Antibodies against the novel coronavirus or its N protein, reagents and kits for detecting the novel coronavirus or its N protein

Abstract

The present invention discloses antibodies against the novel coronavirus or its N protein or reagents and kits for detecting the novel coronavirus or its N protein, and relates to the field of antibodies. The antibodies against the novel coronavirus or its N protein disclosed in the present invention include a heavy chain complementary determining region and a light chain complementary determining region, and the antibodies provide an important source of raw materials for the detection of the novel coronavirus or its N protein, and have improved affinity or activity.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and specifically, to an antibody against the novel coronavirus or its N protein, and a reagent and kit for detecting the novel coronavirus or its N protein. Background Art

[0002] The structural proteins of the novel coronavirus 2019-nCoV are divided into: spike glycoprotein (S protein), envelope glycoprotein (E protein), membrane glycoprotein (M protein) and nucleocapsid protein (N protein), which include multiple antigenic epitopes. The N protein and the viral genomic RNA are entangled to form the viral nucleocapsid, and play an important role in the synthesis of viral RNA. At the same time, the N protein is relatively conservative and accounts for the largest proportion of the structural proteins of the virus. The body can produce high levels of antibodies against the N protein in the early stages of infection. Finally, the N protein is an important marker protein of the novel coronavirus. By using the principle of specific binding between antigen and antibody, the presence of the antigen can be detected by the N protein monoclonal antibody, thereby directly proving that the sample contains the novel coronavirus and realizing the detection of the novel coronavirus.

[0003] The antibodies tested are mainly divided into two categories: IgM and IgG. There is currently a lack of systematic research on the production and duration of these two types of antibodies to the new coronavirus. Generally speaking, IgM antibodies are produced early, and once infected, they are produced quickly, have a short duration, and disappear quickly. A positive test in the blood can reflect that the body is in an acute infection state and can be used as an indicator of early infection. Compared with the nucleic acid detection method, the antibody test sample is serum or plasma, which is less affected by sample sampling, which is conducive to early detection and exclusion of suspected cases. At the same time, the test is fast, convenient, and suitable for large-scale screening.

[0004] The detection of the novel coronavirus or its N protein antigen adopts serological detection methods, mainly including double antibody sandwich ELISA method, immune complex lysis detection method, ultra-sensitive EIA method, enzyme-linked immunofluorescence method, etc. The double antibody sandwich method is currently widely used to detect the novel coronavirus or its N protein antigen, and obtaining antibodies against the novel coronavirus or its N protein is the key to realizing the double antibody sandwich method detection. Therefore, there is a strong demand in this field for antibodies that effectively bind to the novel coronavirus or its N protein and detect it. Summary of the invention

[0005] The present application provides an antibody or a functional fragment thereof, which provides an important source of raw materials for the detection of the new coronavirus or its N protein.

[0006] To achieve the above-mentioned object, according to one aspect of the present invention, an antibody or a functional fragment thereof is provided, wherein the antibody or the functional fragment thereof comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1, HCDR2, HCDR3 comprise / are amino acid sequences consistent with HCDR1, HCDR2, HCDR3 of the heavy chain variable region shown in SEQ ID NO: 17; and the LCDR1, LCDR2, LCDR3 comprise / are amino acid sequences consistent with LCDR1, LCDR2, LCDR3 of the light chain variable region shown in SEQ ID NO: 18.

[0007] In order to achieve the above-mentioned object, according to the second aspect of the present invention, an antibody or a functional fragment thereof is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18.

[0008] To achieve the above object, according to the third aspect of the present invention, an antibody or a functional fragment thereof is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; and the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0009] In order to achieve the above object, according to the fourth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody or a functional fragment thereof.

[0010] In order to achieve the above object, according to the fifth aspect of the present invention, a reagent or a kit is provided, wherein the reagent or the kit comprises the above antibody or a functional fragment thereof or the above antibody conjugate.

[0011] In order to achieve the above-mentioned purpose, according to the sixth aspect of the present invention, a method for detecting the new coronavirus or its N protein is provided, comprising: a) contacting the above-mentioned antibody or its functional fragment, antibody conjugate, or reagent or kit with the new coronavirus or its N protein antigen in the sample to be detected to form an immune complex under conditions sufficient for an antibody / antigen binding reaction to occur; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample.

[0012] In order to achieve the above object, according to the seventh aspect of the present invention, a nucleic acid is provided, which encodes the above antibody or a functional fragment thereof.

[0013] In order to achieve the above object, according to the eighth aspect of the present invention, a vector is provided, wherein the vector comprises the above nucleic acid.

[0014] In order to achieve the above object, according to the ninth aspect of the present invention, a cell is provided, wherein the cell comprises the above nucleic acid, vector or expresses the above antibody or a functional fragment thereof.

[0015] In order to achieve the above object, according to the tenth aspect of the present invention, a method for preparing the above antibody or a functional fragment thereof is provided, the method comprising culturing the above cell.

[0016] In order to achieve the above-mentioned purpose, according to the eleventh aspect of the present invention, there is provided a use of the above-mentioned antibody or its functional fragment, antibody conjugate, reagent or kit in detecting or preparing a product for detecting the new coronavirus or its N protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 The results of reducing SDS-PAGE of Anti-COVN 14C10mut are shown. DETAILED DESCRIPTION

[0019] In a first aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1, HCDR2, HCDR3 comprise / are amino acid sequences consistent with HCDR1, HCDR2, HCDR3 of the heavy chain variable region shown in SEQ ID NO: 17; and the LCDR1, LCDR2, LCDR3 comprise / are amino acid sequences consistent with LCDR1, LCDR2, LCDR3 of the light chain variable region shown in SEQ ID NO: 18.

[0020] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.

[0021] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the highly variable region of the heavy and light chains of the antibody.

[0022] In the present invention, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0023] The definition methods of CDR are well known in the art, and CDR definition methods include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition. As described herein, "Kabat definition" refers to the definition system described by Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" refers to Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that may not strictly follow one of the above schemes, but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or extended based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a specific CDR.

[0024] Table 1: CDR Definition 1

[0025] CDR Kabat <![CDATA[AbM 2 ]]> IMGT HCDR1 31～35 26-35 26-35 HCDR2 50～65 50-58 51-56 HCDR3 95～102 95-102 93-102 LCDR1 24～34 24-34 27-32 LCDR2 50～56 50-56 50-51 LCDR3 89～97 89-97 89-97

[0026] 1 The numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below).

[0027] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0028] Kabat et al. also proposed a numbering system applicable to the variable region sequence of any antibody. A person skilled in the art can clearly correspond the Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself.

[0029] The present invention adopts the Kabat system to define CDR regions, which can be obtained through the Internet. http: / / www.abysis.org / abysis / sequence_input /

[0030] key_annotation / key_annotation.cgi to obtain the CDR region defined by the Kabat system. However, the CDR region defined by other methods also belongs to the protection scope of the present invention.

[0031] In an optional embodiment, the present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises the following complementary determining regions:

[0032] HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0033] HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 2.

[0034] HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3.

[0035] LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:4.

[0036] LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:5.

[0037] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6.

[0038] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region other than the CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0039] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0040] In an optional embodiment, the antibody or a functional fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4;

[0041] The HFR1 comprises / is SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto;

[0042] The HFR2 comprises / is SEQ ID NO:8 or an amino acid sequence having at least 80% identity thereto;

[0043] The HFR3 comprises / is SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto;

[0044] The HFR4 comprises / is SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0045] The LFR1 comprises / is SEQ ID NO:11 or an amino acid sequence having at least 80% identity thereto;

[0046] The LFR2 comprises / is SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0047] The LFR3 comprises / is SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto;

[0048] The LFR4 comprises / is SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto;

[0049] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the antibodies or functional fragments thereof provided by the present invention may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0050] In an optional embodiment, the antibody or its functional fragment has a KD of less than 9.27×10 -9 The affinity of M binds to the new coronavirus or its N protein.

[0051] In an optional embodiment, the antibody or its functional fragment has a KD≤10 -9 M or KD≤10 -10 The affinity of M binds to the new coronavirus or its N protein.

[0052] In an optional embodiment, the antibody or its functional fragment has a KD≤4.14×10 -10 The affinity of M binds to the new coronavirus or its N protein.

[0053] There are many methods for determining antibody affinity (KD), which can be divided into thermodynamic detection methods, kinetic detection methods and dynamic equilibrium detection methods according to the detection principle. Among them, thermodynamic detection methods are commonly used, such as isothermal titration calorimetry (ITC); kinetic detection methods are commonly used, such as surface plasmon resonance (SPR) and biomembrane interferometry (BLI); dynamic equilibrium detection methods are commonly used, such as enzyme-linked immunosorbent assay (ELISA).

[0054] In an alternative embodiment, KD is determined using a kinetic assay; alternatively, a surface plasmon resonance assay, such as by using a surface plasmon resonance assay such as Biosensor system of the system.

[0055] In a second aspect, an embodiment of the present invention provides an antibody or a functional fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 18.

[0056] In an alternative embodiment, the antibody or a functional fragment thereof further comprises a constant region.

[0057] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0058] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and the light chain constant region is selected from the κ type or λ type light chain constant region.

[0059] In an alternative embodiment, the constant region is derived from a species of cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0060] In an alternative embodiment, the species origin of the constant region is mouse.

[0061] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:15, and the light chain constant region (CL) sequence is as shown in SEQ ID NO:16.

[0062] It should be noted that, in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above-mentioned constant region (SEQ ID NO: 15 or 16).

[0063] In an optional embodiment, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0064] The functional fragments of the above antibodies usually have the same binding specificity as the antibodies from which they are derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, a person skilled in the art can easily obtain the above functional fragments.

[0065] The functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0066] In a third aspect, the present invention provides an antibody or a functional fragment thereof, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0067] In a fourth aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate comprises the above-mentioned antibody or a functional fragment thereof.

[0068] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or a functional fragment thereof.

[0069] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody or a functional fragment thereof.

[0070] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0071] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent agents and nanoparticle labels.

[0072] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0073] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0074] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

[0075] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0076] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0077] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0078] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0079] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0080] In an optional embodiment, the colloidal metal is colloidal gold.

[0081] In an optional embodiment, the above-mentioned antibody conjugate further includes a solid phase carrier coupled to the antibody or its functional fragment.

[0082] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0083] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon and nitrocellulose membranes.

[0084] In a fifth aspect, the present invention provides a reagent or a kit, wherein the reagent or the kit comprises the above-mentioned antibody or a functional fragment thereof or the above-mentioned antibody conjugate.

[0085] As mentioned above, the antibodies or functional fragments thereof in some embodiments or examples of the present invention can effectively bind to the novel coronavirus or its N protein antigen, and therefore, the reagent or kit comprising the novel coronavirus or its N protein antibody or its functional fragment can effectively detect the novel coronavirus or its N protein qualitatively or quantitatively. The reagent or kit provided by the present invention can be used, for example, for immunoblotting, immunoprecipitation, and other detections involving the use of the novel coronavirus or its N protein and its antibody-specific binding properties. As mentioned above, the antibodies or functional fragments thereof in some embodiments or examples of the present invention have higher binding activity or affinity with the novel coronavirus or its N protein, and therefore the reagent or kit comprising the antibody or its functional fragment has higher detection sensitivity or specificity.

[0086] In a sixth aspect, the present invention provides a method for detecting a novel coronavirus or its N protein, comprising: a) contacting the above-mentioned antibody or its functional fragment, antibody conjugate, reagent or kit with a novel coronavirus or its N protein antigen in a sample to be detected to form an immune complex under conditions sufficient for an antibody / antigen binding reaction to occur; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample;

[0087] In an optional embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.

[0088] In an optional embodiment, the immune complex further includes a second antibody that binds to the new coronavirus or its N protein antigen.

[0089] In a seventh aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.

[0090] In an eighth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0091] In a ninth aspect, the present invention provides a cell containing the above-mentioned vector.

[0092] In a tenth aspect, the present invention provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing the cell as described above.

[0093] In the eleventh aspect, the present invention provides the use of the above-mentioned antibodies or their functional fragments, antibody conjugates or the above-mentioned reagents or kits in detecting the new coronavirus or its N protein or in preparing products for detecting the new coronavirus or its N protein.

[0094] Based on the amino acid sequence of the antibody or its functional fragment disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment, for example, separating and purifying the antibody or its functional fragment from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment of the present invention, it belongs to the protection scope of the present invention.

[0095] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0097] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0098] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0099] Example 1 Preparation of Anti-COVN-14C10 Monoclonal Antibody

[0100] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TM The RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting the Anti-COVN 14C10 monoclonal antibody was an existing hybridoma cell line, which was revived and used for later use.

[0101] (1) Antibody gene preparation

[0102] mRNA was extracted from the hybridoma cell line secreting Anti-COVN 14C10 monoclonal antibody, and the DNA product was obtained by RT-PCR. The product was inserted into the pMD-18T vector after A addition reaction with rTaq DNA polymerase and transformed into DH5α competent cells. After the colonies grew, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones each were sent to a gene sequencing company for sequencing.

[0103] (2) Sequence analysis of the variable region gene of Anti-COVN 14C10 antibody

[0104] The gene sequences obtained by the above sequencing were placed in the kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 324bp, and there was a 57bp leader peptide sequence in front of it; in the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 366bp, belonging to the VH1 gene family, and there was a 57bp leader peptide sequence in front of it.

[0105] (3) Construction of recombinant antibody expression plasmid

[0106] pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed, which has introduced multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector; according to the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases at both ends, respectively, and the 0.73kb Light Chain gene fragment and the 1.43kb Heavy Chain gene fragment were amplified by the PCR amplification method.

[0107] The Heavy Chain and Light Chain gene fragments were double-digested with HindIII / EcoRI, and the 3.4A vector was double-digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0108] 2. Recombinant Antibody Production

[0109] Resuscitate HEK293 cells in advance, subculture to 200ml system, make the cell density reach 3-5 cell density to reach the selected antibody concentration and cells, cell viability>95%; centrifuge to wash cells, re-dissolve with culture medium, adjust cell density to 2.9, wash cells, re-dissolve with culture medium, and use as cell diluent. Prepare plasmid DNA and transfection reagent diluent with culture medium respectively. Add transfection reagent diluent to plasmid DNA diluent, mix well and let stand at room temperature for 15min; slowly add the mixture to cell diluent within 1min, mix well and count, record and observe the viability of cells after transfection, and place them in a 35℃ constant temperature incubator for culture, speed 120rmp, CO2 content 8%, centrifuge after 13 days. The centrifuged supernatant was affinity purified with proteinA affinity chromatography column. Take 6ug layer of purified antibody for reducing SDS-PAGE, and the electrophoresis is shown in the figure. After reducing SDS-PAGE, two bands were shown, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

[0110] Example 2 Affinity and activity optimization

[0111] Although the Anti-COVN-14C10 monoclonal antibody obtained in Example 1 has the ability to bind to the new coronavirus or its N protein, its affinity and antibody activity are not ideal, so the applicant performs directed mutations on the variable region of the antibody. That is, a computer is used to simulate the structure of the antibody variable region, the structure of the antigen-antibody variable region complex, the key amino acid analysis of the antibody, and the mutation design. According to the mutation scheme, a bidirectional primer covering the mutation site is designed and synthesized, and primers at both ends of the target DNA are synthesized. A high-fidelity PCR reaction is performed, and the PCR product is cloned into a vector, and then the mutant antibody is prepared according to the method described in Example 1. After screening, a monoclonal antibody with significantly improved affinity and antibody activity was obtained, and it was named: Anti-COVN-14C10mut. The heavy chain and light chain amino acid sequences of Anti-COVN-14C10mut are shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

[0112] Example 3 Performance Testing of Antibodies

[0113] 1. Affinity Analysis

[0114] The purified antibody was diluted in advance, and the recombinant antigen of the novel coronavirus N protein (purchased from Feipeng Bio) was diluted in a gradient manner; the binding and dissociation curves of the antigen and antibody were tested on the Biacore 8K+ device using the CM5 chip that had been pre-coupled with the antigen, and the instrument automatically fitted the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., the affinity constant; ka represents the binding rate; and kd represents the dissociation rate).

[0115] Table 2 Affinity data

[0116]

[0117] 2. Activity identification

[0118] The coating solution (main component NaHCO3) was diluted with the recombinant antigen of the new coronavirus N protein (purchased from Feipeng Bio) to 1ug / ml, 100uL per well, and kept overnight at 4°C; the next day, the washing solution (main component Na2HPO4+Nacl) was used to wash twice and pat dry; the blocking solution (20% BSA+80% PBS) was added, 120uL per well, 37°C, 1h, and pat dry; the diluted purified antibody and control antibody were added, 100uL / well, 37°C, 30min; the washing solution was used to wash 5 times and pat dry; the goat anti-mouse IgG-HRP was added, 100uL per well, 37°C, 30min; the washing solution was used to wash 5 times and pat dry; the color developing solution A (50uL / well, containing 1.05g / L citric acid, 0.186g / LEDTA·.1, 0.45g / L TMB and 0.2ml / L concentrated HCl), add colorimetric solution B (50uL / well, containing 1.05g / L citric acid, 0.186g / EDTA, 0.45g / L TMB and 0.2ml / L concentrated HCl), 10min; add stop solution (50uL / well, containing 0.75g / EDTA·.75g and 10.2ml / L concentrated H2SO4); read the OD value at 450nm (reference 630nm) on the microplate reader. The results are shown in the table below:

[0119] Table 3 Activity data

[0120] Sample concentration (ng / ml) 125.00 62.50 31.25 15.63 7.81 0.00 Comparison 1.616 1.302 0.859 0.338 0.156 0.014 Anti-COVN-14C10mut 2.153 1.793 1.175 0.770 0.462 0.036

[0121] 3. Stability assessment

[0122] The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (constant temperature box) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and the activity of the 21-day samples was tested. The results showed that there was no obvious protein state change in the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies are stable. Table 4 below shows the OD results of the enzyme immunoassay activity test for 21 days of antibody test.

[0123] Table 4 Stability data

[0124] Sample concentration (ng / ml) 31.25 15.63 0.00 4℃, 21-day samples 1.135 0.758 0.028 -80℃, 21 days sample 1.166 0.763 0.026 37℃, 21 days sample 1.152 0.779 0.023

[0125] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0126] The partial amino acid sequences involved in this application are as follows:

[0127]

[0128]

Claims

1. An antibody against the novel coronavirus or its N protein, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the light chain variable region comprising LCDR1, LCDR2, and LCDR3, characterized in that: The HCDR1, HCDR2, and HCDR3 are amino acid sequences that are consistent with the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 17, respectively; the LCDR1, LCDR2, and LCDR3 are amino acid sequences that are consistent with the LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 18, respectively; The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are defined by the Kabat, Chothia, IMGT, AbM or Contact systems.

2. An antibody against a novel coronavirus or its N protein, the antibody comprising the following complementary determining regions: HCDR1, which consists of the amino acid sequence shown in SEQ ID NO: 1; HCDR2, which consists of the amino acid sequence shown in SEQ ID NO: 2; HCDR3, which consists of the amino acid sequence shown in SEQ ID NO: 3; LCDR1, which consists of the amino acid sequence shown in SEQ ID NO:4; LCDR2, which consists of the amino acid sequence shown in SEQ ID NO:5; LCDR3 consists of the amino acid sequence shown in SEQ ID NO:

6.

3. The antibody according to any one of claims 1 to 2, characterized in that The antibody further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4; The HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

4. The antibody according to any one of claims 1 to 2, characterized in that The antibody has a KD of <9.27×10 -9 The affinity of M binds to the new coronavirus or its N protein.

5. An antibody against a novel coronavirus or its N protein, comprising a heavy chain variable region and a light chain variable region, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

18.

6. The antibody according to any one of claims 1 to 2 and 5, characterized in that The antibody also comprises a constant region.

7. The antibody according to claim 6, characterized in that The constant region includes a heavy chain constant region and / or a light chain constant region.

8. The antibody according to claim 7, characterized in that The heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

9. The antibody according to claim 6, characterized in that The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

10. The antibody according to claim 6, characterized in that The species origin of the constant region is mouse.

11. The antibody according to claim 7, characterized in that The heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto.

12. The antibody according to claim 7, characterized in that The light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto.

13. A functional fragment of an antibody against a novel coronavirus or its N protein, characterized in that: The antibody is the antibody according to any one of claims 1 to 12, and the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

14. An antibody against a novel coronavirus or its N protein, comprising a heavy chain and a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; the amino acid sequence of the light chain is shown in SEQ ID NO:

20.

15. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody according to any one of claims 1 to 12 and 14 or a functional fragment of the antibody according to claim 13.

16. The antibody conjugate according to claim 15, characterized in that The antibody conjugate further comprises biotin or a biotin derivative coupled to the antibody or a functional fragment of the antibody.

17. The antibody conjugate according to claim 15, characterized in that The antibody conjugate further comprises a label coupled to the antibody or the functional fragment of the antibody.

18. The antibody conjugate according to claim 17, characterized in that The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

19. The antibody conjugate according to claim 15, characterized in that: The antibody conjugate further comprises a solid phase carrier coupled to the antibody or the functional fragment of the antibody.

20. The antibody conjugate according to claim 19, characterized in that The solid support is selected from microspheres, plates and membranes.

21. A reagent or a kit, characterized in that: The reagent or kit comprises the antibody according to any one of claims 1-12 and 14, or the functional fragment of the antibody according to claim 13, or the antibody conjugate according to any one of claims 15-20.

22. Use of the antibody according to any one of claims 1 to 12 and 14, the functional fragment of the antibody according to claim 13, the antibody conjugate according to any one of claims 15 to 20, or the reagent or kit according to claim 21 in the preparation of a product for detecting a novel coronavirus or its N protein, characterized in that: include: a) contacting the antibody of any one of claims 1 to 12 and 14, the functional fragment of the antibody of claim 13, the antibody conjugate of any one of claims 15 to 20, or the reagent or kit of claim 21 with the novel coronavirus or its N protein antigen in the sample to be detected to form an immune complex under conditions sufficient for an antibody / antigen binding reaction to occur; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample.

23. The use according to claim 22, characterized in that The immune complex further comprises a second antibody, which binds to the antibody or a functional fragment of the antibody.

24. The use according to claim 22, characterized in that The immune complex also includes a second antibody, which binds to the new coronavirus or its N protein antigen.

25. A nucleic acid, characterized in that It encodes the antibody according to any one of claims 1 to 12 and 14 or the functional fragment of the antibody according to claim 13.

26. A carrier, characterized in that It contains the nucleic acid according to claim 25.

27. A cell, characterized in that It contains the nucleic acid according to claim 25 or the vector according to claim 26.

28. A method for preparing the antibody according to any one of claims 1 to 12 and 14 or the functional fragment of the antibody according to claim 13, characterized in that: It includes: Cultivate the cell of claim 27.

29. Use of the antibody of any one of claims 1-12 and 14, the functional fragment of the antibody of claim 13, the antibody conjugate of any one of claims 15-20, or the reagent or kit of claim 21 in the preparation of a product for detecting a novel coronavirus or its N protein.

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