WO2022231321A1 - Diagnostic composition or kit comprising sars-cov-2 rbd antigen-specific antibody - Google Patents

Abstract

The present invention relates to: a diagnostic composition comprising an antibody that specifically binds to RBD of a SARS-CoV-2 S protein; or a kit using Sandwich ELISA technology utilizing the composition. The diagnostic composition or kit of the present invention can be used to determine the presence of the SARS-CoV-2 virus in a SARS-CoV-2 infection (COVID-19) patient.

Description

Diagnostic composition or kit comprising SARS-COV-2 RBD antigen-specific antibody

The present invention was made by the project specific number 1711132191 and project number 2020M3A9I2107093 under the support of the Ministry of Science and ICT of the Republic of Korea. The title is "Development of a new clinical candidate material for neutralizing the SARS-CoV2 target", the lead institution is Kookmin University Industry-University Cooperation Foundation, and the research period is 2020.07.01-2022.12.31.

This patent application is based on the Korean Patent Application Nos. 10-2021-0054600 and 10-2021-0054603 submitted to the Korean Intellectual Property Office on April 27, 2021, and the Korean Patent Application filed with the Korean Intellectual Property Office on February 14, 2022. Priority is claimed to Application No. 10-2022-0019199, the disclosure of which is incorporated herein by reference.

The present invention relates to a diagnostic composition or kit comprising an antibody that specifically binds to the RBD of SARS-CoV-2 S protein. More specifically, the present invention relates to a diagnostic composition comprising an antibody that specifically binds to the RBD of SARS-CoV-2 S protein or a kit using the Sandwich ELISA technique using the same.

The COVID-19 infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been prevalent since January 2020, infecting 100 million people worldwide and causing more than 2 million deaths within one year. It is a terrible infectious disease. Due to the high transmission and risk of SARS-CoV-2, many researchers are making great efforts to develop a rapid and efficient method for diagnosing SARS-CoV-2 infection.

In order to detect and confirm SARS-CoV-2 infection, an immunoassay using an antigen-antibody reaction or a molecular diagnostics method for detecting viral nucleic acids (genes) are used. As a research for rapid diagnosis, a diagnostic kit using RT-PCR, one of molecular diagnostic methods, has been developed, but it still takes a long time to make a final diagnosis, and there is a problem that it cannot be diagnosed right away without an expert and a machine for diagnosis. have. On the other hand, immunoassays mainly using Enzyme-Linked ImmunoSorbent Assay (ELISA) enable rapid and efficient detection because they are relatively simple compared to molecular diagnostics and require shorter analysis time.

In particular, the spike protein (S protein) is located on the surface of SARS-CoV-2 and binds to angiotensin-converting enzyme 2 (ACE2), a receptor on the surface of the host cell, a key marker to induce infection. is a molecule Diagnosis of SARS-CoV-2 infection using ELISA is performed by targeting the spike protein, and by using an antibody that specifically binds to it, it is possible to sensitively and accurately confirm whether or not SARS-CoV-2 is infected.

However, in view of the ongoing COVID-19 pandemic caused by SARS-CoV-2, there is an urgent need to develop a more efficient method compared to conventional diagnostic kits. Therefore, it was attempted to develop a diagnostic kit using the Sandwich ELISA technique using an antibody with high affinity and specificity to SARS-CoV-2.

The present inventors made intensive research efforts to develop a method for more rapidly and accurately diagnosing a corona-19 (COVID-19) infectious disease. As a result, a diagnostic composition comprising an antibody that specifically binds to the RBD of SARS-CoV-2 S protein or a kit using the Sandwich ELISA technique was developed. Through this, the present invention was completed by identifying that it is possible to significantly improve the diagnostic efficiency of the COVID-19 infectious disease.

Accordingly, an object of the present invention is to provide a composition for diagnosing SARS-CoV-2 infection (COVID-19) or detecting a SARS-CoV-2 antigen comprising an antibody that specifically binds to the RBD of the SARS-CoV-2 S protein. will do

Another object of the present invention is to provide a kit for diagnosing SARS-CoV-2 infection (COVID-19) or a kit for detecting SARS-CoV-2 antigen.

Another object of the present invention is to provide a method for detecting SARS-CoV-2 virus.

The present inventors have disclosed a diagnostic composition comprising an antibody that specifically binds to the RBD region of SARS-CoV-2 S protein, or the same A kit using the Sandwich ELISA technique was developed.

As used herein, the term "SARS-CoV-2" refers to a coronavirus that causes a worldwide pandemic COVID-19 infectious disease.

SARS-CoV-2 has four major structural proteins, specifically, the Spike; S protein (below, S protein), the envelope (E) protein, the membrane (M) protein, and the nucleocapsid; N) It is a coronavirus with proteins. The N protein is a protein that surrounds the RNA genome, whereas the three structural proteins other than the N protein are components of the viral envelope. The S protein contains an S1 domain that mediates adhesion and an S2 domain that mediates fusion of the viral membrane with the host cell. In particular, the S1 domain includes a receptor binding domain (RBD), which is a serious COVID-19 ( COVID-19) is known to cause infectious diseases.

As used herein, the term “receptor binding domain (hereinafter, RBD)” refers to a target region capable of confirming the presence or absence of SARS-CoV-2 using the diagnostic composition or kit of the present invention.

Therefore, according to one aspect of the present invention, the present invention provides the following antibodies or pair of antigen-binding fragments thereof that specifically bind to the receptor binding domain (RBD) of SARS-CoV-2 S protein. or antigen binding fragments thereof), providing a composition for diagnosing SARS-CoV-2 infection (COVID-19) or detecting SARS-CoV-2 antigen:

(i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5 an antibody or antigen-binding fragment thereof comprising LCDR2 comprising the amino acid sequence of, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; and

(ii) HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13 An antibody or antigen-binding fragment thereof comprising LCDR2 comprising the amino acid sequence of, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 14.

In one embodiment of the present invention, the antibody or antigen-binding fragment thereof of (i) is a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 ( VL), wherein the antibody or antigen-binding fragment thereof of (ii) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16, SARS-CoV -2 To provide a composition for diagnosing infection (COVID-19) or detecting a SARS-CoV-2 antigen.

In one embodiment of the present invention, the antibody or antigen-binding fragment pair of the composition is derived from clones K102.1 and K102.2 selected in Examples of the present invention.

In one embodiment of the present invention, (i) includes the amino acid sequence of SEQ ID NO: 17, and (ii) includes the amino acid sequence of SEQ ID NO: 18, but is not limited thereto.

In one embodiment of the present invention, the anti-SARS-CoV-2 S protein RBD antibody or antigen-binding fragment pair of the composition of the present invention is specific for the receptor binding domain (RBD) of the SARS-CoV-2 S protein. combine with Accordingly, the antibody or antigen-binding fragment pair of the composition can be used to detect SARS-CoV-2 S protein or to diagnose SARS-CoV-2 infection (COVID-19).

In a specific embodiment of the present invention, the antibody or antigen-binding fragment pair of the composition has different epitopes targeting the RBD of the SARS-CoV-2 S protein.

As used herein, the term “epitope” refers to a specific antigenic determinant, recognized as binding by a paratope to an antibody for diagnosis or detection of a subject. Specifically, as a target for diagnosing or detecting SARS-CoV-2 virus in the present specification, antibodies having different antigenic determinants on the RBD of the SARS-CoV-2 S protein were selected, and the use of the antibody pair is faster. and allow for an accurate examination.

In one embodiment of the present invention, the antibody or antigen-binding fragment pair of the composition of the present invention specifically binds to a mutant virus in which the S protein of SARS-CoV-2 is mutated.

In a specific embodiment of the present invention, the antibody or antigen-binding fragment pair of the composition specifically binds to a mutant virus in which the RBD region of the S protein of SARS-CoV-2 is mutated. The amino acid sequence of the RBD region is shown in SEQ ID NO: 19.

In a more specific embodiment of the present invention, the mutant virus in which a mutation in the RBD region of SARS-CoV-2 to which the antibody or antigen-binding fragment pair of the composition specifically binds is A435S mutation at the 435th amino acid position of RBD , N354D mutation at amino acid position 354, G476S mutation at amino acid position 476, V483A mutation at amino acid position 483, F342L mutation at amino acid position 342, V341I mutation at amino acid position 341, N501Y mutation at amino acid position 501, and It is selected from the group consisting of mutant viruses having L452R/T478K mutations at amino acid positions 452 and 478, but is not limited thereto.

In a more specific embodiment of the present invention, the mutant virus in which the RBD region of SARS-CoV-2 is mutated is selected from the group consisting of, but is not limited thereto:

(i) The A435S mutation refers to a SARS-CoV-2 mutant virus including RBD in which Alanine at the 435th amino acid residue of the RBD-wild type represented by the amino acid sequence of SEQ ID NO: 19 is mutated to Serine. The amino acid sequence of the A435S mutant RBD is shown in SEQ ID NO: 20.

(ii) the N354D mutation refers to a SARS-CoV-2 mutant virus including RBD in which the 354 amino acid residue Asparagine of the RBD wild type (RBD-wild type) represented by the amino acid sequence of SEQ ID NO: 19 is mutated to aspartic acid. . The amino acid sequence of the N354D mutant RBD is shown in SEQ ID NO: 21.

(iii) The G476S mutation refers to a SARS-CoV-2 mutant virus including RBD in which the 476th amino acid residue Glycine of the RBD-wild type represented by the amino acid sequence of SEQ ID NO: 19 is mutated to Serine. The amino acid sequence of the G476S mutant RBD is shown in SEQ ID NO: 22.

(iv) The V483A mutation refers to a SARS-CoV-2 mutant virus including RBD in which the 483 amino acid residue Valine of the RBD-wild type represented by the amino acid sequence of SEQ ID NO: 19 is mutated to Alanine. The amino acid sequence of the V483A mutant RBD is shown in SEQ ID NO: 23.

(v) the F342L mutation refers to a SARS-CoV-2 mutant virus including RBD in which the 342 amino acid residue Phenylalanine of the RBD-wild type represented by the amino acid sequence of SEQ ID NO: 19 is mutated to Leucine. The amino acid sequence of the F342L mutant RBD is shown in SEQ ID NO: 24.

(vi) The V341I mutation refers to a SARS-CoV-2 mutant virus including RBD in which the 341 amino acid residue Valine of the RBD-wild type represented by the amino acid sequence of SEQ ID NO: 19 is mutated to Isoleucine. The amino acid sequence of the V341I mutant RBD is shown in SEQ ID NO: 25.

(vii) The N501Y mutation refers to a SARS-CoV-2 mutant virus including RBD in which the 501st amino acid residue Asparagine of the RBD-wild type represented by the amino acid sequence of SEQ ID NO: 19 is mutated to Tyrosine. The amino acid sequence of the N501Y mutant RBD is shown in SEQ ID NO: 26.

And, (viii) the L452R / T478K mutation is that the 452 amino acid residue Leucine of the RBD wild type (RBD-wild type) represented by the amino acid sequence of SEQ ID NO: 19 is mutated to Arginine, and the 478th amino acid residue Threonine is mutated to Lysine It refers to a SARS-CoV-2 mutant virus including RBD. The amino acid sequence of the L452R/T478K mutant RBD is shown in SEQ ID NO: 27.

Antibodies of the present invention can be generated using various phage display methods known in the art [Brinkman et al., 1995, J. Immunol. Methods, 182:41-50]; [Ames et al., 1995, J. Immunol. Methods, 184, 177-186]; [Kettleborough et al. 1994, Eur. J. Immunol, 24, 952-958]; [Persic et al., 1997, Gene, 187, 9-18]; and Burton et al., 1994, Adv. Immunol., 57, 191-280]; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; and WO 95/20401; and US 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743; and 5,969,108.

As used herein, the term "antibody" refers to an antibody that specifically binds to a specific antigen, and includes not only a complete antibody form but also an antigen binding fragment of an antibody molecule. Specifically, as used herein, the term "antibody" refers to an antibody that specifically binds to receptor binding protein (RBD) of SARS-CoV-2 S protein, and includes not only a complete antibody form but also an antigen binding fragment of an antibody molecule. includes

A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The constant region of the antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types. and has subclasses gamma1(γ1), gamma2(γ2), gamma3(γ3), gamma4(γ4), alpha1(α1), and alpha2(α2). The constant region of the light chain has kappa (κ) and lambda (λ) types (Cellular and Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50, W. B. Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, sinauer Associates, Inc., Sunderland, MA (1984)).

As used herein, the term "antigen binding fragment" refers to a fragment having an antigen-binding function, Fab, F(ab'), F(ab') ₂ , chemically linked F(ab') ) ₂ and Fv, and the like. Among antibody fragments, Fab (fragment antigen binding) has a structure having a light chain and heavy chain variable regions, a light chain constant region and a heavy chain first constant region (CH1), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab') ₂ antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and a recombinant technique for generating an Fv fragment is described in PCT International Patent Application Publication Nos. WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086 and WO 88/09344. In a double-chain Fv (tow-chain Fv), the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and single-chain variable fragment (scFv) is generally a heavy chain variable region and a single chain variable region through a peptide linker. The regions may be linked by a covalent bond or linked directly at the C-terminus to form a dimer-like structure like a double-stranded Fv. Such antibody fragments can be obtained by using proteolytic enzymes (for example, by restriction digestion of the whole antibody with papain to obtain Fab, and by digestion with pepsin to obtain F(ab′)2 fragments. ). Alternatively, it can be produced through genetic recombination technology.

In the present invention, the antibody or antigen-binding fragment thereof is specifically in the form of an scFv or a complete antibody. In addition, the heavy chain constant region may be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε). Preferably, the heavy chain constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3) or gamma 4 (IgG4), most specifically gamma 4 (IgG4) isotype. The light chain constant region may be of the kappa (κ) or lambda (λ) type, preferably of the kappa (κ) type. Accordingly, the specific antibody of the present invention is in the form of scFv or IgG4 having a kappa (κ) light chain and a gamma 4 (IgG4) heavy chain, but is not necessarily limited thereto.

As used herein, the term "heavy chain" refers to a hinge with the variable region domain VH and three constant region domains CH1, CH2 and CH3 of an antibody comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. (Hinge) refers to both full-length heavy chains and fragments thereof. Also herein, the term "light chain" refers to a full-length light chain comprising a variable region domain VL and a constant region domain CL of an antibody comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a full-length light chain thereof It means all fragments.

As used herein, the term “complementarity determining region (CDR)” refers to an amino acid sequence of a hypervariable region of an immunoglobulin heavy chain and light chain (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)). Each of the heavy chains (HCDR1, HCDR2 and HCDR3) and light chain (LCDR1, LCDR2 and LCDR3) contains three CDRs, and a framework region (FR) portion exists between these CDRs to support the CDR rings. do The CDR is a ring-shaped region involved in antigen recognition, and determines the specificity of the antibody for the antigen by providing a major contact residue in binding the antibody to an antigen or epitope.

The term “Framework” or “FR” refers to variable domain residues other than hypervariable region residues. The FRs of a variable domain generally consist of the four FR domains FR1, FR2, FR3 and FR4.

As used herein, the term “variable region” or “variable domain” refers to a domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have a similar structure, each domain of which contains four conserved framework regions and three hypervariable regions (HVR) ( Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, an antibody that binds to a specific antigen can be isolated using the VH or VL domain from an antibody that binds the antigen and screens a library of complementary VH or VL domains, respectively.

As used herein, the term "specifically binds" or the like means that an antibody or antigen-binding fragment thereof, or another construct such as an scFv specifically interacts with an antigen to undergo an immunological reaction. Specific binding is at least about 1 x 10 ^-6 M or less (eg, 9 x 10 ^-7 M, 8 x 10 ^-7 M, 7 x 10 ^-7 M, 6 x 10 ^-7 M, 5 x 10 ^-7 M , 4 x 10 ^-7 M, 3 x 10 ^-7 M, 2 x 10 ^-7 M, or 1 x 10 ^-7 M), preferably 1 x 10 ^-7 M or less (eg, 9 x 10 ^-8 M , 8 x 10 ^-8 M, 7 x 10 ^-8 M, 6 x 10 ^-8 M, 5 x 10 ^-8 M, 4 x 10 ^-8 M, 3 x 10 ^-8 M, 2 x 10 ^-8 M, or 1 x 10 ^-8 M), more preferably 1 x 10 ^-8 M or less (eg, 9 x 10 ^-9 M, 8 x 10 ^-9 M, 7 x 10 ^-9 M, 6 x 10 ^-9 M , 5 x 10 ^-9 M, 4 x 10 ^-9 M, 3 x 10 ^-9 M, 2 x 10 ^-9 M, or 1 x 10 ^-9 M) equilibrium dissociation constant (e.g., K _D less than this) indicates a tighter bond). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

As used herein, the term "human antibody" corresponds to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source using human antibody repertoires or other human antibody coding sequences. has an amino acid sequence.

Antibodies or antigen-binding fragments thereof in the composition of the present invention include full-length or intact polyclonal- or monoclonal-antibodies, as well as antigen-binding fragments thereof (eg, Fab, Fab' , F(ab')2, Fab3, Fv and variants thereof), fusion proteins comprising one or more antibody portions, human antibodies, humanized antibodies, chimeric antibodies, minibodies, diabodies, triabodies, tetrabodies, Glycosylation variants of antibodies, antibodies as linear antibodies, single chain antibodies (scFv), scFv-Fc, bispecific antibodies, multispecific antibodies, other modified configurations of immunoglobulin molecules comprising antigen recognition sites of the required specificity amino acid sequence variants and covalently modified antibodies. Specific examples of modified antibodies and antigen-binding fragments thereof include Nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs (bispecific T-cell engagers), TandAbs (tandem diabodies), DAFs (dual acting Fab), tow- These include in-one antibodies, SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs (dual variable domain immunoglobulin), CovX-bodies (peptide modified antibodies), duobodies and triomAbs. The list of such antibodies and antigen-binding fragments thereof is not limited to the above.

Specifically, in the composition of the present invention, the antibody or antigen-binding fragment pair that specifically binds to the RBD of the SARS-CoV-2 S protein comprises a heavy chain variable region and a light chain variable region comprising the CDRs of the above-described sequences. Monoclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies (scFv), Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv) and anti -idiotype (anti-Id) antibodies, and epitope-binding fragments of the antibodies, but are not limited thereto. Preferably, the pair of antibodies or antigen-binding fragments thereof of the composition is an anti-SARS-CoV-2 RBD scFv.

More specifically, the heavy chain variable region and the light chain variable region included in the antibody or antigen-binding fragment thereof of the composition are (Gly-Ser)n, (Gly2-Ser)n, (Gly3-Ser)n or (Gly4-Ser) They are linked by a linker such as n. Here, n is an integer of 1 to 6, specifically 3 to 4, but is not limited thereto. The light chain variable region and the heavy chain variable region of the scFv may exist in, for example, the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region

The definitions of the terms "antibody" and "antigen-binding fragment" used herein are the same as described above.

According to another aspect of the present invention, the present invention provides a pair of antibodies or antigen-binding fragments thereof that specifically bind to a receptor binding domain (RBD) of SARS-CoV-2 S protein. Antiben binding fragments therof) provides a kit for diagnosing SARS-Co-2 infection (COVID-19) or detecting SARS-CoV-2 antigen:

(i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5 an antibody or antigen-binding fragment thereof comprising LCDR2 comprising the amino acid sequence of, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; and

(ii) HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13 An antibody or antigen-binding fragment thereof comprising LCDR2 comprising the amino acid sequence of, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 14.

As used herein, the term “diagnosis” in a broad sense means judging the condition of a patient's disease or infection in all aspects. The content of the judgment is the name of the disease, the type of disease, the severity, the detailed mode of the disease, the presence or absence of complications, and the prognosis. For the purpose of the present invention, diagnosis is to determine whether SARS-CoV-2 virus infection or the possibility of developing COVID-19 due to infection of the virus using the composition or kit of the present invention.

As used herein, the term "detection" refers to confirming whether a subject is infected with a certain disease using the use of the substance or article of the present invention, or confirming the presence of a specific subject in a sample. Specifically, it is to confirm and identify the presence of SARS-CoV-2 virus using the composition or kit of the present invention.

Specifically, the "detection" refers to confirming the presence or absence of the antibody-antigen-antibody complex in the kit of the present invention using various labeling materials. The "antibody-antigen-antibody complex" refers to a combination of a SARS-CoV-2 RBD antigen reacted with an antibody pair of the present invention to confirm the presence or absence of SARS-CoV-2 virus from a sample. In addition, it refers to a combination of the SARS-CoV-2 virus itself and the antibody pair of the present invention. Formation of the antibody-antigen-antibody complex is performed by a colorimetric method, an electrochemical method, a fluorescence method, a luminometry, a particle counting method, and an absorbance measurement. , Spectrometric method, Raman spectroscopic method, Surface Plasmon Resonance method, Visual assessment, and scintillation counting method by a method selected from the group consisting of may be identified, but is not limited thereto.

As used herein, the term “Limit Of Detection (LOD)” refers to a detectable minimum amount or minimum concentration of an analyte in a measurement sample that can be reliably distinguished from an amount or concentration of a value of zero.

As used herein, the term “sensitivity” refers to a ratio to a change in a measured value according to a change in the amount or concentration of an analyte in a measurement sample. The greater the sensitivity (slope of the calibration curve) of the test method, the easier it is to check the small change in the amount or concentration of the analyte.

The term “sample” includes “biological sample” and “non-biological sample”. The "biological sample" includes, but is not limited to, nasal swabs, nasopharyngeal lavage fluid, bronchoalveolar lavage fluid, pleural fluid, sputum, tissue, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid, and urine. The "non-biological sample" includes environmental samples such as soil, water, air, food, and other samples.

SARS-CoV-2 virus can be detected by reacting these samples with a pair of antibodies or antigen-binding fragments thereof included in the composition of the present invention in an manipulated or unmanipulated state, or by using the kit of the present invention.

As used herein, the term “kit” refers to an assembly of similar means such as a solid support plate or test strip for diagnosing or detecting the presence or absence of a target substance. Thus, a kit consists of one or more other component compositions, solutions or devices suitable for diagnostic and analytical methods.

The kit may be manufactured using a method commonly used in the art. In particular, since the kit includes a pair of an antibody or antigen-binding fragment thereof of the present invention, it is basically possible to diagnose or detect SARS-C0V-2 virus through an antigen-antibody binding reaction. Therefore, it can be manufactured suitable for various immunoassays or immunostaining methods. For example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, flow cytometry, immunohistochemical staining, fluorescence immunoassay and It can be measured using the methods of enzyme substrate color development, preferably, enzyme-linked immunoassay (ELISA), most preferably, can be measured through sandwich enzyme-linked immunoassay (sandwich ELISA). The immunoassay or immunostaining method is described in Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; and Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

In addition, the kit includes not only the SARS-CoV-2 virus of the present invention, but also tools and reagents commonly used in the art for immunological analysis. Such tools and reagents include, but are not limited to, suitable carriers, solubilizers, detergents, buffers, stabilizers, and the like. Suitable carriers may be soluble carriers such as physiologically acceptable buffers known in the art, such as PBS, or insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylic It may be ronitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, or a combination thereof, but is not limited thereto.

Specifically, in one embodiment of the present invention, the kit is a kit using the Sandwich ELISA technique, and any one of the antibodies or antigen-binding fragments thereof of (i) and (ii) is a capture antibody, The other is used as a detection antibody.

As used herein, the term “Sandwich ELISA” refers to a method using two types of antibodies that specifically bind to a target substance in order to detect or quantitatively analyze the target substance. As described above, the present invention provides a composition comprising a pair of antibodies or antigen-binding fragments thereof having different epitopes of RBD of SARS-CoV-2 S protein, and any of the pairs of antibodies or antigen-binding fragments thereof One is used as a capture antibody, and the other is used as a detection antibody to form a sandwich-type antibody-antigen-antibody complex.

When the present invention is carried out in the Sandwich ELISA method, a specific embodiment of the present invention comprises the steps of (i) coating a capture antibody on the surface of a solid support; (ii) reacting the capture antibody with the SARS-CoV-2 RBD antigen in the sample; (iii) reacting the antigen on the capture antibody-antigen complex of step (ii) with the detection antibody; (iv) further reacting the result of step (iii) (capture antibody-antigen-detection antibody) with an antibody for signal detection to which a signal generating label is bound; and (v) measuring a signal generated from the labeling material.

As used herein, the term “capture antibody” refers to an antibody that is immobilized on a solid support that can be used as a reaction vessel and first binds to a target material.

Suitable as the solid support are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most particularly microtiter plates.

As used herein, the term "detection antibody" refers to an antibody capable of forming a sandwich-type complex of capture antibody-target material (antigen)-detection antibody in response to an immobilized target material by binding to the capture antibody. it means. In addition, it is an antibody that can be indirectly detected through another labeled antibody, and by forming the sandwich-type complex, a target substance can be more easily detected.

For the detection of the sandwich-type complex, an "antibody for signal detection" to which a label generating a signal is bound is additionally added to react with the label conjugated to the detection antibody, and then the signal is detected This is done by measuring the signal generated from the antibody. Accordingly, the "antibody for signal detection" refers to an antibody that can be directly detected through a label to which it is bound. The labeling material is a chemical (eg, biotin), an enzyme (eg, alkaline phosphatase, β-galactosidase), horseradish peroxidase (HRP) and cytochrome _P450 (cytochrome P ₄₅₀ )), radioactive material (eg C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ), fluorescent material (eg fluorescein), luminescent material, chemiluminescent and FRET ( fluorescence resonance energy tansfer), but is not limited thereto. Various labeling materials and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999.

Specifically, in one embodiment of the present invention, the label of the antibody for signal detection includes, but is not limited to, an enzyme that catalyzes a color reaction, a fluorescence reaction, a luminescence reaction, or an infrared reaction. For example, the labeling material is β-galactosidase (β-galactosidase), horseradish peroxidase (HRP), basic dephosphorylation enzyme (alkaline phosphatase), colloid gold (colloid gold), FITC (poly L-lysine fluorescein isothiocyanate) ), rhodamine-B-isothiocyanate _{(RITC), luciferase, and cytochrome P 450 may} include, but are not limited thereto. When basic dephosphorylation enzyme is used as the enzyme binding to the antibody for signal detection, BCIP (5-bromo-4-chloro-3-indolyl-phosphate), NBT (nitro blue tetrazolium), naphthol-AS- A chromogenic substrate such as B1-phosphate (naphthol-AS-B1-phosphate) and ECR (enhanced chemifluorescence) is used, and when HRP is used, chloronaphthol, AEC (3-amino-9-ethylcarbazole), DAB (3,3'-o-diaminobenzidine), D-luciferin (D-luciferin), lucigenin (bis-N-methylacridinium nitrate; lucigenin), luminol, resorufin benzyl ether, Amplex ^® Red (10-acetyl-3,7-dihydroxyphenoxazine; ADHP), HYP (p-phenylenediamine-HC1 and pyrocatechol), TMB (3,3′,5,5′- Tetramethylbenzidine), ABTS (2,2'-azino) -bis(3-ethylbenzothiazoline-6-sulfonic acid)), O-Phenylenediamine (OPD) and naphthol/pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) ) and substrates such as m-PMS (phenzaine methosulfate) can be used.

In one embodiment of the present invention, the concentration of the capture antibody and the detection antibody included in the kit is 0.1 to 15 μg/ml, more specifically, 0.1 to 12 μg/ml, 0.1 to 10 μg/ml, 0.1-8 μg/ml, 0.1-7 μg/ml, 0.1-6 μg/ml, 0.1-5 μg/ml, 0.1-4 μg/ml, 0.1-3 μg/ml, 0.1-2 μg/ml, 0.1 to 1 μg/ml, 0.5 to 10 μg/ml, 0.5 to 8 μg/ml, 0.5 to 7 μg/ml, 0.5 to 6 μg/ml, 0.5 to 5 μg/ml, 0.5 to 4 μg/ml, 0.5 to 3 μg/ml, 0.5-2 μg/ml, 0.5-1 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6 It may be μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, or 10 μg/ml, but is not limited thereto.

In another embodiment of the present invention, the concentration of the capture antibody included in the kit may be higher than the concentration of the detection antibody.

In one embodiment of the present invention, the concentration of the capture antibody included in the kit is 5 μg/ml, and the concentration of the detection antibody is 1 μg/ml.

According to another aspect of the present invention, the present invention provides a method for detecting a SARS-CoV-2 antigen from a sample using the SARS-CoV-2 antigen detection kit of the present invention.

Since the method for detecting SARS-CoV-2 antigen from a sample of the present invention includes the kit for detecting SARS-CoV-2 antigen, which is another aspect of the present invention, overlapping contents to avoid excessive complexity of the present specification , and the description thereof is omitted.

The present invention provides a composition for diagnosing SARS-CoV-2 infection (COVID-19) or detecting a SARS-CoV-2 antigen comprising a pair of an antibody and antigen fragment thereof that specifically binds to the RBD of SARS-CoV-2 S protein; Alternatively, a kit for diagnosing COVID-19 infection using the Sandwich ELISA technique using the same or a kit for detecting SARS-CoV-2 virus is provided.

As a result, the composition or diagnostic kit according to the present invention forms a sandwich complex of an antibody-antigen-antibody between a sample sample and an antibody pair that specifically binds to SARS-CoV-2 RBD of the present invention. It is faster than diagnostic kits and has higher sensitivity and specificity. Therefore, the composition or kit of the present invention can be usefully used for more rapid and accurate diagnosis of Corona 19 (COVID-19) infectious disease or detection of SARS-CoV-2 virus.

1 shows the selection process of RBD antigen-specific human antibody of SARS-CoV-2 virus from scFv antibody library using phage display technology.

FIG. 2 shows the results of RBD-binding scFv and phage ELISA for selection of RBD antigen-specific human antibody against SARS-CoV-2 virus.

3 shows the SDS-PAGE results of four selected SARS-CoV-2 RBD-specific IgG antibodies.

4 is a result of measuring the K _D value for the SARS-CoV-2 RBD antigen using Surface Plasmon Resonance (SPR) of four selected SARS-CoV-2 RBD-specific IgG antibodies.

5 shows the results of a competition ELISA for K102.1-HRP and four types of SARS-CoV-2 RBD specific IgG selection antibodies to select antibody pairs for Sandwich ELISA.

6 shows the results of Sandwich ELISA for confirming whether a pair of selection antibodies of the present invention are paired.

7 shows the results of the SPR-based competition binding assay for confirming the presence or absence of different epitopes of the selection antibody pair of the present invention.

8 shows a schematic diagram of a Sandwich ELISA assay using a SARS-CoV-2 RBD antigen-specific selection antibody pair.

9 shows optimization of capture antibody concentration among selection antibody pairs for Sandwich ELISA.

10 shows the optimization of the detection antibody concentration among the selection antibody pairs for the Sandwich ELISA.

11 shows a calibration curve for determining a limit of detection (LOD) of SARS-CoV-2 RBD.

12 shows CV values and recovery rates of intra- and inter-assays for the optimized Sandwich ELISA assay.

13 shows the results of confirming the detection ability of the Sandwich ELISA assay optimized for each concentration of eight SRAS-CoV-2 RBD variants.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (vol/vol) %.

Example

Example 1. Selection of RBD antigen-specific human antibody of SARS-CoV-2 virus using phage display technology

SARS-CoV-2 RBD antigen-specific human antibodies were selected from the synthesized human single-chain variable fragment (scFv) antibody library. As shown in FIG. 1 , a screening process using a phage display technique was performed.

First, using 4 μg of recombinant SARS-CoV-2 RBD antigen-coated magnetic beads (Dynabeads M-270 epoxy, Invitrogen), bio-panning was performed over 5 rounds to specific SARS-CoV-2 RBD antigen. Selection of hostile scFv clones was performed. Thereafter, 96 clones were randomly selected from the plate on which the colony was generated, and human antibody clones having excellent reactivity to the SARS-CoV-2 RBD antigen and specific for the antigen were selected through phage ELISA. Phage ELISA results are shown in FIG. 2 .

Example 2. IgG Conversion, Production and Purification of Selection Antibodies

2.1. Conversion of SARS-CoV-2 RBD antigen-specific scFv antibodies to IgG

After securing insert DNA for each of the heavy and light chains of the selected RBD domain-specific four scFv antibodies through PCR, cloning was performed with mammalian expression vector pcDNA3.1 for the production of IgG antibodies. Recombinant DNA of each of the four scFvs was obtained using a Maxi-prep kit to obtain a large amount of high-purity DNA for IgG antibody production in Expi293 cells. The purity of the DNA was confirmed using a nanodrop 2000 spectrophotometer (Thermo Fisher Scientific).

2.2. Mass production and purification of RBD-specific IgG antibody

To use the temporary expression method using the Expi293 system (Invitrogen), 4 types of IgG antibodies were transfected into Expi293 cells using the ExpiFectamine 293 transfection kit (Gibco), and then cultured for 5 days. After centrifugation of the culture medium for the purification of the antibody, the precipitated cell pellet was removed and a culture medium (media) was finally obtained.

In addition, the purification of the selection antibody was performed using affinity chromatography using protein A sepharose beads (Repligen, Waltha, MA, USA).

Example 3. Analysis of physical/chemical properties of selection antibody

: Analysis of the purity and molecular weight of the selection antibody

The present inventors loaded the selected four SARS-CoV-2 RBD-specific antibodies (K102.1, K102.2, K102.3 and K102.4) in equal amounts on polyacrylamide gel, and then SDS-PAGE was performed. After that, it was confirmed that all four antibodies had a purity of 90% or more through Coomassie Brilliant Blue staning, and the molecular weight size was also about 50 kDa and 25 kDa for the heavy chain and light chain of the antibody, respectively (FIG. 3) .

In addition, as a result of measuring the affinity (K _D ) of the selected antibody, it was confirmed that K102.1 had an affinity of 1.643 x 10 ^-9 M and K102.2 had an affinity of 2.465 x 10 ^-9 M ( FIG. 4 ).

Example 4. Selection of monoclonal pairs binding to different epitopes of SARS-CoV-2 RBD

4.1. Conjugation of label to selection antibody (K102.1)

To use the antibody K102.1 produced in Example 2 as a detection antibody for sandwich ELISA, Horseradish peroxidase (HRP) was conjugated as a labeling material. EZ-Link ^TM Plus Activated Peroxidase Kit (Thermo Fisher Scientific) was used according to the manufacturer's instructions.

4.2. Selection of antibody pairs that recognize different epitopes of SARS-CoV-2 RBD

In order to select an antibody pair recognizing different epitopes of SARS-CoV-2 RBD from the selected SARS-CoV-2 RBD-specific antibody group, the HRP-conjugated antibody (K102.1-HRP) was used. A competition ELISA was performed on four antibody groups.

As a result, as shown in FIG. 5 , it was confirmed that K102.1 had a different binding site for SARS-CoV-2 RBD compared to K102.2 and K102.3. In addition, as a result of SPR analysis, as shown in FIG. 4 , K102.2 exhibited about 5 times higher affinity to SARS-CoV-2 RBD than K102.3.

Therefore, K102.1 and K102.2 were selected as antibody pairs that could be used for the development of the Sandwich ELISA assay.

Example 5. Selection Antibody Pair Analysis for Sandwich ELISA

5.1. Assessment of suitability of selection antibody pairs

By confirming the pairing of the selection antibody pair (K102.1 and K102.2) identified in Example 4, suitability in the Sandwich ELISA assay of the present invention was evaluated. Sandwich ELISA was performed using K102.1 or K102.2 as a capture antibody and K102.1-HA or K102.2-HA as a detection antibody.

As a result, as shown in FIG. 6 , it was confirmed that K102.1 and K102.2 can be paired with a capture antibody or a detection antibody, respectively, as an antibody pair for the Sandwich ELISA test. .

5.2. Confirmation of different epitope for SARS-CoV-2 RBD of the selection antibody pair

In addition, the present inventors tried to confirm that the selected antibody pair has different epitopes for SARS-CoV-2 RBD through SPR (Surface Plasmon Resonance)-based competition binding assay. By measuring the real-time binding between SARS-CoV-2 RBD and selection antibody using iMSPR mini-instrument (icluebio, South Korea), unique or overlapping K102.1 or K102.2-HA through competition binding assay It was evaluated whether or not it has a binding site.

Briefly, 128 nM K102.1 in hepes buffered steinberg's solution containing 0.005% (v/v) tween-20 was added to SARS-CoV for 240 s at a flow rate of 50 μl/min. -2 RBD (about 1,000RU) was injected onto the surface. Then, 128 nM of K102.2-HA was applied to the surface to which K102.1 and SARS-CoV-2 RBD were bound under the same conditions. The curve representing the result was obtained as a sensorgram using the iMSPR analysis software.

As a result, as shown in FIG. 7 , it was confirmed that K102.1 had an epitope different from that of K102.2 for SARS-CoV-2 RBD.

Example 6. Development of a Sandwich ELISA assay using a selection antibody pair

As a SARS-CoV-2 specific monoclonal antibody, a Sandwich ELISA test method that can detect SARS-CoV-2 was developed using K102.1 (capture antibody) and K102.2 (detection antibody). A schematic diagram of the Sandwich ELISA assay of the present invention is shown in FIG. 8 .

6.1. Condition Optimization for Sandwich ELISA Assays

6.1.1. Confirmation of optimal concentration of selection antibody pair

In order to determine the optimal conditions for the Sandwich ELISA, the ELISA was performed by increasing the concentration of the capture antibody or the detection antibody.

As a result, it was confirmed that 5 μg/ml of capture antibody (K102.1) and 1 μg/ml of detection antibody (K102.2-HA) were optimal concentrations for the Sandwich ELISA assay ( FIGS. 9 and 10 ).

6.1.2. Calibration curve for Limit Of Detection (LOD)

A calibration curve was derived to determine the detection limit of SARS-CoV-2 RBD in the Sandwich ELISA of the present invention. The reproducibility of the calibration curve was demonstrated through six independent analyzes, and the linear dynamic range of the derived calibration curve was 0 ng/ml to 12 ng/ml of SARS-CoV-2 RBD (0 pM to 480 pM). was determined (FIG. 11).

The limit of detection (LOD) of the Sandwich ELISA of the present invention was derived by Equation 1 below by calculating the standard deviation (SD) and the slope of the calibration curve (S), and the derived limit of detection (LOD) was calculated. ) was determined to be 0.55 ng/ml (22 pM).

[Equation 1]

Limit of detection (LOD) = 3 X (standard deviation (SD)/slope of calibration curve (S))

In addition, the sensitivity was derived by Equation 2 below by calculating the standard deviation (SD) and the mean of the blank sample, and the derived sensitivity was determined to be 0.48 ng/ml (19.2 pM).

[Equation 2]

sensitivity = 3 X standard deviation (SD) + mean of blank

Table 1 compares the performance of the Sandwich ELISA of the present invention with two types of Sandwich ELISA kits that have been developed and approved and sold on the market.

Company Name	product name	Cat. No.	Sensitivity	Source
Eaglebio	GENLISATM SARS-CoV-2 (2019-nCoV) Spike RBD Antigen Quantitative ELISA	KBVH015-12	12 ng/ml	https://eaglebio.com/wp-content/uploads/2021/06/KBVH015-12-SARS-CoV-2-Spike-RBD-Antigen-Quantitative-ELISA-Package-Insert.pdf
Biozol	SARS-CoV-2 Spike S1 Protein ELISA Kit	ACM-55030	0.58 ng/ml	https://www.biozol.de/en/product/acm-55030

As shown in Table 1, the sensitivity of the kit of the present invention is 0.48 ng/ml (19.2 pM), and the sensitivity of the conventionally approved and commercially available kits are 12 ng/ml (Eaglebio) and 0.58 ng/ml (Biozol), respectively. ), it was confirmed that the sensitivity of the kit of the present invention was more excellent.

6.2. Sandwich ELISA assay validation

: Coefficient of Variation (CV) value and recovery measurement

In order to verify the Sandwich ELISA test method developed using the selection antibody pair of the present invention, intra- and inter-assays were performed to measure the coefficient of variation (CV) and recovery. Intra-assay was determined by measuring 6 samples in 3 replicates within the same experiment. Inter-assay was determined by measuring the sample by performing 3 replicates of 6 individual experiments.

Each mean (mean) and standard deviation (SD) was calculated, and the coefficient of variation (CV) value was derived by Equation 3 below.

[Equation 3]

Coefficient of variation (CV)(%) = (standard deviation (SD)/mean) X 100

In addition, the recovery for the Sandwich ELISA assay of the present invention was derived using Equation 4 below.

[Equation 4]

Recovery (%) = [average measured concentration / expected concentration] X 100

As a result, as shown in FIG. 12 , the intra- and Inter-CV values for SARS-CoV-2 RBD at 5 ng/ml were 8.46% and 9.52%, respectively, and the recovery rates were 105.57% and 98.56%, respectively. appear.

Therefore, it was confirmed that the Sandwich ELISA assay of the present invention is a sensitive, accurate and reliable technique for measuring SARS-CoV-2 RBD.

Experimental example

Experimental Example 1. Performance analysis of Sandwich ELISA test method

: Confirmation of detection ability of sandwich ELISA test for 8 types of RBD mutants

By performing the detection of SRAS-CoV-2 RBD mutants using the optimized sandwich ELISA assay of the present invention, it was intended to confirm the detection ability of the assay.

With respect to SRAS-CoV-2, 8 representative RBD variants worldwide classified by country (A435S; Finland, N354D; China, G476S and V483A; USA (USA), F342L, V341I and N501Y) Antigens were obtained for England (UK), L452R/T478K; India (India). Sandwich ELISA was performed by increasing the antigen concentration to 32 pM, 96 pM and 200 pM.

First, a 96-well high-binding microplate (Corning) was coated with a capture antibody, K102.1, and then PBS containing 3% bovine serum albumin (w/v) was used at 37°C for 2 hours. Blocking was performed. Next, 100 μl of each increasing concentration of SARS-CoV-2 mutant (A435S, N354D, G476S, V483A, F342L, V341I, N501Y, L452R/T478K) RBD was added to each well, and the microplate was incubated at 37°C for 3 incubated for hours. After washing the plate 3 times with 0.05% (v/v) PBST, it was incubated with HA-tagged K102.2 (K102.2-HA) as a detection antibody in blocking buffer at 37° C. for 1 hour. Then, it was washed three times with 0.05% (v/v) PBST, and the detection of K102.2-HA was carried out using an HRP-conjugated anti-HA antibody. To this end, after three washes, TMB substrate solution (Thermo Fisher Scientific) was added to each well and left to react with HRP for 15 minutes. To stop the reaction, 1M H ₂ SO ₄ (100 μl/well) was added, and absorbance was measured in a 450 nm wavelength region using a spectrophotometer (BioTek).

As a result, as shown in FIG. 13 below, it was confirmed that all 8 types of RBD mutant protein antigens could be detected in the picomolar range using the optimized sandwich ELISA assay of the present invention.

Claims (10)

1. SARS-CoV comprising the following antibodies or antigen binding fragments thereof that specifically bind to a receptor binding domain (RBD) of SARS-CoV-2 S protein, SARS-CoV -2 Composition for diagnosing infection (COVID-19) or detecting SARS-CoV-2 antigen:

   (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5 an antibody or antigen-binding fragment thereof comprising LCDR2 comprising the amino acid sequence of, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; and

   (ii) HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13 An antibody or antigen-binding fragment thereof comprising LCDR2 comprising the amino acid sequence of, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 14.
2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof of (i) comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8 A composition comprising, wherein the antibody or antigen-binding fragment thereof of (ii) comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16.
3. The composition of claim 1, wherein the antibody or antigen-binding fragment pair has different epitopes targeting the RBD of the SARS-CoV-2 S protein.
4. The composition of claim 1, wherein the antibody or antigen-binding fragment pair thereof specifically binds to a mutant virus in which the RBD region of the SARS-CoV-2 S protein is mutated.
5. 5. The method of claim 4, wherein the mutant virus in the RBD region of the SARS-CoV-2 S protein has A435S mutation at amino acid position 435, N354D mutation at amino acid position 354, G476S mutation at amino acid position 476, A mutant virus with V483A mutation at amino acid position 483, F342L mutation at amino acid position 342, V341I mutation at amino acid position 341, N501Y mutation at amino acid position 501, and L452R/T478K mutation at amino acid positions 452 and 478 A composition selected from the group consisting of.
6. SARS-CoV- containing the following antibodies or antigen binding fragments thereof that specifically binds to a receptor binding domain (RBD) of SARS-CoV-2 S protein (pair of antibodies or antigen binding fragments thereof) 2 Kits for diagnosing infectious disease (COVID-19) or detecting SARS-CoV-2 antigen:

   (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5 an antibody or antigen-binding fragment thereof comprising LCDR2 comprising the amino acid sequence of, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; and

   (ii) HCDR1 comprising the amino acid sequence of SEQ ID NO: 9, HCDR2 comprising the amino acid sequence of SEQ ID NO: 10, HCDR3 comprising the amino acid sequence of SEQ ID NO: 11, LCDR1 comprising the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13 An antibody or antigen-binding fragment thereof comprising LCDR2 comprising the amino acid sequence of, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 14.
7. 7. The method of claim 6, wherein the kit is a Sandwich ELISA kit, one of the antibodies or antigen-binding fragments of (i) and (ii) is a capture antibody, and the other is a detection antibody. which will be used as a kit.
8. The kit according to claim 6, wherein the kit comprises a solid support on which the capture antibody is immobilized.
9. The kit according to claim 7, wherein the kit further comprises an antibody for signal detection to which a label that binds to the detection antibody is bound.
10. A method for detecting a SARS-CoV-2 antigen from an isolated sample using the kit of any one of claims 6 to 9.

Images