A highly sensitive detection kit for novel coronavirus nucleocapsid protein

Abstract

The present invention discloses a highly sensitive detection kit for the nucleocapsid protein of the novel coronavirus. The detection kit comprises the capture antibody 6C3-D8 and the detection antibody 5F2-C11, which specifically target the novel coronavirus N protein. The present invention provides a novel method for detecting the nucleocapsid protein of the novel coronavirus in the art, which has high specificity and sensitivity and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to a highly sensitive detection kit for the nucleocapsid protein of a novel coronavirus. Background Art

[0002] The novel coronavirus nucleocapsid protein (N) stabilizes the virus by binding to its genomic RNA and has a low mutation frequency. The N protein is one of the most important structural proteins within the novel coronavirus capsid, making up the largest proportion of the virus's structural proteins.

[0003] The novel coronavirus's N protein, which entangles with the viral genomic RNA to form the viral nucleocapsid, plays a crucial role in viral RNA synthesis. The N protein is relatively conserved among novel coronaviruses, and high levels of anti-N protein antibodies are produced early in infection. Therefore, the N protein has the potential to establish a rapid method for detecting serum antibodies against the novel coronavirus, and further research is underway to develop monoclonal antibodies. Currently, the field lacks a highly sensitive and specific method for detecting the novel coronavirus's N protein and related detection products. Summary of the Invention

[0004] In order to overcome the technical problems currently existing in this field, the purpose of the present invention is to provide a highly sensitive detection kit for the new coronavirus nucleocapsid protein in the field.

[0005] The present invention uses N protein as the target molecule, applies traditional hybridoma technology to prepare paired monoclonal antibodies against N protein, and constructs a double-antibody sandwich ELISA method. On this basis, a biotin-streptavidin amplification system is added, so that the sensitivity of the detection system basically reaches the level of RT-PCR.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0007] In one aspect, the present invention provides monoclonal antibodies to the novel coronavirus N protein.

[0008] Furthermore, the monoclonal antibody is 6C3-D8 or 5F2-C11;

[0009] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 in the 6C3-D8 heavy chain variable region are shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively;

[0010] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region of 6C3-D8 are shown in SEQ ID NO: 11, LVS, and SEQ ID NO: 15, respectively;

[0011] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region of 5F2-C11 are shown in SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24, respectively;

[0012] The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region of 5F2-C11 are shown in SEQ ID NO: 29, RAS, and SEQ ID NO: 32, respectively.

[0013] Furthermore, the amino acid sequences of FR-H1, FR-H2, FR-H3, and FR-H4 in the heavy chain variable region of 6C3-D8 are shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, respectively;

[0014] Optionally, the amino acid sequences of FR-L1, FR-L2, FR-L3, and FR-L4 in the light chain variable region of 6C3-D8 are shown as SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively;

[0015] Optionally, the amino acid sequences of FR-H1, FR-H2, FR-H3, and FR-H4 in the heavy chain variable region of 5F2-C11 are shown in SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25, respectively;

[0016] Optionally, the amino acid sequences of FR-L1, FR-L2, FR-L3, and FR-L4 in the light chain variable region of 5F2-C11 are shown in SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33, respectively;

[0017] Optionally, the amino acid sequence of the 6C3-D8 heavy chain variable region is shown in SEQ ID NO: 8;

[0018] Optionally, the amino acid sequence of the 6C3-D8 light chain variable region is shown in SEQ ID NO: 17;

[0019] Optionally, the amino acid sequence of the 5F2-C11 heavy chain variable region is shown in SEQ ID NO: 26;

[0020] Optionally, the amino acid sequence of the 5F2-C11 light chain variable region is shown in SEQ ID NO:34.

[0021] In the present invention, the CDR refers to the complementarity determining region (CDR) within the variable sequence of an antibody. Three CDRs are present in each variable region of the heavy and light chains, referred to as CDR1, CDR2, and CDR3 for each variable region. The exact boundaries of these CDRs can be defined differently according to different systems. The CDRs described herein are not limited to the specific amino acid sequences described above; any CDR definition scheme or system known in the art (e.g., Kabat, IMGT, or Chothia numbering systems) defined for the monoclonal antibodies 6C3-D8 and 5F2-C11 described herein fall within the scope of protection of the present invention.

[0022] The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system that can be applied to any antibody variable region, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs.

[0023] Chothia (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987); and Chothia et al., Nature, 342: 877-883 (1989)) discovered that certain subsegments within the Kabat CDRs adopt nearly identical peptide backbone conformations despite having great diversity at the amino acid sequence level. These subsegments are referred to as L1, L2, and L3, or H1, H2, and H3, where L and H refer to the light and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs.

[0024] Other boundaries defining CDRs that overlap with Kabat CDRs have been described by Padlan, FASEB J., 9: 133-139 (1995) and MacCallum, J. Mol. Biol., 262(5): 732-745 (1996). Other CDR boundary definitions may not strictly follow one of the systems herein, but will still overlap with Kabat's CDRs, although they may be shortened or lengthened, based on predictions or experimental findings that particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. The methods used herein can utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs.

[0025] In the present invention, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as the novel coronavirus, belongs to the genus Betacoronavirus and is an enveloped, single-stranded, positive-sense RNA virus. The genome sequence of the novel coronavirus is known to those skilled in the art and can be found, for example, in GenBank: MN908947.

[0026] In some embodiments, the novel coronavirus is a wild-type strain of the novel coronavirus or a mutant strain of the novel coronavirus. Exemplarily, the mutant strain of the novel coronavirus includes but is not limited to: Alpha variant, Beta variant, Delta variant, Omicron variant, Gamma variant, Lambda variant, Mu variant, BQ.1 strain, XBB strain and / or JN.1 variant.

[0027] In the present invention, the monoclonal antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as κ and λ (light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a J region of about 12 or more amino acids, and the heavy chain also contains a D region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in the binding of the antibody to the antigen. Although immunoglobulins are not specifically targeted to the immune system, they exhibit a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into regions of high variability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL region consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding site.

[0028] In another aspect, the present invention provides a nucleic acid molecule encoding the monoclonal antibody of the first aspect of the present invention;

[0029] Optionally, the nucleotide sequence of the 6C3-D8 heavy chain variable region is shown in SEQ ID NO: 9;

[0030] Optionally, the nucleotide sequence of the 6C3-D8 light chain variable region is shown in SEQ ID NO: 18;

[0031] Optionally, the nucleotide sequence of the 5F2-C11 heavy chain variable region is shown in SEQ ID NO: 27;

[0032] Optionally, the nucleotide sequence of the 5F2-C11 light chain variable region is shown in SEQ ID NO:35.

[0033] In another aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the present invention as described above.

[0034] In some embodiments, the expression vector can be introduced into a host cell via transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), bacteriophages (e.g., lambda phage or M13 phage), and animal viruses.

[0035] In some embodiments, animal viruses that can be used as expression vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). An expression vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, expression vectors may also contain a replication origin site.

[0036] In another aspect, the present invention provides a host cell comprising the expression vector of the present invention as described above.

[0037] In some embodiments, the host cell refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0038] In another aspect, the present invention provides a novel coronavirus N protein detection kit.

[0039] Furthermore, the detection kit comprises a capture antibody and a detection antibody;

[0040] The capture antibody is 6C3-D8 as described above in the present invention, and the detection antibody is 5F2-C11 as described above in the present invention.

[0041] Furthermore, the detection antibody is a biotin-labeled detection antibody.

[0042] In some embodiments, the test kit further comprises a reagent for processing a sample to be tested. Exemplarily, the sample to be tested includes, but is not limited to, a blood sample, feces, oral or nasal secretions, or bronchoalveolar lavage fluid from a subject.

[0043] In some embodiments, the subject is a mammal, and in preferred embodiments, the subject is a human.

[0044] In some embodiments, the detection antibody comprises a detectable label, illustratively, the detectable label includes but is not limited to: biotin, an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein) or a radionuclide.

[0045] In some embodiments, the antibodies contained in the kit are detectably labeled. In the present invention, the detectable label can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. In preferred embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32 P, etc.), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds), magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.), and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0046] In some embodiments, the detection kit of the present invention comprises a capture antibody and a detection antibody, wherein the capture antibody is 6C3-D8 as described above in the present invention, and the detection antibody is 5F2-C11 as described above in the present invention. As long as the kit comprises these two antibodies, it will fall within the scope of protection of the present invention.

[0047] In another aspect, the present invention provides any of the following products:

[0048] (1) A group of antibody combinations, comprising a capture antibody and a detection antibody, wherein the capture antibody is 6C3-D8 as described above in the present invention, and the detection antibody is 5F2-C11 as described above in the present invention;

[0049] (2) A detection reagent comprising the monoclonal antibody described above;

[0050] (3) A detection product comprising the monoclonal antibody or the detection reagent as described above;

[0051] (4) A pharmaceutical composition comprising the monoclonal antibody of the present invention as described above;

[0052] (5) A biological preparation comprising the monoclonal antibody or the pharmaceutical composition as described above.

[0053] In some embodiments, the pharmaceutical composition or biologic further comprises a pharmaceutically acceptable carrier and / or excipient.

[0054] In some embodiments, the pharmaceutically acceptable carrier and / or excipient refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, as is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffer. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and their analogs. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, and sorbic acid. Stabilizers have the meaning commonly understood by those skilled in the art and are capable of stabilizing the desired activity of the active ingredient in the pharmaceutical, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid and glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate). In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable liquid is selected from water for injection, bacteriostatic water for injection, sodium chloride solution, glucose solution, solutions containing surfactants, pH buffered solutions, Ringer's solution, and any combination thereof.

[0055] On the other hand, the present invention provides a method for detecting the novel coronavirus N protein in a test sample for non-diagnostic and non-therapeutic purposes, the method comprising: contacting the test sample with the monoclonal antibody, the detection kit, the detection reagent or the detection product as described above, and detecting the formation of an immune complex between the novel coronavirus N protein and the monoclonal antibody.

[0056] In another aspect, the present invention provides any of the following applications:

[0057] (1) Use of the monoclonal antibody described above in the preparation of a reagent for detecting a novel coronavirus;

[0058] (2) Use of the monoclonal antibody of the present invention as described above or the detection reagent of the present invention as described above in the preparation of diagnostic products for diagnosing and / or assisting in the diagnosis of novel coronavirus infection diseases;

[0059] (3) Use of the monoclonal antibody of the present invention or the detection reagent of the present invention in the preparation of a detection kit for detecting the novel coronavirus;

[0060] (4) Use of the monoclonal antibodies described above in the present invention in the preparation of drugs for preventing and / or treating novel coronavirus infections;

[0061] Optionally, the novel coronavirus includes the original strain of the novel coronavirus, Alpha variant, Beta variant, Delta variant, Omicron variant, Gamma variant, Lambda variant, Mu variant, BQ.1 strain, XBB strain and / or JN.1 variant.

[0062] In addition, the present invention also provides a method for detecting whether a sample to be tested from a subject contains the new coronavirus N protein or diagnosing whether a subject has a new coronavirus infection disease.

[0063] Furthermore, the method includes: using the monoclonal antibody, detection kit, detection reagent or detection product as described above of the present invention to contact the test sample derived from the subject, detecting whether the test sample contains the new coronavirus N protein or the level of the new coronavirus N protein contained in it, so as to diagnose whether the subject has a new coronavirus infection disease.

[0064] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0065] The present invention provides a novel high-sensitivity detection kit for the novel coronavirus nucleocapsid protein in the art. The detection kit comprises the capture antibody 6C3-D8 and the detection antibody 5F2-C11 that specifically target the novel coronavirus N protein. The present invention provides a novel method for detecting the novel coronavirus nucleocapsid protein in the art, which has high specificity and sensitivity and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is the result diagram of the antibody pairing sensitivity test;

[0067] Figure 2 Specificity test results for antibody pairing;

[0068] Figure 3 This is the result graph corresponding to the improvement of the sensitivity of antibody pairing detection by biotin / streptavidin;

[0069] Figure 4 :The detection results of clinical specimens using the double antibody sandwich ELISA method based on N protein. DETAILED DESCRIPTION

[0070] The present invention will be further described below in conjunction with specific examples. The following specific examples are only used to explain the present invention and are not to be construed as limiting the present invention. Those of ordinary skill in the art will appreciate that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The reagents and raw materials used in the present invention are readily available to those of ordinary skill in the art and are commercially available unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are typically tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and are not to be construed as limiting the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0071] Example 1 Preparation of monoclonal antibody against N antigen

[0072] 1. Immunization of mice with recombinant N protein

[0073] Five-week-old female Balb / c mice were immunized with recombinant N protein (100 μg / mouse). For the first immunization, 100 μg of antigen was mixed with an equal volume of Freund's complete adjuvant and injected intraperitoneally. After the third week, an equal amount of antigen was mixed with Freund's incomplete adjuvant and injected intraperitoneally. The third immunization was performed on the fifth week without adding adjuvant.

[0074] 2. Fusion of spleen cells and myeloma cells

[0075] One week before fusion, mouse myeloma cells sp2 / 0 were revived in OPTI-MEM medium (containing 10% fetal bovine serum) and cultured in a 37°C, 5% CO2 incubator. Three days before fusion, the cells were passaged once. On the day of fusion, myeloma cells were harvested, counted, and 5.0×10 7 Myeloma cells were washed twice with serum-free medium and set aside. Three to five days after the third immunization, mice were enucleated, bled, and sacrificed. The spleens were removed aseptically, placed on sterile plates, and spleen cells were isolated, counted, and set aside.

[0076] Combine splenocytes equivalent to half a mouse spleen with myeloma cells and centrifuge at 1300 rpm for 5 minutes. Remove as much supernatant as possible. Add 1.5 mL of 50% PEG over 1.5 minutes, shaking thoroughly. Then, add 20 mL of serum-free medium over 8.5 minutes, shaking thoroughly.

[0077] The PEG-fused cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and 150 mL of HAT selection medium was added to resuspend the cells. The fused cells were inoculated into a sterile 96-well plate at 150 μL / well and cultured in a 37°C, 5% CO2 incubator for 4 days. 100 μL of selection medium was added to each well.

[0078] 3. Screening and cloning of hybridoma cells

[0079] Ten days after fusion, 50 μL of supernatant was aspirated from each well and added to a 96-well ELISA plate coated with recombinant N protein (blocked with 1% BSA). Incubate at room temperature for 1.0 hour and wash five times. Dilute horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody at a dilution of 1:4000 and add 50 μL to each well. Incubate at room temperature for 0.5 hour and wash five times. Add 100 μL of HRP substrate (H2O2 + TMB) to each well and incubate at room temperature for 10 minutes. Then, add 50 μL of 0.5 M H2SO4 to each well and measure A. 450nm value.

[0080] Collect cells from positive wells, resuspend them in HT selection medium, dilute them by limiting dilution, and plate them in 96-well cell culture plates. After 5 days, observe and identify wells with only one cell clone by ELISA. Perform limiting dilution of cells from positive wells and isolate and culture them 3-4 times until a stable hybridoma clone is obtained. Culture the hybridoma cells in large quantities, collect the culture supernatant containing the antibody, purify it using a protein G affinity chromatography column, dialyze it into PBS, measure the concentration, and freeze it at -20°C until needed.

[0081] Example 2: Selecting the best antibody pair using double antibody sandwich ELISA

[0082] Ten candidate monoclonal antibodies were screened and used to identify the optimal pairing. Using a double-antibody sandwich ELISA, these 10 antibodies were paired with each other to select the optimal combination for N protein detection, as described below.

[0083] Monoclonal antibody coating: dilute the antibody concentration to 1.0 μg / mL with 0.1 M NaHCO3 / Na2CO3 buffer (pH 9.6), add to a 96-well ELISA plate at 100 μL / well, coat overnight at 4°C, wash once with PBST, block with 5% milk at 4°C overnight, and wash once with PBST for later use.

[0084] Conjugation of monoclonal antibody to HRP: Dialyze the monoclonal antibody into PBS and adjust the concentration to 1 mg / mL. Use activated HRP from Beijing Taitianhe Biotechnology Co., Ltd. for conjugation, following the manufacturer's instructions. Add 50% glycerol to the labeled antibody and freeze at -20°C until ready for use.

[0085] ELISA operation: add 50 μL of 5% milk powder and 50 μL of N protein solution (20 ng / mL) to each well, incubate at 37°C for 1 hour, wash 5 times, then add 100 μL of enzyme-labeled antibody (1:1000) to each well, incubate at 37°C for 0.5 hour, wash 5 times, add 100 μL of TMB+H2O2 substrate to each well, incubate at room temperature for 10 minutes, add 100 μL of 0.5M sulfuric acid to each well to terminate the reaction, and measure A 450nm The specific results are shown in Table 1.

[0086] Table 1 Capture antibody and detection antibody array titration results

[0087]

[0088] The results showed that the three combinations of c6C3-D8 / d5F2-C11, c5F2-C11 / d6C3-D8, and c5F2-C11 / d12H5-D11 had higher OD 450 value.

[0089] The sequence information of the capture antibody 6C3-D8 and the detection antibody 5F2-C11 are shown in Tables 2 and 3 below, respectively. Furthermore, it has been verified that the monoclonal antibodies 6C3-D8 and 5F2-C11 have high affinity and specificity for the novel coronavirus N protein.

[0090] Table 2 Sequence information of capture antibody 6C3-D8

[0091]

[0092] Table 3 Sequence information of detection antibody 5F2-C11

[0093]

[0094] Example 3 Sensitivity analysis of double antibody sandwich ELISA based on N protein

[0095] 1. Sensitivity test of antibody pairing

[0096] To achieve higher sensitivity, we increased the capture antibody concentration from 1.0 μg / mL to 4.0 μg / mL. The specific experimental method is as follows:

[0097] Monoclonal antibody coating: dilute antibody 6C3-D8 to a concentration of 4.0 μg / mL with 0.1 M NaHCO3 / Na2CO3 buffer (pH 9.6), add 100 μL / well to a 96-well ELISA plate, coat overnight at 4°C, wash once with PBST, block with 5% skim milk powder at 4°C overnight, and wash once with PBST before use.

[0098] Conjugation of monoclonal antibody 5F2-C11 to HRP: Dialyze the monoclonal antibody into PBS and adjust the concentration to 1 mg / mL. Use activated HRP from Beijing Taitianhe Biotechnology Co., Ltd. for conjugation, following the manufacturer's instructions. Add 50% glycerol to the labeled antibody and freeze at -20°C until ready for use.

[0099] Washing solution: PBST, i.e., PBS + 0.05% Tween 20.

[0100] Substrates: TMB and H2O2.

[0101] Purified N protein was serially diluted in PBS containing 5% skim milk powder (100, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 ng / mL), with 100 μL added to each well. A blank control was also established, and duplicate wells were set up for each dilution. The cells were incubated at 37°C for 1 hour and washed five times with PBST. HRP-labeled antibody was diluted 1:1000 in PBS containing 5% skim milk powder, with 100 μL added to the corresponding wells. The cells were incubated at 37°C for 0.5 hour, washed five times with PBST, and 100 μL of TMB+H2O2 substrate was added to each well. The cells were incubated at room temperature for 10 minutes. The reaction was terminated by adding 100 μL of 0.5 M sulfuric acid to each well, and the OD value was measured at 450 nm. A reaction exceeding twice the OD value of the blank well was considered positive.

[0102] 2. Experimental results

[0103] The results showed that c6C3-D8 / d5F2-C11 was the best pairing, with a detection limit of less than 0.78 ng / mL ( Figure 1 ), that is, this antibody pairing has higher sensitivity.

[0104] Example 4 Specificity test of antibody pairing

[0105] 1. Specificity test of antibody pairing

[0106] This example further tests the specificity of antibody pairing, and the specific experimental method is as follows:

[0107] Add 50 μL of 5% skim milk powder to each well. Simultaneously, add 50 μL of each of the following samples: MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-229E, the original strain of novel coronavirus, the alpha variant, the beta variant, the delta variant, and the omicron variant. Each sample was replicated. Control groups were set up: recombinant protein novel coronavirus N was used as a positive control, and 5% skim milk powder was used as a negative control. Absorbance (A) at 450 nm was measured according to the ELISA protocol described above.

[0108] 2. Experimental results

[0109] The results showed that the double antibody sandwich ELISA composed of this antibody pair could not sandwich coronaviruses such as MERS-CoV, HCoV-OC43, HCoV-NL63 and HCoV-229E, but could sandwich the original strain of the new coronavirus and variants such as Alpha, Beta, Delta and Omicron ( Figure 2 ), that is, the antibody pairing has high specificity.

[0110] Example 5: Effect of the biotin / streptavidin system on improving detection sensitivity

[0111] 1. Experimental methods

[0112] In this example, the detection antibody 5F2-C11 in the antibody pair was biotinylated and streptavidin-horseradish peroxidase conjugate (SA-HRP) was used as the detection substance. The specific experimental method is as follows:

[0113] The detection monoclonal antibody 5F2-C11 in this detection system was biotinylated using the EZ-Linksulfo-NHS-LC-Biotinylation kit from Thermo.

[0114] The plate was coated with antibody 6C3-D8 at a concentration of 1µg / mL and incubated at 4°C overnight. After washing once, the plate was blocked with 5% skim milk powder, incubated at 4°C overnight, and washed once. Diluted recombinant N protein was added at 100µL / well and incubated at 37°C for 1 hour. After washing five times, HRP-labeled streptavidin (SA-HRP) (Shanghai Bioengineering Co., Ltd.) was added at a dilution of 1:1000 at 100µL / well and incubated at 37°C for 0.5 hour. After washing five times, 100µL TMB+H2O2 substrate was added to each well and incubated at room temperature for 10 minutes. 100µL 0.5M sulfuric acid was added to each well to terminate the reaction. A was measured. 450nm value.

[0115] 2. Experimental results

[0116] By biotinylating the detection antibody 5F2-C11 in this antibody pair and using streptavidin-horseradish peroxidase conjugate (SA-HRP) as the detector, the detection sensitivity was increased from 1.0 ng / mL to 0.15 ng / mL, a 6.7-fold increase ( Figure 3 ).

[0117] Example 6 Clinical specimen testing

[0118] 1. Experimental methods

[0119] First, 50 μL of 5% skim milk powder was added to each well. Then, 50 μL of throat swab samples from 48 novel coronavirus infected patients were added to each well. The samples were incubated at 37°C for 1 hour and washed 5 times with PBST. 100 μL of enzyme-labeled antibody (dilution ratio 1:1000) was added to each well. The samples were incubated at 37°C for 0.5 hour and washed 5 times with PBST. 100 μL of TMB+H2O2 substrate was added to each well. The samples were incubated at room temperature for 10 minutes. 100 μL of 0.5 M sulfuric acid was added to each well to terminate the reaction. A 450nm value.

[0120] 2. Experimental results

[0121] Preliminary application of the double antibody sandwich ELISA method based on N protein (with added biotin / streptavidin). Figure 4 As shown, 46 of the 48 throat swab samples from people confirmed to be infected by RT-PCR were tested positive, with a sensitivity of 95.8%.

[0122] Example 7 Cloning of the Heavy and Light Chain Variable Region Genes of Monoclonal Antibody 6C3-D8 / 5F2-C11

[0123] 1. Experimental methods

[0124] Total RNA was extracted from hybridoma cells in the logarithmic growth phase using Invitrogen's Trizol. Reverse transcription was performed using oligo(dT)20 as a primer to generate cDNA. PCR was then performed using specific primers to amplify the heavy and light chain variable region genes. The PCR products were purified by electrophoresis and inserted into the pMD-18T vector via TA cloning. Sequencing was then performed for sequence analysis.

[0125] 2. Experimental results

[0126] The sequence information corresponding to the monoclonal antibodies 6C3-D8 and 5F2-C11 are shown in Tables 2 and 3 as mentioned above, respectively.

Claims

1. A monoclonal antibody targeting the novel coronavirus N protein, characterized in that: The monoclonal antibody is 6C3-D8 or 5F2-C11; The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 in the 6C3-D8 heavy chain variable region are shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively; The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region of 6C3-D8 are shown in SEQ ID NO: 11, LVS, and SEQ ID NO: 15, respectively; The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region of 5F2-C11 are shown in SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24, respectively; The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region of 5F2-C11 are shown in SEQ ID NO: 29, RAS, and SEQ ID NO: 32, respectively.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequences of FR-H1, FR-H2, FR-H3, and FR-H4 in the 6C3-D8 heavy chain variable region are shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, respectively.

3. The monoclonal antibody according to claim 1, characterized in that The amino acid sequences of FR-L1, FR-L2, FR-L3, and FR-L4 in the 6C3-D8 light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively.

4. The monoclonal antibody according to claim 1, characterized in that The amino acid sequences of FR-H1, FR-H2, FR-H3, and FR-H4 in the heavy chain variable region of 5F2-C11 are shown in SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25, respectively.

5. The monoclonal antibody according to claim 1, characterized in that The amino acid sequences of FR-L1, FR-L2, FR-L3, and FR-L4 in the light chain variable region of 5F2-C11 are shown in SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 33, respectively.

6. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the 6C3-D8 heavy chain variable region is shown in SEQ ID NO:

8.

7. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the 6C3-D8 light chain variable region is shown in SEQ ID NO:

17.

8. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the 5F2-C11 heavy chain variable region is shown in SEQ ID NO:

26.

9. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the 5F2-C11 light chain variable region is shown in SEQ ID NO:

34.

10. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 9.

11. The nucleic acid molecule according to claim 10, characterized in that The nucleotide sequence of the 6C3-D8 heavy chain variable region is shown in SEQ ID NO:

9.

12. The nucleic acid molecule according to claim 10, characterized in that The nucleotide sequence of the 6C3-D8 light chain variable region is shown in SEQ ID NO:

18.

13. The nucleic acid molecule according to claim 10, characterized in that The nucleotide sequence of the 5F2-C11 heavy chain variable region is shown in SEQ ID NO:

27.

14. The nucleic acid molecule according to claim 10, characterized in that The nucleotide sequence of the 5F2-C11 light chain variable region is shown in SEQ ID NO:

35.

15. An expression vector comprising the nucleic acid molecule according to any one of claims 10 to 14.

16. A host cell comprising the expression vector according to claim 15.

17. A novel coronavirus N protein detection kit, characterized in that: The detection kit comprises a capture antibody and a detection antibody; The capture antibody is 6C3-D8 described in claim 1, and the detection antibody is 5F2-C11 described in claim 1.

18. The detection kit according to claim 17, characterized in that The detection antibody is a biotin-labeled detection antibody.

19. A group of antibody combinations, characterized in that The antibody combination comprises a capture antibody and a detection antibody, wherein the capture antibody is 6C3-D8 described in claim 1, and the detection antibody is 5F2-C11 described in claim 1.

20. A detection reagent, characterized in that The detection reagent comprises the monoclonal antibody according to any one of claims 1 to 9.

21. A method for detecting novel coronavirus N protein in a sample for non-diagnostic and non-therapeutic purposes, the method comprising: The sample to be tested is contacted with the monoclonal antibody according to any one of claims 1 to 9, the detection kit according to claim 17 or 18, or the detection reagent according to claim 20 to detect the formation of an immune complex between the novel coronavirus N protein and the monoclonal antibody.

22. Use of the monoclonal antibody according to any one of claims 1 to 9 in the preparation of a detection reagent for detecting a novel coronavirus; The novel coronavirus is the original strain, Alpha variant, Beta variant, Delta variant and / or Omicron variant of the novel coronavirus.

23. Use of the monoclonal antibody according to any one of claims 1 to 9 or the detection reagent according to claim 20 in the preparation of a diagnostic product for diagnosing and / or assisting in the diagnosis of a novel coronavirus infection; The novel coronavirus is the original strain, Alpha variant, Beta variant, Delta variant and / or Omicron variant of the novel coronavirus.

24. Use of the monoclonal antibody according to any one of claims 1 to 9 or the detection reagent according to claim 20 in the preparation of a detection kit for detecting a novel coronavirus; The novel coronavirus is the original strain, Alpha variant, Beta variant, Delta variant and / or Omicron variant of the novel coronavirus.

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