Novel high-sensitivity detection kit for coronavirus nucleocapsid protein

Abstract

The invention discloses a novel coronavirus nucleocapsid protein high-sensitivity detection kit which comprises a capture antibody 6C3-D8 and a detection antibody 5F2-C11 which specifically target novel coronavirus N. The invention provides a brand-new method for detecting novel coronavirus nucleocapsid protein for the field. And the kit has relatively high specificity and sensitivity and wide 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] Although the global pandemic caused by the novel coronavirus has ended, the virus is still in the process of continuous evolution under the influence of human immune pressure and various factors in nature. Sporadic or local outbreaks caused by a mutant of the virus are still foreseeable events. The novel coronavirus nucleocapsid protein (N) plays a stabilizing role by binding to the viral genome RNA and has a low mutation frequency. The N protein is one of the most important structural proteins in the novel coronavirus capsid and accounts for the largest proportion of the virus's structural proteins.

[0003] The novel coronavirus N protein and the viral genomic RNA are entangled to form the viral nucleocapsid, which plays an important role in the synthesis of viral RNA. The N protein is relatively conservative in the novel coronavirus, and the body can produce high levels of antibodies against the N protein in the early stages of infection. Therefore, the N protein has the potential to establish a rapid method for detecting serum antibodies against the novel coronavirus, and further research on monoclonal antibody preparation can also be carried out. At present, this field lacks a novel coronavirus N protein detection method with high sensitivity and specificity and related detection products. Summary of the invention

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

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

[0009] The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region of 6C3-D8 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 in 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 as 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 as shown in SEQ ID NO:34.

[0021] In the present invention, the CDR refers to the complementary determining region in the variable sequence of the antibody. There are three CDRs in each variable region of the heavy chain and the light chain, which are called CDR1, CDR2 and CDR3 for each variable region. The exact boundaries of these CDRs can be defined differently according to different systems. The CDR of the present invention is not limited to the above-mentioned specific amino acid sequence, and the CDR obtained by defining the monoclonal antibodies 6C3-D8 and 5F2-C11 as described above of the present invention using any scheme or system of CDR definition known in the prior art (for example, Kabat, IMGT or Chothia numbering system) will fall within the protection scope 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 the exact 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)) found that certain sub-portions within the Kabat CDR adopt almost identical peptide backbone conformations despite having great diversity at the amino acid sequence level. These sub-portions are referred to as L1, L2, and L3, or H1, H2, and H3, where L and H refer to the light chain and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, which have borders 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 in light of 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 (severe acute respiratory syndrome coronavirus 2), also known as a novel coronavirus, belongs to the genus beta coronavirus and is a single-stranded positive RNA virus containing an envelope. 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 strains of the novel coronavirus include but are not limited to: Alpha variants, Beta variants, Delta variants, Omicron variants, Gamma variants, Lambda variants, Mu variants, BQ.1 strains, XBB strains and / or JN.1 variants.

[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 the isotype of the antibody is defined 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. The VH and VL regions can be further subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively.

[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 as shown in SEQ ID NO:9;

[0030] Optionally, the nucleotide sequence of the 6C3-D8 light chain variable region is as 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 as 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 by transformation, transduction or transfection, so that the genetic material elements carried by the expression vector are expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids, phagemids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)), phages (such as lambda phage or M13 phage) and animal viruses, etc.

[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 (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as 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. In addition, the expression vector may also contain a replication initiation 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] On the other hand, the present invention provides a novel coronavirus N protein detection kit.

[0039] Further, 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, excrement, oral or nasal secretions, or alveolar 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 agent (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, spectral, 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, fluorescent immunoassay, chemiluminescent 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-mentioned 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, the antibody combination comprising a capture antibody and a detection antibody, 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, the detection reagent comprising the monoclonal antibody as described above in the present invention;

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

[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, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. 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 the like. 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), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, 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, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection, bacteriostatic water for injection, sodium chloride solution, dextrose solution, a solution containing a surfactant, a pH buffered solution, Ringer's solution, and any combination thereof.

[0055] On the other hand, the present invention provides a method for detecting a 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 of the present invention as described above, the detection kit of the present invention as described above, the detection reagent or the detection product of the present invention 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 detection reagent for detecting a novel coronavirus;

[0058] (2) Use of the monoclonal antibody or the detection reagent of the present invention as described above in the preparation of a diagnostic product for diagnosing and / or assisting in the diagnosis of a disease infected by a novel coronavirus;

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

[0060] (4) Use of the monoclonal antibody described above in the preparation of a drug for preventing and / or treating a disease caused by a novel coronavirus;

[0061] Optionally, the new coronavirus includes the original strain of the new 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 the 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 in the present invention to contact the test sample from the subject, and 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 nucleocapsid protein of the novel coronavirus in the art. The detection kit comprises a capture antibody 6C3-D8 and a detection antibody 5F2-C11 that specifically targets 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. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0067] Figure 2 The specificity test result diagram for antibody pairing;

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

[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 is further described below in conjunction with specific examples. The following specific examples are only used to explain the present invention and cannot be construed as limiting the present invention. It can be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and 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 easily available to those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually 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 should not limit 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 then immunized 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 and counted. 5.0×10 7 Myeloma cells were washed twice with serum-free medium for later use. 3-5 days after the third immunization, the mice were bled and killed by removing the eyeballs. The spleens of the mice were removed by aseptic operation and placed in sterile dishes, and the spleen cells were separated, counted, and set aside.

[0076] Mix spleen cells equivalent to 1 / 2 of the mouse spleen with myeloma cells, centrifuge at 1300rpm for 5 minutes, and remove as much supernatant as possible. Add 1.5mL of 50% PEG within 1.5 minutes, shaking while adding; then add 20mL of serum-free culture medium within 8.5 minutes, shaking while adding.

[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, and 100 μL of selection medium was added to each well.

[0078] 3. Screening and cloning of hybridoma cells

[0079] 10 days after fusion, aspirate 50 μL of supernatant from each well and add to a 96-well ELISA plate coated with recombinant N protein (blocked with 1% BSA), incubate at room temperature for 1.0 hour; wash 5 times. Dilute horseradish peroxidase (HRP)-labeled goat anti-mouse 1:4000, add 50 μL to each well, incubate at room temperature for 0.5 hour; wash 5 times. Add 100 μL of HRP substrate (H2O2+TMB) to each well, incubate at room temperature for 10 minutes, add 50 μL of 0.5M H2SO4 to each well, and measure A 450nm value.

[0080] Collect the cells in the positive wells, resuspend them in HT selection medium, dilute the cells by limiting dilution, and plant them in a 96-well cell culture plate. Observe after 5 days, and identify the wells that have only one cell clone growing by ELISA. Perform limiting dilution on the cells in the positive wells, and culture them by single cell separation 3-4 times until a stable hybridoma cell clone is obtained. Culture hybridoma cells in large quantities, collect the culture supernatant containing antibodies, purify them with protein G affinity chromatography column, dialyze them into PBS, measure the concentration, and freeze them at -20℃ for later use.

[0081] Example 2: Using double antibody sandwich ELISA to select the best antibody pairing

[0082] Ten candidate monoclonal antibodies were screened and used to determine the best pairing. The double antibody sandwich ELISA method was used to pair the above ten antibodies with each other and select the best combination to facilitate the detection of N protein, 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 96-well ELISA plate, 100 μL / well, coat overnight at 4°C, wash once with PBST, add 5% milk and block overnight at 4°C, wash once with PBST for use.

[0084] Conjugation of monoclonal antibody with 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. For specific operations, refer to the company's instructions. Add 50% glycerol to the labeled antibody and freeze at -20°C for later 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 0.5 M 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]

[0089] 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.

[0090] Among them, the sequence information corresponding to the capture antibody 6C3-D8 and the detection antibody 5F2-C11 are shown in Table 2 and Table 3 below, respectively. In addition, it has been verified that the monoclonal antibodies 6C3-D8 and 5F2-C11 have high affinity and specificity for the new coronavirus N protein.

[0091] Table 2 Sequence information of capture antibody 6C3-D8

[0092]

[0093]

[0094] Table 3 Sequence information of detection antibody 5F2-C11

[0095]

[0096]

[0097] Example 3 Double Antibody Sandwich ELISA Sensitivity Analysis Based on N Protein

[0098] 1. Sensitivity test of antibody pairing

[0099] In order to obtain higher sensitivity, we increased the concentration of capture antibody from 1.0 μg / mL to 4.0 μg / mL. The specific experimental method is as follows:

[0100] 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 to a 96-well ELISA plate, 100 μL / well, coat overnight at 4°C, wash once with PBST, add 5% skim milk powder and block overnight at 4°C, wash once with PBST for later use.

[0101] Conjugation of monoclonal antibody 5F2-C11 with 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. For specific operations, refer to the company's instructions. Add 50% glycerol to the labeled antibody and freeze at -20°C for later use.

[0102] Washing solution: PBST, i.e., PBS+0.05% Tween 20.

[0103] Substrates: TMB and H2O2.

[0104] Dilute the purified N protein (100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78) ng / mL in PBS containing 5% skim milk powder, add 100 μL to each well, set up a blank control, set up duplicate wells for each dilution, incubate at 37°C for 1 hour, and wash 5 times with PBST. Dilute the HRP enzyme-labeled antibody (1:1000) with PBS containing 5% skim milk powder, add 100 μL of enzyme-labeled antibody to the corresponding wells, incubate at 37°C for 0.5 hour, wash 5 times with PBST, add 100 μL 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 the OD value at 450nm (a value greater than 2 times the OD value of the blank well is considered positive).

[0105] 2. Experimental results

[0106] 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 a higher sensitivity.

[0107] Example 4 Specificity test of antibody pairing

[0108] 1. Specificity test of antibody pairing

[0109] This example further tests the specificity of antibody pairing, and the specific experimental method is as follows:

[0110] Add 50 μL of 5% skim milk powder to each well, and add 50 μL of the following samples to each well: MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-229E, novel coronavirus original strain, Alpha variant, Beta variant, Delta and Omicron variant, and set up duplicate wells for each sample. Set up a control group: use the recombinant protein novel coronavirus N as a positive control and 5% skim milk powder as a negative control, and measure the absorbance (A) value at 450 nm according to the above ELISA operation method.

[0111] 2. Experimental results

[0112] 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 a higher specificity.

[0113] Example 5 Effect of Biotin / Streptavidin System on Improving Detection Sensitivity

[0114] 1. Experimental methods

[0115] 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:

[0116] The detection monoclonal antibody 5F2-C11 in this detection system was biotinylated using the EZ-Linksulfo-NHS-LC-Biotinylation kit from Thermo Corporation.

[0117] The ELISA plate was coated with antibody 6C3-D8 at a concentration of 1 μg / mL, incubated at 4°C overnight, washed once, blocked with 5% skim milk powder, incubated at 4°C overnight, and washed once. Diluted recombinant N protein was added at 100 μL / well, incubated at 37°C for 1 hour, washed 5 times, and HRP-labeled streptavidin (SA-HRP) (Shanghai Shenggong Company) was added at a dilution of 1:1000, 100 μL / well, incubated at 37°C for 0.5 hour, washed 5 times, and 100 μL TMB+H2O2 substrate was added to each well, incubated at room temperature for 10 minutes, and 100 μL 0.5M sulfuric acid was added to each well to terminate the reaction, and A was measured. 450nm value.

[0118] 2. Experimental results

[0119] By biotinylating the detection antibody 5F2-C11 in the 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, an increase of 6.7 times ( Figure 3 ).

[0120] Example 6 Clinical specimen testing

[0121] 1. Experimental methods

[0122] 50 μL of 5% skim milk powder was first added to each well, and then 50 μL of throat swab samples from 48 novel coronavirus-infected patients were added to each well, incubated at 37°C for 1 hour, washed 5 times with PBST, 100 μL of enzyme-labeled antibody (dilution ratio 1:1000) was added to each well, incubated at 37°C for 0.5 hour, washed 5 times with PBST, 100 μL of TMB+H2O2 substrate was added to each well, incubated at room temperature for 10 minutes, 100 μL of 0.5 M sulfuric acid was added to each well to terminate the reaction, and A was measured. 450nm value.

[0123] 2. Experimental results

[0124] Preliminary application of the double antibody sandwich ELISA method based on N protein (adding biotin / streptavidin). The test results are as follows 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%.

[0125] Example 7 Cloning of the heavy and light chain variable region genes of monoclonal antibody 6C3-D8 / 5F2-C11

[0126] 1. Experimental methods

[0127] Take the hybridoma cells in the logarithmic growth phase, use Invitrogen's Trizol to extract total RNA, use oligo (dT) 20 as primer, reverse transcribe to generate cDNA. Then use specific primers to PCR amplify the heavy and light chain variable region genes respectively. After electrophoresis purification, the PCR product is inserted into the pMD-18T vector by TA cloning, sequenced, and sequence analysis is performed.

[0128] 2. Experimental results

[0129] The sequence information corresponding to the monoclonal antibodies 6C3-D8 and 5F2-C11 are shown in Table 2 and Table 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 heavy chain variable region of 6C3-D8 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 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; 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 in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, respectively; 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; 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; Optionally, the amino acid sequence of the 6C3-D8 heavy chain variable region is shown in SEQ ID NO: 8; Optionally, the amino acid sequence of the 6C3-D8 light chain variable region is as shown in SEQ ID NO: 17; Optionally, the amino acid sequence of the 5F2-C11 heavy chain variable region is shown in SEQ ID NO: 26; Optionally, the amino acid sequence of the 5F2-C11 light chain variable region is as shown in SEQ ID NO:

34.

3. A nucleic acid molecule encoding the monoclonal antibody according to claim 1 or 2; Optionally, the nucleotide sequence of the 6C3-D8 heavy chain variable region is shown in SEQ ID NO:9; Optionally, the nucleotide sequence of the 6C3-D8 light chain variable region is as shown in SEQ ID NO: 18; Optionally, the nucleotide sequence of the 5F2-C11 heavy chain variable region is shown in SEQ ID NO: 27; Optionally, the nucleotide sequence of the 5F2-C11 light chain variable region is as shown in SEQ ID NO:

35.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the expression vector of claim 4.

6. 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 or 2, and the detection antibody is 5F2-C11 described in claim 1 or 2.

7. The detection kit according to claim 6, characterized in that The detection antibody is a biotin-labeled detection antibody.

8. Any of the following products, characterized in that: The products include: (1) A group of antibody combinations, comprising a capture antibody and a detection antibody, wherein the capture antibody is 6C3-D8 as described in claim 1 or 2, and the detection antibody is 5F2-C11 as described in claim 1 or 2; (2) A detection reagent comprising the monoclonal antibody according to claim 1 or 2; (3) A detection product, comprising the monoclonal antibody or the detection reagent according to claim 1 or 2; (4) A pharmaceutical composition comprising the monoclonal antibody according to claim 1 or 2; (5) A biological agent comprising the monoclonal antibody or the pharmaceutical composition according to claim 1 or 2.

9. A method for detecting the novel coronavirus N protein in a sample for non-diagnosis and non-treatment purposes, the method comprising: The sample to be tested is contacted with the monoclonal antibody described in claim 1 or 2, the detection kit described in claim 6 or 7, the detection reagent or detection product described in claim 8 to detect the formation of an immune complex between the new coronavirus N protein and the monoclonal antibody.

10. Any one of the following applications, characterized in that: The applications include: (1) Use of the monoclonal antibody described in claim 1 or 2 in the preparation of a test reagent for detecting a novel coronavirus; (2) Use of the monoclonal antibody described in claim 1 or 2 or the detection reagent described in claim 8 in the preparation of a diagnostic product for diagnosing and / or assisting in the diagnosis of a disease infected by a novel coronavirus; (3) Use of the monoclonal antibody described in claim 1 or 2 or the detection reagent described in claim 8 in the preparation of a detection kit for detecting the novel coronavirus; (4) Use of the monoclonal antibody according to claim 1 or 2 in the preparation of a drug for preventing and / or treating a disease infected by a novel coronavirus; Optionally, the new coronavirus includes the original strain of the new 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.

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