV-2 NP antigen were capable of recognizing the SARS-CoV-2 NP protein with high sensitivity, similar to or higher than commercially available antibodies, as well as 5B6 and 3E10. It was confirmed that the antibody had high sensitivity to recognize CoV NP.

Antibody EC ₅₀ (ng/mL) to
SARS-CoV-2 NPs EC ₅₀ (ng/mL) to
SARS-CoV NPs 3E10 2 2 3F10 369 10 5B6 4 4 5B6.8 437 1316 B46F (Commercial mAb) 68 43 4B21 (Commercial mAb) 12690 60

In addition, as a result of conducting the experiment using SARS-CoV-2 lysate in the same manner as in Example 5, all four novel antibodies (3E10, 3F10, 5B6, 5B6.8) were commercially available antibodies (4B21, B46F) Higher binding to SARS-CoV-2 lysate (FIG. 14c), EC50 values were also 3E10 (4 ng/mL), 5B6 (10 ng/mL), 3F10 (10 ng/mL), 5B6.8 (1056 ng /mL), B46F (1246 ng/mL), and 4B21 (20760 ng/mL) in order (Table 6).

Antibody EC ₅₀ (ng/mL) to
SARS-CoV-2 viral lysates 3E10 4 3F10 10 5B6 10 5B6.8 1056 B46F (Commercial mAb) 1246 4B21 (Commercial mAb) 20760

As a result of confirming the epitope recognition specificity of SARS-CoV-2 of the four antibodies through Western blotting in Example 6, it was confirmed that the four antibodies detected proteins at the SARS-CoV-2 NP antigen protein position (FIG. 15a) , it was confirmed to recognize the linear epitope of the SARS-CoV-2 NP protein. In addition, it was confirmed by the method of Example 7 that the SARS-CoV2 NP protein expressed in cells of four anti-SARS-CoV-2 antibodies derived from inactivated SARS-CoV-2 immunized mice was confirmed. The results are shown in FIG. 15B. The result of verifying the immunoprecipitation ability in the same manner as in Example 8 is shown in FIG. 15C.

As a result of CDR analysis in the same manner as in Example 10, it was confirmed that 3E10, 3F10, 5B6, and 5B6.8 had sequences as shown in FIGS. 16a to 16d. Among them, 3E10, 3F10, and 5B6 were selected and used in Examples 11 to 14.

Example 11. Additional analysis of epitopes

Based on the information confirmed in Example 9, an experiment was performed to identify the epitope of the anti-NP antibody in more detail. SARS-CoV-2 NP full-length (amino acids 1-419) protein and 7 partially defective recombinant proteins were cloned, and the defective proteins were NP-NTD at 47-174; NP-CTD numbers 248-364; NP-p1 is 1-174; NP-p2 is numbered 1-248; NP-p3 is numbered 1-305; 101-419 for NP-p4; NP-p5 has amino acids 248-419 (FIG. 17A). The obtained NP full-length or partially deleted gene and pCMV3 vector (Sino Biological) were treated with restriction enzymes (HindIII/EcoRV) at 37 ° C for 15 hours, and then the cut gene and vector were mixed at a certain ratio to ligase (Roche g) was added, and a ligation reaction was performed at 16° C. for 16 hours. The conjugation reaction mixture was transformed into DH5α, spread on LB-restricted medium plates containing kanamycin antibiotic, and incubated at 37°C for 15 hours. The colony grown on the plate was selected and cultured in LB-limited liquid medium to isolate the amplified recombinant plasmid. After confirming the nucleotide sequence of the gene introduced into the isolated plasmid, the expression vector was transferred to human embryonic kidney cells (HEK293T) using PEI. transduced. After 72 hours, cells were collected, dissolved in RIPA buffer, and used as protein analysis samples.

Protein quantification of the cell lysate was performed by the Bradford method. Prepared protein samples were developed in reducing 12% SDS-PAGE conditions by 20 μg, and transferred to a PVDF membrane. The membrane onto which the protein was transferred was blocked by soaking in a TBS solution containing 5% (w/v) skim milk, and then purified anti-NP antibody (0.5 μg/mL) or mouse anti-6xHis antibody (1 :5000, Qiagen) was diluted in a blocking solution and treated with a primary antibody. After treatment with the primary antibody, it was thoroughly washed with TBST, treated with anti-mouse IgG-HRP as the secondary antibody, and the antibody-reactive protein band was confirmed by enhanced chemiluminescence.

As a result, it was confirmed that the 364 to 419 amino acid region of the NP protein was the epitope of the 1G6 antibody (FIG. 17B). On the other hand, it was confirmed that the epitope of the 3F10 antibody was the 1st to 47th amino acid region of the NP protein, and it was confirmed that the 47th to 101st amino acid region of the NP protein was the epitope of 3E10 and 5B6.

Example 12. Sandwich ELISA and SARS-CoV-2 detection using antibody pairs

Sandwich ELISA is a method widely used in the diagnostic industry. An antibody (capture antibody, Capture Ab) for an antigen is first attached to the bottom of a plate, an antigen is bound to the antibody, and then another antigen (detection antibody, Detection Ab) is used. to be detected either directly or indirectly. Since this method uses two antibodies specific to an antigen, an antibody pair having different antigenic determining regions is used, and it is most widely used due to its high sensitivity.

In order to confirm whether the anti-NP antibody obtained through this study is applicable to sandwich ELISA, 100 ng per well was treated with 1G6 as a capture antibody on the bottom of a MaxiSorp 96-well plate (Thermo Scientific) and incubated at 4 ° C for 12 hours. Attached. In order to remove non-adhered substances, the plate was washed three times with TBS (TBST) containing 0.05% Tween-20, and then treated with 5% BSA-added TBST at 37 ° C for 2 hours to prevent non-specific reactions. did 300 ng of SARS-CoV-2 NP recombinant protein (Fig. 18 left) or virus lysate (Fig. 18 right) was added per well, incubated at 37 ° C. for 2 hours, and washed three times with TBST to remove the residue. . An HRP-conjugated antibody used as a detection antibody was prepared using an HRP conjugation kit (Abcam), and 10 ng of HRP-conjugated anti-NP antibody was added per well and reacted at 37° C. for 1 hour. Finally, after washing the plate 5 times with TBST, the reaction mixture was developed with chromogenic tetramethylbenzidine substrate (TMB) and the reaction was terminated with 2N H2SO4. Results were analyzed as optical density (OD) values at 450 nm using a VICTOR Nivo plate reader (PerkinElmer).

Among various antibody pairs, it was confirmed that 1G6 and 3E10 were the most effective capture and detection antibody pairs in the sandwich ELISA method for detecting SARS-CoV-2 NP antigen protein (Fig. 18 left). It was confirmed that the most effective antibody pairs in the sandwich ELISA method for detecting SARS-CoV-2 virus lysate were 1G6 and 3F10, and 1G6 and 3E10 (Fig. 18 right). No detection was seen when influenza NP-HRP was used as a negative control. Through the above results, it was confirmed that 1G6 and 3E10 were the best antibody pairs for detecting SARS-CoV-2 NP as well as virus lysates in the Sandwich ELISA method.

Example 13. Confirmation of binding ability to SARS-CoV-2 mutant virus NP

Viruses are characterized by genetic mutations, and amino acid mutations have also been reported in SARS-CoV-2 mutant virus NP (Fig. 19 left). Therefore, since the binding ability of anti-NP antibodies may change, the binding ability of the 1G6 antibody to SARS-CoV-2 mutant virus NP was confirmed through ELISA. Anti-NP antibodies were serially diluted and added to wells coated with the SARS-CoV-2 mutant virus NP antigen, and bound antibodies were detected with an anti-mouse IgG-HRP antibody. It was confirmed that the 1G6 antibody had binding ability to the NP of the alpha, beta, and gamma mutant SARS-CoV-2 viruses.

Specifically, 100 ng per well was coated on a maxisorp ELISA plate using SARS-CoV-2 alpha, beta, gamma and delta mutant NP recombinant proteins (Sino Biological), blocked with 5% BSA in PBS, and then continuously 3-fold diluted monoclonal antibodies (3E10, 3F10, 5B6) were added and incubated for 2 hours. After washing with PBST, anti-mouse IgG-HRP was added and incubated for 1 hour. Finally, the plate was washed 5 times with TBST, and the reaction was developed using chromogenic tetramethylbenzidine substrate (TMB) and 2N H ₂ SO ₄ The reaction was terminated with Results were analyzed as optical density (OD) values at 450 nm using a VICTOR Nivo plate reader (PerkinElmer).

As a result of the experiment, it was confirmed that the 1G6 antibody bound well to SARS-CoV-2 alpha, beta, gamma, and delta mutant NP proteins and had the ability to detect the variants (right side of FIG. 19).

Example 14. Detection of sandwich ELISA and SARS-CoV-2 mutant virus using antibody pairs

Based on the results of Example 13, the NP detection ability of the SARS-CoV-2 mutant virus using sandwich ELISA was confirmed.

The specific experimental method was the same as in Example 13, and the ELISA plate was coated with 3E10 capture antibody, blocked, and then SARS-CoV-2 mutant recombinant NP protein was added and incubated for 2 hours. After washing the plate, 1G6-HRP was used as a detection antibody. Influenza H1N1 NP and BSA were used as negative controls. Experimental results are shown in FIG. 20 .

In the sandwich ELISA loaded with 3E10 and 1G6 antibody pairs, it was confirmed that all NPs of alpha, beta, and gamma mutant SARS-CoV-2 viruses were detected. It was confirmed that the negative control, HRP-bound influenza NP antibody or BSA was not detected at all. That is, it was confirmed that the 3E10-1G6 antibody pair of the present invention can be usefully used to detect SARS-CoV-2 mutant virus.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Claims (14)

An antibody or antigen-binding fragment thereof that specifically binds to the C-terminus of a coronavirus nucleocapsid protein, wherein the epitope of the antibody or antigen-binding fragment thereof is 364 to 419 from the N-terminus of SEQ ID NO: 19 Which is contained in the amino acid sequence of Burn, antibody or binding fragment thereof.
The method of claim 1, wherein the antibody or antigen-binding fragment thereof
HCDR1 of SEQ ID NO: 2;
HCDR2 of SEQ ID NO: 4, and
a heavy chain variable region comprising HCDR3 of SEQ ID NO: 6; and
LCDR1 of SEQ ID NO: 10;
LCDR2 of SEQ ID NO: 12, and
An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising LCDR3 of SEQ ID NO: 14.
The method of claim 2, wherein the antibody or antigen-binding fragment thereof
HFR (heavy chain framework region (FR)) of SEQ ID NO: 1;
HFR2 of SEQ ID NO: 3;
HFR3 of SEQ ID NO: 5, and
a heavy chain variable region comprising HFR4 of SEQ ID NO: 7; and
LFR (light chain FR) 1 of SEQ ID NO: 9;
LFR2 of SEQ ID NO: 11;
LFR3 of SEQ ID NO: 13, and
The antibody or antigen-binding fragment thereof comprising a light chain variable region comprising LFR4 of SEQ ID NO: 15.
The method of claim 3, wherein the antibody or antigen-binding fragment thereof
Further comprising the heavy chain tail domain of SEQ ID NO: 8, the antibody or antigen-binding fragment thereof.
The method of claim 3, wherein the antibody or antigen-binding fragment thereof
Further comprising a light chain tail domain of SEQ ID NO: 16, the antibody or antigen-binding fragment thereof.
The method of claim 3, wherein the antibody or antigen-binding fragment thereof
The antibody or binding fragment thereof comprising the heavy chain variable region of SEQ ID NO: 17 and the light chain variable region of SEQ ID NO: 18.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the coronavirus is SARS-CoV-2.
A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.
An expression vector comprising the polynucleotide of claim 8.
A host cell comprising the expression vector of claim 9.
A composition for diagnosing coronavirus infection, comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 6.
12. The method of claim 11, wherein the composition
(i) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 28, HCDR2 of SEQ ID NO: 30, and HCDR3 of SEQ ID NO: 32; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising LCDR1 of SEQ ID NO: 36, LCDR2 of SEQ ID NO: 38, and LCDR3 of SEQ ID NO: 40;
(ii) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 46, HCDR2 of SEQ ID NO: 48, and HCDR3 of SEQ ID NO: 50; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising LCDR1 of SEQ ID NO: 54, LCDR2 of SEQ ID NO: 56, and LCDR3 of SEQ ID NO: 58; and
(iii) a heavy chain variable region comprising HCDR1 of SEQ ID NO: 64, HCDR2 of SEQ ID NO: 66, and HCDR3 of SEQ ID NO: 68; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising LCDR1 of SEQ ID NO: 72, LCDR2 of SEQ ID NO: 74, and LCDR3 of SEQ ID NO: 76; Including any one or more selected from, the composition.
A kit for diagnosing coronavirus infection, comprising the composition of claim 11.
Claims 1 to 6 of any one of the antibody or antigen-binding fragment thereof; Or a composition for diagnosing infection comprising the same; Or a method for providing information for diagnosing coronavirus infection, comprising the step of detecting coronavirus present in a biological sample isolated from a subject suspected of being infected with coronavirus using a kit containing the composition.

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