WO2022193980A1 - Antibody or antigen-binding fragment thereof for novel coronavirus nucleocapsid protein, and application thereof - Google Patents

Abstract

The present invention provides an antibody or an antigen-binding fragment thereof for novel coronavirus nucleocapsid protein, and an application thereof. The antibody is selected from any one of mAb6 antibody, mAb7 antibody, mAb8 antibody and mAb9 antibody; and the antibody is secreted by hybridoma cells having the accession numbers of CCTCC NO: C2020236, CCTCC NO: C2020237, CCTCC NO: C2020238 or CCTCC NO: C2020239. The presence of a novel coronavirus or an antigen thereof in an environmental sample and/or a biological sample can be detected by using the antibody. In addition, the present invention also provides a novel coronavirus detection kit prepared by using the antibody, capable of detecting the nucleocapsid protein in the early stage of infection, thereby providing a means to make the clinical detection of a novel coronavirus fast and precise.

Description

Antibody or antigen-binding fragment thereof against nucleocapsid protein of novel coronavirus and its application technical field

The invention belongs to the technical field of biomedicine, and in particular relates to an antibody against a novel coronavirus, in particular to an antibody against a novel coronavirus nucleocapsid protein or an antigen-binding fragment thereof and an application thereof.

Background technique

The novel coronavirus (SARS-CoV-2) belongs to the genus Betacoronavirus, and both the MERS virus known to cause Middle East Respiratory Syndrome and the SARS virus known to cause Severe Acute Respiratory Syndrome belong to this genus. The acute respiratory infectious disease caused by the novel coronavirus is novel coronavirus pneumonia (COVID-19). Its clinical manifestations are mainly fever, fatigue, and dry cough. The upper respiratory tract symptoms such as nasal congestion and runny nose are rare, and hypoxia and hypoxia state may occur. .

At present, the existing clinical novel coronavirus detection reagents include enzyme-linked immunosorbent assay for serum antibody detection and RT-PCR method for viral genetic material detection. Since the production of antibodies after infection requires a window period of 1-3 weeks, based on the current epidemiological investigation, the incubation period of the new coronavirus is generally 3-7 days, and the longest is not more than 14 days, so antibody detection is not meaningful for early diagnosis. In addition, the current antibody detection has a high false positive rate, and the specificity cannot meet the needs.

Nucleic acid detection has high specificity and sensitivity, but this detection method has high technical requirements, is prone to false negatives, requires special handling of specimens, and requires professional equipment such as PCR amplifiers and gel electrophoresis. It takes a long time to test and requires professional technicians to operate and judge the test results. It cannot be used for early preliminary screening in communities, grass-roots hospitals, airports, customs and even families. Therefore, the antigen diagnosis method for the new coronavirus, especially the rapid, sensitive and specific antigen diagnosis method, has irreplaceable significance in the diagnosis of COVID-19.

During virion assembly, the nucleocapsid protein (N protein) binds to the viral RNA and leads to the formation of the helical nucleocapsid. Nucleocapsid protein is a highly immunogenic phosphoprotein involved in viral genome replication and regulation of cellular signaling pathways. Nucleocapsid protein is also the most abundant protein in coronaviruses. Due to the sequence conservation and strong immunogenicity of nucleocapsid proteins, nucleocapsid proteins are often used as diagnostic tools for coronaviruses.

Therefore, there is an urgent need to develop a product that can detect the presence of novel coronavirus antigens in samples at an early stage of infection to meet clinical needs.

SUMMARY OF THE INVENTION

In view of the deficiencies in the prior art, the purpose of the present invention is to provide an antibody or an antigen-binding fragment thereof against the nucleocapsid protein of the novel coronavirus and its application. Using the novel coronavirus nucleocapsid protein as an antigen to immunize mice, obtain a mouse with the highest immune serum titer, and obtain hybridoma cells and store them; the antibodies or their antigen-binding fragments secreted by the obtained hybridoma cells can specifically bind to the nucleocapsid capsid protein, which can then be made into a kit for the detection of the new coronavirus.

For this purpose, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides an antibody against a novel coronavirus nucleocapsid protein or an antigen-binding fragment thereof, the antibody is selected from any one of mAb6 antibody, mAb7 antibody, mAb8 antibody and mAb9 antibody;

Wherein, the mAb6 antibody is secreted by the hybridoma cell line #mAb6 with the deposit number CCTCC NO: C2020236, the mAb7 antibody is secreted by the hybridoma cell line #mAb7 with the deposit number CCTCC NO: C2020237, and the mAb8 antibody is secreted by the deposit The hybridoma cell line #mAb8 with the number CCTCC NO: C2020238 is secreted, and the mAb9 antibody is secreted by the hybridoma cell line #mAb9 with the deposit number CCTCC NO: C2020239.

It is found in the present invention that the specific antibody against the SARS-CoV-2 virus N protein can be used to determine the presence or content of the SARS-CoV-2 virus or its corresponding antigen in a sample, and can achieve high sensitivity and stability sex.

The monoclonal antibody of the present invention can be obtained using the nucleocapsid protein of the novel coronavirus as an immunogen. The amino acid sequence of nucleocapsid protein is well known, and the amino acid sequence is shown in SEQ ID NO:1. The base sequence of the nucleic acid encoding the amino acid sequence shown in SEQ ID NO:1 is shown in SEQ ID NO:2. Likewise, the monoclonal antibody of the present invention can be obtained using a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 as an immunogen. It can also be a natural mutant of the amino acid sequence of SEQ ID NO:1.

In addition, it should be noted that, by decomposition with papain or trypsin, antibody fragments (referred to as "antigen-binding fragments" in the present invention) having binding properties to the corresponding antigens such as Fab fragments or F(ab')2 fragments can be obtained. ), the antigen-binding fragment of the present invention can also be used in the same manner as the monoclonal antibody of the present invention, and is included in the scope of the present invention.

In a second aspect, the present invention provides a hybridoma cell line that secretes the antibody or its antigen-binding fragment according to the first aspect, wherein the hybridoma cell line has a deposit number of CCTCC NO: C2020236, CCTCC NO: C2020237, CCTCC Either NO: C2020238 or CCTCC NO: C2020239.

The biological material preservation information of the present invention is as follows:

The hybridoma cell line with the deposit number CCTCC NO: C2020236 has been registered and deposited in the China Collection of Type Cultures (CCTCC) (Address: Wuhan University, Wuhan, China) on November 26, 2020, and the classification name is: Hybridoma cell line #mAb6.

The hybridoma cell line with the deposit number CCTCC NO: C2020237 has been registered and deposited in the China Center for Type Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China) on November 26, 2020, and the classification name is: Hybridoma cell line #mAb7.

The hybridoma cell line with the deposit number CCTCC NO: C2020238 has been registered and deposited in the China Center for Type Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China) on November 26, 2020, and the classification name is: Hybridoma cell line #mAb8.

The hybridoma cell line with the deposit number CCTCC NO: C2020239 has been registered and deposited in the China Center for Type Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China) on November 26, 2020, and the classification name is: Hybridoma cell line #mAb9.

The hybridoma of the present invention can be produced by a cell fusion technique known in the art: immunizing an animal with an immunogen by a conventional method, cell fusion of an anti-nucleocapsid protein antibody-producing cell obtained from the animal and a tumor cell, thereby obtaining hybridomas to produce the above antibodies. Animals immunized to produce hybridomas of the present invention are not particularly limited and include, but are not limited to, goats, sheep, guinea pigs, mice, rats and rabbits. Preferred among them are mice.

The above-mentioned hybridoma can be obtained, for example, by subcutaneously administering the nucleocapsid protein of the novel coronavirus together with Freund's complete adjuvant to animals such as mice by subcutaneous multiple injections at intervals of 2 to 3 weeks. immunity. Next, an antibody-producing cell derived from a spleen or the like obtained from an immunized animal is fused with a tumor cell, and the tumor cell is a myeloma cell selected from an immortalized cell line such as a myeloma cell line. cultured tumor cells. The above-mentioned fusion method can be, for example, according to the conventional method of Khler and Milstein (G. Kohler and C. Milstein, Nature, 1975, 256: 495-497), using the polyethylene glycol method, or the Sendai virus method or the like.

The monoclonal antibody of the present invention can be produced by the hybridoma of the present invention. The method for producing the monoclonal antibody of the present invention from the hybridoma of the present invention can be conventional or known methods in the art, for example, it can be obtained from the tissue culture medium for culturing the hybridoma of the present invention, or it can be inoculated into mice Proliferated in vivo, isolated from collected ascites fluid or serum. The culturing of the hybridomas of the present invention can be carried out according to conventional or known methods in the art. The methods of inoculating, collecting ascites fluid or serum, and isolating the monoclonal antibody of the present invention from ascites fluid or serum may be conventional or known methods in the art.

In a third aspect, the present invention provides the application of the antibody or antigen-binding fragment thereof described in the first aspect or the hybridoma cell line described in the second aspect in the preparation of a novel coronavirus detection kit.

The application field of the monoclonal antibody or the antigen-binding fragment thereof in the present invention is not particularly limited, and can especially be applied to immunoassays for judging the infection status of the SARS-CoV-2 virus.

In a fourth aspect, the present invention also provides a non-disease diagnosis and treatment purpose, a method for detecting the presence of a novel coronavirus or its corresponding antigen in a sample, the method comprising the following steps:

The sample to be tested is mixed with the antibody or its antigen-binding fragment as described in the first aspect, incubated, and the presence of the novel coronavirus or its corresponding antigen is detected.

The present invention provides an immunoassay method for detecting or quantifying the presence or quantification of SARS-CoV-2 virus or its antigen in biological samples and/or environmental samples by using the above-mentioned antibodies; the method comprises combining the biological samples to be tested and /or the environmental sample is incubated with the anti-SARS-CoV-2 virus N protein monoclonal antibody of the present invention or its antigen-binding fragment to form an antigen-antibody complex, and qualitative detection and quantitative determination of the formed binding complex are performed. The presence or amount of SARS-CoV-2 virus is indicative of the presence or amount.

The immunoassay method itself is well-known, and any well-known immunoassay method can be used, which need not be repeated in this specification. That is, if it is classified by the measurement format, there are sandwich methods, competition methods, aggregation methods, Western blotting methods, etc., and if they are classified by the labels used, there are fluorescence methods, enzymatic methods, radioactive methods, biotin methods, etc. can be used. Diagnosis can also be made by immunohistostaining. When a labeled antibody is used in the immunoassay method, the method for labeling the antibody itself is known, and any known method can be used.

The immunoassay of the present invention is performed using at least one monoclonal antibody or antigen-binding fragment thereof secreted by the above-mentioned hybridoma cell line. Immunoassays for detecting antigens are well known in the art. The monoclonal antibodies or antigen-binding fragments thereof according to the present invention may be independent of the label used (eg, enzyme, fluorescence, etc.) and independent of the detection mode (eg, fluorescence immunoassay, enzyme-linked immunosorbent assay, or chemiluminescence assay). method, etc.) or assay principles (eg sandwich method, competition method, etc.) are used in the above immunoassay methods.

The above immunoassays include enzyme immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescence immunoassays, Western blotting, immunochromatography, latex agglutination assays, etc.; The method uses a marker-labeled antigen or antibody to determine the target antigen in a biological sample and/or an environmental sample.

Among the various immunoassays described above, enzyme immunoassays, fluorescent immunoassays and chemiluminescence immunoassays are preferred. The competitive method is based on the detection of SARS-CoV-2 virus in the specimen and a known amount of labeled SARS-CoV-2 virus N protein and the monoclonal antibody of the present invention or its antigen-binding fragment for quantitative competitive binding reaction; namely, the present invention Including qualitative or quantitative determination of the presence or content of SARS-CoV-2 virus in biological components using assay color, fluorescence, time-resolved fluorescence, chemiluminescence, electrochemiluminescence, or radioactivity. The sandwich method is to immobilize the antibody or antigen-binding fragment of the present invention as a first antibody as a solid phase, react with the biological sample to be tested and/or an environmental sample, rinse it, and then react it with the second antibody, and measure the difference after rinsing. Solid phase bound secondary antibody. The second antibody bound to the solid phase can be measured by labeling the second antibody with an enzyme, a fluorescent substance, a radioactive substance, biotin, or the like. In the aggregation method, the antibody or antigen-binding fragment thereof of the present invention is immobilized on particles such as latex, reacted with a sample, and the absorbance is measured. Through the above method, a plurality of standard substances of known concentration are measured, and a standard curve is prepared according to the relationship between the measured labeled amount and the content of the standard substance. Quantification of SARS-CoV-2 viral antigens in samples.

For SARS-CoV-2 immunoassays according to the competition method and the sandwich method described above, the solid phase needs to be washed sufficiently to measure the activity of binding to the label.

When the label is a radioisotope, the measurement is performed with a pore counter or a liquid scintillation counter. For example, when the label is an enzyme, the substrate is added and the enzyme activity is measured colorimetrically or fluorometrically after color development. When the label is a fluorescent substance, a phosphorescent substance, or a coloring substance, it can be measured by methods known in the art, respectively.

Through the above method, a plurality of standard substances of known concentration are measured, and a standard curve is prepared according to the relationship between the measured labeled amount and the content of the standard substance. Quantification of SARS-CoV-2 viral antigens in samples.

As a preferred technical solution of the present invention, the samples to be tested include biological samples and/or environmental samples.

The sample to be supplied to the above-mentioned immunoassay is not particularly limited as long as it contains the nucleocapsid protein of the SARS-CoV-2 virus. For example, it can be derived from human and animal serum, plasma, whole blood, and others. Nasal swab (nasal swab), nasal aspiration, throat swab (pharyngeal swab) and other body fluid extracts, respiratory secretions, cell or tissue homogenate, etc.

Wherein, the biological sample includes any one or a combination of at least two of plasma, whole blood, mouthwash, throat swab, urine, feces or bronchial perfusate.

As a preferred technical solution of the present invention, the method includes a sandwich method or a competition method. In simple terms, the sandwich method is to fix the antibody or antigen-binding fragment of the present invention as a first antibody on a solid phase, react with the sample to be tested, rinse it, and then react it with a second antibody, and measure the solid phase after rinsing. conjugated secondary antibody. The second antibody bound to the solid phase can be measured by labeling the second antibody with an enzyme, a fluorescent substance, a radioactive substance, biotin, or the like.

Wherein, the sandwich method comprises the steps:

(1) binding the antibody or its antigen-binding fragment as described in the first aspect to a solid-phase carrier as a first antibody;

(2) mixing the solid-phase carrier obtained in step (1) with the sample to be tested, incubating, and comparing with the control sample;

(3) after the incubation is completed, wash the solid phase carrier, add a second antibody labeled with a marker, and incubate again, the second antibody binds to the novel coronavirus or its corresponding antigen;

(4) washing the solid phase carrier again, and detecting the signal value of the marker bound to the second antibody;

(5) Compare the measured signal value with the signal value of the control sample to determine the presence and relative amount of the novel coronavirus in the sample to be tested.

Preferably, the sandwich method further comprises: in step (3), the second antibody is first labeled with biotin, and then combined with the enzyme-labeled avidin or streptavidin;

Then, in step (4), the substrate of the enzyme is added to develop color.

The above sandwich method is based on the fact that the monoclonal antibody or its antigen-binding fragment of the present invention as a capture antibody (or solid-phase antibody) and the labeled antibody that can be used in combination can specifically bind to the SARS-CoV-2 virus in the sample to be tested, The amount of SARS-CoV-2 virus in the sample was determined by quantifying the labeled antibody. Specifically, the sandwich method binds the specific monoclonal antibody against the SARS-CoV-2 virus N protein or its antigen-binding fragment of the present invention to a solid-phase carrier to form a solid-phase antibody (also called capture antibody or primary antibody) , and then add the sample to be tested and the control sample to the coated solid phase carrier and incubate for a long enough time under appropriate conditions;

After the reaction, the solid phase is thoroughly washed, and an appropriate amount of labeled secondary antibody that can bind to the SARS-CoV-2 virus N protein is added and incubated again; Detecting the signal value of the label bound to the secondary antibody; comparing the measured signal value with the signal value of a predetermined amount of control samples measured in parallel to determine the presence of SARS-CoV-2 virus in the sample and its relative quantity.

In addition, the competition law includes the following steps:

(1') binding a predetermined amount of the antibody or antigen-binding fragment thereof as described in the first aspect to a solid-phase carrier;

(2') adding the sample to be tested and a predetermined amount of the marker-labeled novel coronavirus or its corresponding antigen to the solid-phase carrier obtained in step (1'), incubating, and comparing with the control sample;

(3') washing the solid phase carrier after the reaction, and detecting the signal value of the marker bound to the novel coronavirus or its corresponding antigen;

(4') Compare the measured signal value with the signal value of the control sample to determine the presence and relative amount of the novel coronavirus in the sample to be tested.

Labeled virus monoclonal antibodies can be prepared by binding an anti-SARS-CoV-2 virus monoclonal antibody to a label. Labels can be enzymes, colloidal metal particles, colored latex particles, fluorescent latex particles, luminescent substances, fluorescent substances, and the like. The enzyme can be various enzymes used in enzyme-linked immunoassay (EIA), such as alkaline phosphatase, peroxidase, β-D-galactosidase, etc.; colloidal metal particles such as colloidal gold particles, colloidal Selenium particles, etc.

Among them, the binding method of the marker and the anti-SARS-CoV-2 virus monoclonal antibody can use a known method for generating covalent bonds or non-covalent bonds. For example, the binding methods include: glutaraldehyde method, periodate method, maleimide method, pyridyl·disulfide method, methods using various cross-linking agents, etc. (for example, refer to "Protein Nucleic Acid Enzymes", 1985, Separate volume No. 31, pages 37-45).

Among them, in the bonding method using a cross-linking agent, for example, N-succinimidyl-4-maleimidobutyric acid (GMBS), N-succinimidyl-6-maleimide can be used as the cross-linking agent. Aminocaproic acid, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, etc. In the method of covalent bonding, the functional groups present in the antibody can be used depending on the use of functional groups. In addition, functional groups such as thiol group, amino group, carboxyl group, and hydroxyl group can be introduced into the antibody according to conventional methods, and then the above-mentioned binding method can be used. The functional group is combined with the label, thereby preparing a labeled anti-SARS-CoV-2 virus monoclonal antibody. There are also physical adsorption methods through non-covalent bonding methods.

As the substrate, various chromogenic substrates, fluorescent substrates, luminescent substrates, and the like can be used corresponding to the enzymes of the labels and shown below. E.g:

(a) Chromogenic substrate: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3′,5, 5'-tetramethylbenzidine (TMB), diaminobenzidine (DAB) for peroxidase; 5-bromo-4-chloro-3-indolyl phosphate (BCIP), p-nitrophenyl phosphate (p-NPP), 5-bromo-4-chloro-3-indolyl sodium phosphate (BCIP·Na) was used for alkaline phosphatase.

(b) Fluorescent substrates: 4-methylumbelliferyl phenyl phosphate (4-MUP) for alkaline phosphatase; 4-methylumbelliferyl phenyl-β-D-galactoside (4MUG) for in β-D-galactosidase.

(c) Luminescent substrate: 3-(2′-Spiroadamantane)-4-methoxy-4-(3′-phosphoryloxy)phenyl-1,2-dioxetane·2 Sodium salt (AMPPD) for alkaline phosphatase; 3-(2'-spiroadamantane)-4-methoxy-4-(3'-β-D-galactopyranosyl)phenyl-1, 2-Dioxetane (AMGPD) was used for β-D-galactosidase; luminol, isoluminol obtained in combination with hydrogen peroxide were used for peroxidase.

In a preferred embodiment of the present invention, a double-antibody sandwich ELISA detection kit is provided, and a method for using the kit to detect the presence of SARS-CoV-2 virus in a sample is provided, including:

(1) Provide a monoclonal antibody or its antigen-binding fragment (primary antibody or solid-phase antibody) that can bind to the N protein of SARS-CoV-2 virus and coat the solid-phase carrier with the first antibody;

(2) adding the sample to be tested and the control sample (or standard) to the solid phase carrier coated in step (1) and incubating under appropriate conditions;

(3) After the reaction, wash thoroughly to remove any unbound samples, and add an appropriate amount of biotin-labeled monoclonal antibody or its antigen-binding fragment that can bind to another epitope of the SARS-CoV-2 virus N protein (section 1). secondary antibody or labeled antibody) and incubated again;

(4) After the reaction, it is thoroughly washed to remove any unbound secondary antibody, the biotinylated secondary antibody is combined with the enzyme-labeled avidin or streptavidin, and then the enzyme substrate is added for color development, and then Use a microplate reader to determine the corresponding absorbance value;

(5) The measured absorbance value is compared with the absorbance of the known quantity standard measured in parallel to determine the presence and relative amount of SARS-CoV-2 virus in the sample.

Preferably, the sample to be tested is a biological sample, further, the sample is plasma, serum and whole blood; the solid phase carrier is a microtiter plate; the avidin or streptavidin is expressed in horseradish Peroxidase label; the enzyme substrate is TMB.

In a fifth aspect, the present invention also provides a novel coronavirus detection kit, the novel coronavirus detection kit includes the antibody or its antigen-binding fragment as described in the first aspect.

Diagnosis of SARS-CoV-2 virus infection can be performed by assaying various biological samples and/or environmental samples from humans or animals using the monoclonal antibodies of the present invention against the nucleoprotein of SARS-CoV-2 virus. Using the monoclonal antibody of the present invention, the nucleocoat of SARS-CoV-2 virus in various body fluids, cells, tissues, etc. and/or environmental samples from humans or animals can be directly determined by immunochemical methods or immunohistochemical methods. shell protein.

As a preferred technical solution of the present invention, the novel coronavirus detection kit further includes a solid-phase carrier, and the antibody or its antigen-binding fragment is embedded in the solid-phase carrier.

The kit is a kit for detection using a competitive method, wherein the immunoassay reagent can be prepared, for example, into a certain amount of viral antigens labeled with enzymes, colloidal metal particles, colored latex particles, luminescent substances, fluorescent substances, radioactive substances, and the like. Using this reagent, a competitive reaction can be carried out with, for example, a sample containing a certain amount of the monoclonal antibody of the present invention, the above-mentioned labeled viral antigen and the antigen to be determined, and the amount of the labeled viral antigen bound or unbound to the antibody is relative. Immunoassays are performed by measuring the amount of antigen in a sample for quantification.

Alternatively, the novel coronavirus detection kit further includes a solid-phase carrier and a second antibody, the monoclonal antibody or its antigen-binding fragment is bound to the solid-phase carrier as a first antibody, and the second antibody carries a label with primary antibody combination.

This detection kit is a kit for detection by a sandwich method. In the immunoassay reagent of the sandwich method, a monoclonal antibody can be used as a solid-phase antibody and a labeled antibody (for example, when the antigen is a polymer), but it is usually preferred Two or more antibodies are used that can recognize two different epitopes of the antigen to be assayed, respectively. In addition, any solid-phase antibody and labeled antibody can be selected and used in combination from two or more monoclonal antibodies.

In the present invention, the following reagents can be used. For example, two types of monoclonal antibodies of the present invention can be prepared, one of which is the labeled antibody and the other is a solid-phase antibody bound to a solid-phase carrier.

First, a sample containing an antigen to be measured is reacted with the solid-phase antibody, and then a labeled antibody (secondary antibody) is reacted with the antigen captured on the solid-phase antibody to detect the presence of the label bound to the insoluble carrier. or activity, immunoassays can be performed. Similarly, a sample containing the antigen to be assayed is reacted with a solid-phase antibody, and then a labeled antibody (secondary antibody) is reacted with the antigen captured on the solid-phase antibody, and the presence of the label bound to the insoluble carrier or the Activity, ie, quantification of the amount of antigen to be assayed by the amount of labeled antibody, allows immunoassays to be performed.

Preferably, the second antibody is selected from any one of the antibodies of the present invention that can cooperate with the first antibody, that is, a monoclonal antibody or an antigen-binding fragment thereof that can be paired for use. It should be noted that, in the context of the present invention, antibodies that "can be matched" and "can be used in pairs" mean that the antibodies are directed against different epitopes in the N protein antigen of the novel coronavirus, and are different from the N protein antigen. Antibody combinations that do not interfere or antagonize each other in their binding.

In addition, the second antibody can also be other polyclonal antibody.

As a preferred technical solution of the present invention, the first antibody and the second antibody are selected from any one of the following combinations:

Combination I: the first antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb7, and the second antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb6;

Combination II: the first antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb7, and the second antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb8;

Combination III: the first antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb7, and the second antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb9.

As a preferred technical solution of the present invention, the label includes any one or a combination of at least two of radioisotopes, enzymes, enzyme substrates, phosphorescent substances, fluorescent substances, biotin or coloring substances.

Preferably, the radioisotope includes any one or a combination of at least two ^of125I , ^3H , ^{14C or32P} .

Preferably, the enzyme comprises any one or a combination of at least two of alkaline phosphatase, horseradish peroxidase, beta-galactosidase, urease or glucose oxidase.

Preferably, the fluorescent substance includes any one or a combination of at least two of fluorescein derivatives, rhodamine derivatives, rare earth elements or rare earth element complexes.

Preferably, the phosphorescent substance comprises acridine ester and/or isoluminol.

As a preferred technical solution of the present invention, the solid phase carrier comprises: any one or at least two of nitrocellulose membrane, latex particles, magnetic particles, colloidal gold, glass, fiberglass, polymer or fiber optical sensor combination.

By using the above-described monoclonal antibody of the present invention, the antibody can be used as at least one of a solid-phase antibody and a labeled antibody to prepare a SARS-CoV-2 virus immunoassay reagent.

Various solid phases used in conventional immunoassays can be used as the solid phase bound to the above-mentioned monoclonal antibodies, such as ELISA plates, latex, gelatin particles, magnetic particles, polystyrene, glass and other solid phase beads. Insoluble carriers such as liquid bases, etc. In addition, labeled antibodies can be prepared by labeling antibodies with enzymes, colloidal metal particles, colored latex particles, luminescent substances, fluorescent substances, radioactive substances, and the like. By combining reagents such as these solid-phase antibodies and/or labeled antibodies, reagents for use in enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, and the like can be prepared. These assay reagents are reagents for measuring the target antigen in a sample by a sandwich method or a competitive binding assay.

In addition, another aspect of the present invention provides the application of the above immunoassay reagent in diagnosing diseases caused by SARS-CoV-2 virus infection.

Preferably, the disease is novel coronavirus pneumonia (COVID-19).

Compared with the prior art, the beneficial effects of the present invention are:

(1) The monoclonal antibody of the present invention or its antigen-binding fragment is secreted by four hybridoma cell lines CCTCC NO: C2020236, CCTCC NO: C2020237, CCTCC NO: C2020238 or CCTCC NO: C2020239, the monoclonal antibody or its antigen The binding fragment can specifically recognize the N protein of SARS-CoV-2 virus with an affinity up to pM level, with very high sensitivity and specificity;

(2) The present invention also provides a SARS-CoV-2 virus detection kit, which can detect the presence of SARS-CoV-2 virus by using a variety of immunoassays, especially the sandwich method and the competition method. The detection kit for SARS-CoV-2 virus, because the monoclonal antibody or its antigen-binding fragment used has high affinity and specificity, the obtained detection result is relatively accurate; wherein, the present invention also provides a double-antibody sandwich ELISA detection The kit is constructed with the best paired three groups of antibodies (mAb7/Bio-mAb6, mAb7/Bio-mAb8 and mAb7/Bio-mAb9), the detection limit is 12.5pg/mL, and its repeatability is good;

(3) The double-antibody sandwich ELISA immunoassay method constructed in the present invention is a very sensitive virus detection technology, which is more specific than other serological methods when detecting most viruses, and has high ELISA sensitivity and easy operation. The development of supporting instruments and equipment standardizes and automates operating procedures, thereby further improving stability. Therefore, the novel coronavirus antigen detection kit provided by the present invention can detect the nucleocapsid protein in the early stage of infection of the patient, solves the problem of rapid clinical diagnosis of the novel coronavirus infection, and has high accuracy, It provides a rapid and accurate diagnostic method for clinical detection of the disease.

Description of drawings

FIG. 1 is a graph showing the results of measuring the titer of mouse immune serum in Example 1. FIG.

Fig. 2 is the cross-reactivity determination result curve diagram of the antibody against the new coronavirus nucleocapsid protein and SARS virus in embodiment 3.

3 is a graph showing the results of cross-reactivity determination between the antibody against the new coronavirus nucleocapsid protein and MERS virus in Example 3.

FIG. 4 is a graph showing the results of the double-antibody sandwich ELISA test of the capture antibody mAb7 and the labeled antibody Bio-mAb6 in Example 5. FIG.

FIG. 5 is a graph showing the results of the double-antibody sandwich ELISA test of the capture antibody mAb7 and the labeled antibody Bio-mAb8 in Example 5. FIG.

FIG. 6 is a graph showing the results of the double-antibody sandwich ELISA test of the capture antibody mAb7 and the labeled antibody Bio-mAb9 in Example 5. FIG.

Detailed ways

The technical solutions of the present invention are further described below in conjunction with the accompanying drawings and through specific embodiments, but the following examples are only simple examples of the present invention, and do not represent or limit the protection scope of the present invention. The request shall prevail.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment and materials used in the embodiments, according to the mastery of the prior art by those skilled in the art and the description of the present invention, the methods, equipment and materials described in the embodiments of the present invention can also be used Any methods, devices and materials similar or equivalent to those of the prior art can be used to implement the present invention.

Unless otherwise specified, the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. conventional technology. These techniques have been well described in the existing literature, see for example: Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Press, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons , New York, 1987 and periodic updates; METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third Edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M. Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999 and METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 and other related literature.

Example 1 Acquisition of hybridoma cell lines and preparation of monoclonal antibodies

This embodiment is used to obtain hybridoma cell lines and prepare monoclonal antibodies, and the specific steps are as follows:

1.1 Animal Immunization

After the SARS-CoV-2 N protein antigen (Sino Biological, 40588-V08B) was fully emulsified with complete Freund's adjuvant, male Balb/C mice (Shanghai Slack Laboratory Animal Co., Ltd.) were immunized by multi-point immunization, 50 μg / only, the immunization cycle is once every three weeks;

On the 10th day after the third immunization, blood was collected from the eye socket, and serum antibody titers were tested according to the indirect ELISA method to monitor the degree of immune response in mice.

1.2 Determination of immune serum titer

An indirect ELISA method was established to determine the titer of immune serum: 96-well polystyrene microplates were coated with 0.1 μg/mL recombinant SARS-CoV-2 N protein, 100 μL/well, overnight at 4°C; the next day, the mass fraction was 1% Blocking solution of bovine serum albumin (BSA, Sigma), 0.3 mg/well, overnight at 4°C, the coated strips were treated with 10 mM PBS buffer containing 10% sucrose the next day, vacuum-dried and then vacuum-packed in aluminum film bags Store at 4°C for the determination of mouse immune serum titer;

Orbital blood was collected 10 days after the third immunization, and the collected mouse serum was diluted with 1:100 in 10mM PBS containing 1.5% BSA for a series of concentration gradients, and then added to a 96-well plate, 100 μL/well, 30 minutes at 37°C, wash the plate 3 times with 10mM PBS containing 0.1% Tween-20 washing solution, add a 1:5000-fold dilution of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Jackson Laboratory Inc, Cat. No. 115- 035-07), 100 μL/well, 30 minutes at 37°C;

After washing the plate as above, add citrate buffer containing 0.05% (w/v) TMB and 0.06% (w/v) hydrogen peroxide pH5.0, 100 μL/well, and protect from light at room temperature for 10 minutes, and stop by adding 0.2M H ₂ SO ₄ Reaction, 100 μL/well, the microplate reader reads the absorption value at the dual wavelengths of 450/620 nm, with the mouse serum before immunization as the negative control, and the ratio of the measured value to the control value ≥ 2.0 is positive to judge the titer of the immune serum ;

The four mice all produced strong specific immune responses after the third immunization. The response results are shown in Figure 1. The titers of the immune sera of mice M51, M52, M53 and M54 were all higher.

1.3 Hybridoma preparation

Three days before fusion, the mouse with the highest antibody titer was boosted once; three days later, the mice were sacrificed, and the mouse spleen was taken under aseptic conditions to prepare a spleen cell suspension, which was then mixed with 2 in a ratio of 1:1. ×10 ⁸ mouse myeloma Sp2/0 cell lines in logarithmic growth phase were mixed and then fused in a solution containing 50% polyethylene glycol (molecular weight 1450) and 5% dimethyl sulfoxide (DMSO) ;

Use Iscove's medium (containing 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.1 mM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine) to adjust the spleen cell number to 5 x 10 ⁵ /mL, add 0.3mL to the well of a 96-well culture plate, and place it in a 37°C, 5% CO ₂ incubator for cultivation;

After 1 day, add 100 μL of screening medium containing hypoxanthine and aminopterin-thymidine (HAT, Sigma) to each well, and then use this screening medium to change the culture medium every 3 days until the colony. cell formation.

1.4 Screening of hybridoma cells secreting antibodies against the N protein of 2019-nCoV

In order to screen positive clones that produce antibodies, the cell culture supernatant was detected by indirect ELISA, which is briefly described as follows:

The hybridoma cell culture supernatant was added to the ELISA plate pre-coated with antigen, 100 μL/well, the SP2/0 cell culture supernatant was used as a negative control, and the immune polyantibody serum was used as a positive control, one hour at room temperature; PBST washed 3 times; Add working concentration of HRP-labeled goat anti-mouse IgG antibody (Jackson Laboratory Inc, Cat. No. 115-035-07), 100 μL/well, 30 minutes at 37°C, wash the plate as above, add substrate TMB, 100 μL/well, avoid at room temperature. Light for 10 minutes, add 0.2M H ₂ SO ₄ at 100 μL/well to stop the reaction, and the microplate reader reads the absorbance at dual wavelengths of 450/620 nm. If the OD 450/620nm reading of the tested well is more than twice the negative control, it is judged as positive;

1.5 Cloning of positive hybridoma cells

The screened strong positive cell clones were subcloned 2 to 3 times by the limiting dilution method, and a total of 6 hybridoma cell lines that stably secreted antibodies were obtained, named #mAb6, #mAb7, #mAb8, #mAb9, # mAb10 and #mAb11, and 4 of them were bio-deposited, #mAb6, #mAb7, #mAb8 and #mAb9 correspond to the deposit numbers CCTCC NO: C2020236, CCTCC NO: C2020237, CCTCC NO: C2020238 and CCTCC NO: C2020239 of hybridoma cell lines.

1.6 Purification of antibodies

The positive hybridoma cell clones #mAb6, #mAb7, #mAb8, #mAb9, #mAb10 and #mAb11 obtained from the above screening were cultured in RPMI 1640 medium supplemented with 10% FCS.

When the cell density reached approximately ⁵ x 105 cells/mL, the medium was replaced with serum-free medium. After incubation, centrifuge and collect the culture supernatant, then use Protein G affinity chromatography column to purify the antibody, dialyze the monoclonal antibody eluate with 150mM NaCl, and filter sterilize the dialyzed solution through a 0.2 μm filter to obtain purified Antibody samples.

1.7 Antibody labeling

Biotin labeling: NN-hydroxysuccinimide ester (NHS) is the most common biotin labeling reagent, NHS-activated biotin can react with primary amine groups (-NH ₂ ) in alkaline buffer to form stable amide bond. Proteins (such as antibodies) often have many primary amine groups and thus can be targeted for biotin labeling;

In this example, the biotin derivative NHS-LC-Biotin (Thermo Scientific, Item No. 21435) was used for antibody labeling, and the monoclonal antibody was labeled with biotin according to the method described in the manual of Thermo Scientific EZ-Link Biotin Labeling Kit to obtain Biotinylated antibodies Bio-mAb6, Bio-mAb7, Bio-mAb8, Bio-mAb9, Bio-mAb10 and Bio-mAb11.

The monoclonal antibody of the present invention can also be labeled by other methods known in the art, such as HRP (horseradish peroxidase) labeling, and the specific method is as follows:

8-10 mg of the purified antibody obtained in the above step was put into a dialysis bag, and dialyzed overnight at 4°C with 0.01M carbonate buffer at pH 9.6. Take 4 mg of HRP, dissolve it in 1 mL of pure water, slowly add 0.1 M NaIO ₄ 0.1 mL, stir at room temperature in the dark for 20 minutes, put it into a dialysis bag, and dialyze it with 0.001 M pH4.4 acetate buffer at 4°C overnight ;

Add 0.05 mL of 0.2M Na ₂ CO ₃ to HRP to adjust the pH to 9.6, then mix with the antibody, stir in the dark for 2 hours, add 0.1 mL of 4 mg/mL NaBH ₄ , stand at 4°C for 2 hours, and dialyze against PBS at 4°C overnight.

An equal volume of saturated ammonium sulfate was added, stirred for 30 minutes, allowed to stand at 4°C for 2 hours, centrifuged at 3000 rpm for 20 minutes, and the supernatant was discarded. The resulting precipitate was repeated with 50% saturated ammonium sulfate. The obtained precipitate was dissolved in 1 mL of PBS and dialyzed with PBS at 4°C for 48 hours to obtain enzyme-labeled monoclonal antibodies HRP-mAb6, HRP-mAb7, HRP-mAb8, HRP-mAb9, HRP-mAb10 and HRP-mAb11.

Example 2 Screening of the best paired monoclonal antibody by double-antibody sandwich ELISA

Monoclonal hybridoma cells produce antibodies against only one epitope of the antigen, and the "double antibody sandwich method" is used to screen the best paired solid-phase antibody and labeled antibody.

A 6×6 matrix was used for pairwise screening of antibodies, and the above 6 monoclonal antibodies were coated as capture antibodies, respectively, and the 6 biotin-labeled monoclonal antibodies Bio-mAb6, Bio-mAb7, Bio-mAb8, Bio-mAb9, Bio-mAb10, and Bio-mAb11 were paired for rapid screening of captured and labeled monoclonal antibody pairs in sandwich ELISA.

By comparing the detection sensitivities, it was shown that the monoclonal antibodies mAb7 or mAb11 as capture antibodies, when paired with Bio-mAb6, Bio-mAb8, Bio-mAb9 and Bio-mAb10, respectively, could produce stronger signals.

Example 3 Functional identification of anti-N protein monoclonal antibody

3.1 Monoclonal antibody affinity constant determination

The binding affinity constants of purified mouse monoclonal antibodies mAb6, mAb7, mAb8, mAb9 and the N protein of 2019-nCoV were determined by biofilm interferometry (BLI). The assay was performed using the ForteBio Octet RED&QK platform of PALL Company, and the method was referred to the instruction manual of the platform.

First, the biotinylated SARS-CoV-2 N protein was immobilized on the surface of the SA sensor, and the aforementioned monoclonal antibody against SARS-CoV-2 N protein was used as the analyte. The data were processed and fitted with _a 1:1 binding model of the analysis software. The fitted data basically overlapped with the experimental data to obtain the association and dissociation rate constants Ka and K _{d . Divide Ka by K d to} obtain the equilibrium dissociation constant K _D (see Table 1).

The results showed that the mouse mAb mAb7 had the highest affinity, while the affinity of mAb6, mAb8 and mAb9 were comparable, and the K _D values were all lower than the pM level.

Table 1

Antibody name	K D (M)	K a (1/Ms)	K d (1/s)
mAb6	3.86E-12	2.133+05	8.24+07
mAb7	<1.0E-12	2.174E+05	<1.0E-07
mAb8	7.797E-11	2.117E+05	1.65E-05
mAb9	1.017E-11	2.450E+05	2.492E-06

3.2 Cross-reactivity of monoclonal antibodies with SARS and MERS viruses

In this example, the cross-reactivity of the above-mentioned anti-SARS-CoV-2 monoclonal antibodies mAb6, mAb7, mAb8, mAb9, mAb10 and mAb11 to human SARS and MERS coronavirus nucleocapsid proteins was also detected.

Dilute SARS virus N protein (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., Item No. 40143-V08B) or MERS virus N protein (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., Item No. 40068-V08B) with PBS buffer to 0.1 μg/ mL, added to a 96-well plate at a volume of 100 μL/well, and placed at 4°C for 20 h. Aspirate the PBS buffer in the 96-well plate, wash the plate once with PBST (pH 7.4, PBS containing 0.05% Tween 20) buffer, add 200 μL/well PBST/1% nonfat dry milk, and incubate at room temperature for 1 h to block.

Remove the blocking solution, wash the plate three times with PBST buffer, dilute the monoclonal antibody with an initial concentration of 10 μg/mL at a ratio of 1:4 to a gradient concentration, add 100 μL/well, and incubate at room temperature for 1.5 h. The reaction system was removed, and after washing the plate three times with PBST, 50 μL/well of HRP-labeled goat anti-mouse IgG Fc secondary antibody (Jackson Laboratory Inc, Cat. No. 115-035-07) diluted 1:5000 was added, and incubated at room temperature for 1 hour. After washing the plate three times with PBST, add 100 μL/well of TMB, and after incubation at room temperature, add 50 μL/well of 0.2M sulfuric acid to stop the reaction; the microplate reader reads the absorbance at dual wavelengths of 450/620 nm.

As shown in Figure 2, all of the above-mentioned 6 kinds of monoclonal antibodies can specifically bind to the N protein antigen of SARS virus. In addition, none of the six mAbs could specifically bind to the MERS virus N protein (see Figure 3).

Example 4 Establishment of a double-antibody sandwich ELISA kit for detecting SARS-CoV-2 antigen with anti-SARS-CoV-2 N protein monoclonal antibody

Taking the antibody mAb7 provided by the present invention and the biotin-labeled monoclonal antibody Bio-mAb8 of the present invention as examples, a double-antibody sandwich ELISA immunoassay method for SARS-CoV-2 virus was established.

The washing solution is PBS-Tween20, pH 7.4 (PBST); the blocking solution is PBST containing 1.5% bovine serum albumin (BSA); the standard and test sample dilution solution is PBST containing 1.5% BSA; biotin-labeled antibody The diluent is PBST containing 1.5% BSA; Streptavidin-HRP (Thermo Scientific, Lot: 21130) is prepared into a working solution with PBST containing 1.5% BSA according to the method of its matching instructions.

The detection method is as follows:

(1) Dilute the antibody mAb7 with 1×PBS to a final concentration of 5 μg/mL, coat 100 μL of each well of a polystyrene micro-reaction plate, shake at room temperature for 1 hour, 500 rpm, wash with washing solution, and pat dry;

(2) Add blocking solution, 200 μL/well, and let stand for 1 hour at room temperature.

(3) Dilute the SARS-CoV-2 N protein standard at different concentrations as the standard curve (take the standard and dilute it with PBST solution of 1.5% BSA to the following concentrations: 400pg/mL, 200pg/mL, 100pg/mL, 50pg/mL , 25pg/mL, 12.5pg/mL), 100μL per well was added to the reaction plate; the sample to be tested was diluted 100 times and added to different wells, with 1.5% BSA-PBS solution for negative control, stand at room temperature for 1 hour, wash Wash 3 times and pat dry.

(4) Add 100 μL of biotin-labeled secondary antibody Bio-mAb8 (diluted with 1.5% BSA-PBS solution at a ratio of 1:5000) to each well, let stand for 1 hour at room temperature, wash three times with washing solution, and pat dry.

(5) Add the prepared Streptavidin-HRP solution, 100 μL per well, and let stand for 1 hour at room temperature.

(6) Wash three times with washing solution, pat dry, then add prepared TMB, 100 μL/well, and develop color at room temperature for 20-30 minutes.

(7) Add 0.2M H ₂ SO ₄ to stop, 50 μL/well.

(8) The microplate reader reads the absorption value at the dual wavelengths of 450/620nm, draws a standard curve according to the measured OD value of the sample and the concentration of the standard substance, and obtains a linear regression equation. The content of SARS-CoV-2 N protein in the sample can be obtained by substituting the OD value of the tested sample.

Example 5 ELISA method and performance of its kit

We used the best paired 3 groups of antibodies screened in Example 2 above, mAb7/Bio-mAb6, mAb7/Bio-mAb8, mAb7/Bio-mAb9, established to measure the SARS-CoV-2 virus content of the double antibody Sandwich ELISA method and kit for performance evaluation.

5.1 Detection range

It can be seen from Figure 4-Figure 6, in which Figure 4 is mAb7/Bio-mAb6, Figure 5 is mAb7/Bio-mAb8, Figure 6 is mAb7/Bio-mAb9, with the increase of SARS-CoV-2 N protein (NP) concentration, The OD value also increased. When the NP concentration range was 1-400 ng/mL, the OD value measured by the established sandwich method was linearly positively correlated with the NP concentration (R ² ≥0.988). The detection limits of the three best paired antibodies screened above were all as low as 12.5 pg/mL.

5.2 Specificity

The reaction of the ELISA kit composed of the three antibody pairs screened above with several other serum substances was measured, and the specificity of the reaction was observed. Coated capture antibody 4μg/mL, human serum albumin and bovine serum albumin 100ng/ml, 5 SARS-CoV-2 N protein negative serum samples (NP negative serum) diluted 1:100, NP positive control pure product 100ng/ml mL, biotin-labeled antibody (1:5000 dilution), after color development, the judgment result is shown in Table 2.

Table 2

sample	human albumin	bovine albumin	NP negative serum (5 copies)	Positive control (NP pure)
mAb7/Bio-mAb6	-	-	- - - - -	+
mAb7/Bio-mAb8	-	-	- - - - -	+
mAb7/Bio-mAb9	-	-	- - - - -	+

Remarks: "+" indicates that the test result of the sample is positive, and "-" indicates that the test result of the sample is negative.

5.3 Sensitivity

According to the experimental method established above, the detection of the reference substance/standard substance is carried out. The detection limit was 12.5pg/mL, and the condition satisfies the OD value>3 times SD of the OD value of the blank control, and the OD value>2 times the OD value of the blank control. It shows that the method of the present invention has good sensitivity.

5.4 Repeatability

Repeated detection of one sample, a total of 4 times, the CV value of 8 results in the same plate at the same time is less than 4%, and the CV value of the average value of 4 results of different operations by different people is 1.63%, it can be seen that according to the method of the present invention. The kit has good reproducibility for the determination of the N protein of 2019-nCoV.

The above double-antibody sandwich enzyme-linked immunosorbent assay can be used to detect the SARS-CoV-2 N protein antigen in human or animal serum or plasma samples for early diagnosis of SARS-CoV-2 infection to achieve early detection, early isolation, and avoidance of purpose of dissemination. After labeling with the antibody of the present invention for fluorescence, the immunofluorescence method is used to detect viral antigens in biological samples and/or environmental samples infected with SARS-CoV-2, which can also be used to identify the existence of novel coronaviruses.

The applicant declares that the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should Changes or substitutions that can be easily conceived within the technical scope all fall within the protection scope and disclosure scope of the present invention.

Claims (13)

1. An antibody against the nucleocapsid protein of the novel coronavirus or an antigen-binding fragment thereof, wherein the antibody is selected from any one of mAb6 antibody, mAb7 antibody, mAb8 antibody and mAb9 antibody;

   Wherein, the mAb6 antibody is secreted by the hybridoma cell line #mAb6 with the deposit number CCTCC NO: C2020236, the mAb7 antibody is secreted by the hybridoma cell line #mAb7 with the deposit number CCTCC NO: C2020237, and the mAb8 antibody is secreted by the deposit The hybridoma cell line #mAb8 with the number CCTCC NO: C2020238 is secreted, and the mAb9 antibody is secreted by the hybridoma cell line #mAb9 with the deposit number CCTCC NO: C2020239.
2. A hybridoma cell line that secretes the antibody or its antigen-binding fragment as claimed in claim 1, wherein the hybridoma cell line has a deposit number of CCTCC NO: C2020236, CCTCC NO: C2020237, CCTCC NO: C2020238 or any of CCTCC NO: C2020239.
3. Application of the antibody or its antigen-binding fragment as claimed in claim 1 or the hybridoma cell strain as claimed in claim 2 in the preparation of a novel coronavirus detection kit.
4. A non-disease diagnosis and treatment purpose, a method for detecting the presence of a novel coronavirus or its corresponding antigen in a sample, wherein the method comprises the following steps:

   Mixing the sample to be tested with the antibody or its antigen-binding fragment as claimed in claim 1, incubating, and detecting the presence of the novel coronavirus or its corresponding antigen.
5. The method according to claim 4, wherein the sample to be tested comprises a biological sample and/or an environmental sample.
6. The method of claim 4, wherein the method comprises a sandwich method or a competition method;

   The sandwich method comprises the following steps:

   (1) binding the antibody or antigen-binding fragment thereof as claimed in claim 1 to the solid-phase carrier as the first antibody;

   (2) mixing the solid-phase carrier obtained in step (1) with the sample to be tested, incubating, and comparing with the control sample;

   (3) after the incubation is completed, wash the solid phase carrier, add a second antibody labeled with a marker, and incubate again, the second antibody binds to the novel coronavirus or its corresponding antigen;

   (4) washing the solid phase carrier again, and detecting the signal value of the marker bound to the second antibody;

   (5) comparing the measured signal value with the signal value of the control sample to determine the presence of the novel coronavirus in the sample to be tested and its relative amount;

   The competition law includes the following steps:

   (1') binding a predetermined amount of the antibody or antigen-binding fragment thereof as claimed in claim 1 to a solid-phase carrier;

   (2') adding the sample to be tested and a predetermined amount of the marker-labeled novel coronavirus or its corresponding antigen to the solid-phase carrier obtained in step (1'), incubating, and comparing with the control sample;

   (3') washing the solid phase carrier after the reaction, and detecting the signal value of the marker bound to the novel coronavirus or its corresponding antigen;

   (4') Compare the measured signal value with the signal value of the control sample to determine the presence and relative amount of the novel coronavirus in the sample to be tested.
7. The method according to claim 6, wherein the sandwich method further comprises:

   In step (3), the second antibody is first labeled with biotin, and then combined with enzyme-labeled avidin or streptavidin;

   Then, in step (4), the substrate of the enzyme is added to develop color.
8. A novel coronavirus detection kit, characterized in that the novel coronavirus detection kit comprises the antibody or its antigen-binding fragment as claimed in claim 1.
9. The novel coronavirus detection kit according to claim 8, wherein the novel coronavirus detection kit further comprises a solid-phase carrier, and the antibody or its antigen-binding fragment is embedded in the solid-phase carrier;

   Alternatively, the novel coronavirus detection kit further includes a solid-phase carrier and a second antibody, the monoclonal antibody or its antigen-binding fragment is bound to the solid-phase carrier as a first antibody, and the second antibody carries a label with Combination of primary antibodies.
10. The novel coronavirus detection kit according to claim 9, wherein the first antibody and the second antibody are selected from any one of the following combinations:

    Combination I: the first antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb7, and the second antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb6;

    Combination II: the first antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb7, and the second antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb8;

    Combination III: the first antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb7, and the second antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb9.
11. The novel coronavirus detection kit according to claim 9, wherein the second antibody is a polyclonal antibody.
12. The novel coronavirus detection kit according to claim 9, wherein the label comprises: any one of radioisotopes, enzymes, enzyme substrates, phosphorescent substances, fluorescent substances, biotin or coloring substances or a combination of at least two;

    The radioisotope includes any one or a combination of at least two ^of125I , ^3H , ^{14C or32P} ;

    The enzyme comprises any one or a combination of at least two of alkaline phosphatase, horseradish peroxidase, beta-galactosidase, urease or glucose oxidase;

    The fluorescent substance includes any one or a combination of at least two of fluorescein derivatives, rhodamine derivatives, rare earth elements or rare earth element complexes;

    The phosphorescent substance includes acridine ester and/or isoluminol.
13. The novel coronavirus detection kit according to claim 9, wherein the solid phase carrier comprises: nitrocellulose membrane, latex particles, magnetic particles, colloidal gold, glass, fiberglass, polymer or fiber optic sensor any one or a combination of at least two.

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