阅读说明：本技术 针对新型冠状病毒核衣壳蛋白的抗体或其抗原结合片段及其应用 (Antibodies or antigen binding fragments thereof against novel coronavirus nucleocapsid proteins and uses thereof ) 是由 李强 武翠 翁仕强 张晓峰 周宇 于 2021-03-15 设计创作，主要内容包括：本发明提供了一种针对新型冠状病毒核衣壳蛋白的抗体或其抗原结合片段及其应用,所述抗体选自mAb6抗体、mAb7抗体、mAb8抗体和mAb9抗体中的任意一种；且所述抗体由保藏号为CCTCC NO：C2020236、CCTCC NO：C2020237、CCTCC NO：C2020238或CCTCC NO：C2020239的杂交瘤细胞分泌。利用所述抗体能够检测环境样品和/或生物样品中新型冠状病毒或者其抗原的存在情况。此外,本发明还提供了利用所述抗体制备得到的新型冠状病毒检测试剂盒,能够在感染病毒早期就检测出核衣壳蛋白,为临床检测新型冠状病毒提供了快速、准确的手段。(The invention provides an antibody or an antigen-binding fragment thereof against a novel coronavirus nucleocapsid protein and application thereof, wherein the antibody is selected from any one of mAb6 antibody, mAb7 antibody, mAb8 antibody and mAb9 antibody; and the antibody is prepared from a polypeptide with a preservation number of CCTCC NO: c2020236, CCTCC NO: c2020237, CCTCC NO: c2020238 or CCTCC NO: c2020239 hybridoma cell secretion. The use of said antibodies enables the detection of the presence of a novel coronavirus, or an antigen thereof, in an environmental sample and/or a biological sample. In addition, the invention also provides a novel coronavirus detection kit prepared by using the antibody, which can detect the nucleocapsid protein at the early stage of virus infection and provides a rapid and accurate means for clinically detecting the novel coronavirus.)

1. An antibody against a novel coronavirus nucleocapsid protein 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 consists of a nucleic acid sequence with a preservation number of CCTCC NO: c2020236 hybridoma cell strain # mAb6 is secreted, and the mAb7 antibody is expressed by a nucleic acid sequence with the preservation number of CCTCC NO: c2020237 hybridoma cell strain # mAb7, wherein the mAb8 antibody is prepared from a hybridoma cell strain with a preservation number of CCTCC NO: c2020238 hybridoma cell strain # mAb8 is secreted, and the mAb9 antibody is expressed by a nucleic acid sequence with the preservation number of CCTCC NO: and C2020239 hybridoma cell strain # mAb 9.

2. A hybridoma cell strain secreting the antibody or antigen binding fragment thereof according to claim 1, wherein the hybridoma cell strain has a preservation number of CCTCC NO: c2020236, CCTCC NO: c2020237, CCTCC NO: c2020238 or CCTCC NO: any one of C2020239.

3. Use of the antibody or antigen-binding fragment thereof according to claim 1 or the hybridoma cell line according to claim 2 for the preparation of a novel coronavirus detection kit.

4. A method for detecting the presence of a novel coronavirus, or a corresponding antigen thereof, in a sample for non-disease diagnostic and therapeutic purposes, said method comprising the steps of:

mixing a sample to be tested with the antibody or antigen-binding fragment thereof of claim 1, incubating, and detecting the presence of the novel coronavirus or the corresponding antigen thereof.

5. The method of 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 an antibody or antigen-binding fragment thereof as described in claim 1 as a first antibody to a solid support;

(2) mixing the solid phase carrier obtained in the step (1) with a sample to be detected, incubating, and performing comparison by using a comparison sample;

(3) after the incubation is completed, washing the solid phase carrier, adding a second antibody labeled with a labeling substance, and incubating again, wherein the second antibody is combined with the novel coronavirus or the corresponding antigen thereof;

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

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

the competition method comprises the following steps:

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

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

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

(4') comparing the measured signal value with the signal value of a control sample to determine the presence and relative amount of the novel coronavirus in the test sample.

7. The method of claim 6, further comprising:

labeling the second antibody in the step (3) by using biotin, and then combining the labeled second antibody with enzyme-labeled avidin or streptavidin;

then, a substrate for the enzyme is added in step (4) to develop color.

8. A novel coronavirus detection kit comprising the antibody or antigen-binding fragment thereof of claim 1.

9. The novel coronavirus detection kit according to claim 8, wherein the novel coronavirus detection kit further comprises a solid support, and the antibody or the antigen-binding fragment thereof is embedded in the solid support;

or, the novel coronavirus detection kit further comprises a solid-phase carrier and a second antibody, wherein the monoclonal antibody or the antigen binding fragment thereof is bound on the solid-phase carrier as a first antibody, and the second antibody carries a marker to be combined with the first antibody.

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 an antigen binding fragment thereof secreted by a hybridoma cell strain # mAb7, and the second antibody is a monoclonal antibody or an antigen binding fragment thereof secreted by a hybridoma cell strain # mAb 6;

combination II: the first antibody is a monoclonal antibody or an antigen binding fragment thereof secreted by a hybridoma cell strain # mAb7, and the second antibody is a monoclonal antibody or an antigen binding fragment thereof secreted by a hybridoma cell strain # mAb 8;

combination III: the first antibody is a monoclonal antibody or an antigen-binding fragment thereof secreted by a hybridoma cell strain # mAb7, and the second antibody is a monoclonal antibody or an antigen-binding fragment thereof secreted by a hybridoma cell strain # mAb 9.

11. The novel coronavirus detection kit according to claim 9, wherein the second antibody is a polyclonal antibody.

12. The novel coronavirus detection kit of claim 9, wherein the marker comprises: any one or a combination of at least two of a radioisotope, an enzyme substrate, a phosphorescent substance, a fluorescent substance, biotin, or a coloring substance;

the radioactive isotope comprises¹²⁵I、³H、¹⁴C or³²Any one or a combination of at least two of P;

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

the fluorescent substance comprises any one or the combination of at least two of fluorescein derivative, rhodamine derivative, rare earth element or rare earth element compound;

the phosphorescent substance comprises acridine ester and/or isoluminol.

13. The novel coronavirus detection kit according to claim 9, wherein the solid support comprises: any one or a combination of at least two of nitrocellulose membrane, latex particles, magnetic particles, colloidal gold, glass, fiberglass, polymer, or fiber optic sensor.

Technical Field

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

Background

The novel coronavirus (SARS-CoV-2) belongs to the genus beta coronavirus, and both MERS virus known to cause respiratory syndrome in the middle east and 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), the clinical manifestations of the coronavirus pneumonia mainly comprise fever, hypodynamia and dry cough, the upper respiratory symptoms such as nasal obstruction, watery nasal discharge and the like are rare, and the coronavirus pneumonia can generate an anoxic and hypoxic state.

At present, the existing clinical novel coronavirus detection reagents comprise an enzyme-linked immunosorbent assay method for detecting serum antibodies and an RT-PCR method for detecting virus genetic materials. Since a window period of 1-3 weeks is required for antibody production after infection, the latency period of the novel coronavirus is generally 3-7 days and not more than 14 days at most based on current epidemiological investigation, so that antibody detection is not meaningful for early diagnosis. In addition, the existing antibody detection has higher false positive rate, and the specificity cannot meet the requirement.

Nucleic acid detection has higher specificity and sensitivity, but the detection method has high technical requirements, false negative easily occurs, samples need special treatment, professional instruments and equipment such as a PCR amplification instrument and gel electrophoresis are required, the detection time of the novel coronavirus needs to be long, and professional technicians need to operate and judge detection results, so that the method cannot be applied to early primary screening of communities, primary hospitals, airports, customs, even families and other primary screening. Therefore, antigen diagnostic methods against the novel coronaviruses, especially rapid, sensitive, specific antigen diagnostic methods, have no substitutable significance in the COVID-19 diagnosis.

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

Therefore, there is an urgent need to develop a product that can determine the presence of a novel coronavirus antigen in a sample at an early stage of infection, thereby satisfying clinical needs.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide an antibody or an antigen binding fragment thereof aiming at a novel coronavirus nucleocapsid protein and application thereof. Immunizing a mouse by using the novel coronavirus nucleocapsid protein as an antigen to obtain a mouse with the highest immune serum titer, and obtaining and preserving hybridoma cells; the antibody or the antigen binding fragment thereof secreted by the hybridoma cell can be specifically bound with the nucleocapsid protein, and then the antibody or the antigen binding fragment thereof can be prepared into a kit for detecting the novel coronavirus.

In order to achieve the purpose, the invention adopts the following technical scheme:

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

wherein, the mAb6 antibody consists of a nucleic acid sequence with a preservation number of CCTCC NO: c2020236 hybridoma cell strain # mAb6 is secreted, and the mAb7 antibody is expressed by a nucleic acid sequence with the preservation number of CCTCC NO: c2020237 hybridoma cell strain # mAb7, wherein the mAb8 antibody is prepared from a hybridoma cell strain with a preservation number of CCTCC NO: c2020238 hybridoma cell strain # mAb8 is secreted, and the mAb9 antibody is expressed by a nucleic acid sequence with the preservation number of CCTCC NO: and C2020239 hybridoma cell strain # mAb 9.

The invention discovers that the specific antibody aiming at the N protein of the SARS-CoV-2 virus can be used for determining the existence condition or the content of the SARS-CoV-2 virus or the corresponding antigen thereof in a sample, and can achieve high sensitivity and stability.

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 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. Similarly, 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. Also natural mutants of the amino acid sequence of SEQ ID NO. 1.

Further, papain or trypsin is used to obtain an antibody fragment (referred to as "antigen-binding fragment" in the present invention) having binding properties to a corresponding antigen, such as an Fab fragment or an F (ab')2 fragment, and the antigen-binding fragment of the present invention can 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 strain secreting the antibody or the antigen binding fragment thereof according to the first aspect, wherein the hybridoma cell strain has a preservation number of CCTCC NO: c2020236, CCTCC NO: c2020237, CCTCC NO: c2020238 or CCTCC NO: any one of C2020239.

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

the preservation number is CCTCC NO: the hybridoma cell strain of C2020236 has been registered and preserved in China center for type culture Collection (address: Wuhan university in Wuhan city) at 26.11.2020, and is classified and named as: hybridoma cell line # mAb 6.

The preservation number is CCTCC NO: the hybridoma cell strain of C2020237 has been registered and preserved in China center for type culture Collection (address: Wuhan university in Wuhan city) at 26.11.2020, and is classified and named as: hybridoma cell line # mAb 7.

The preservation number is CCTCC NO: the hybridoma cell strain of C2020238 has been registered and preserved in China center for type culture Collection (address: Wuhan university in Wuhan city) at 26.11.2020, and is classified and named as: hybridoma cell line # mAb 8.

The preservation number is CCTCC NO: the hybridoma cell strain of C2020239 has been registered and preserved in China center for type culture Collection (address: Wuhan university in Wuhan city) at 26.11.2020, and is classified and named as: hybridoma cell line # mAb 9.

The hybridomas of the present invention can be produced using cell fusion techniques known in the art: immunization of an animal with an immunogen an anti-nucleocapsid protein antibody-producing cell and a tumor cell obtained from the animal are subjected to cell fusion by a conventional method, and the antibody is produced by a hybridoma thus obtained. The animal immunized to produce the hybridoma of the present invention is not particularly limited, and includes, but is not limited to, goats, sheep, guinea pigs, mice, rats, and rabbits. Among them, preferred is a mouse.

The hybridoma can be obtained, for example, by the following method: the nucleocapsid protein of the novel coronavirus is administered to an animal such as a mouse by subcutaneous multi-point injection several times at intervals of 2 to 3 weeks together with Freund's complete adjuvant, thereby immunizing. Then, antibody-producing cells derived from spleen or the like obtained from an immunized animal and tumor cells that can be cultured in vitro, such as myeloma cells that are immortalized cells selected from myeloma cell lines or the like, are fused. The fusion method can be performed, for example, by the conventional method of Khler and Milstein (G. Kohler and C. Milstein, Nature, 1975, 256: 495-.

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 may be a method which is conventional or known in the art, for example: can be obtained from a tissue culture medium for culturing the hybridoma of the present invention, or can be obtained by inoculating the hybridoma into a mouse and proliferating the hybridoma or separating the hybridoma from collected ascites or serum. The cultivation of the hybridoma of the present invention may be performed according to a method conventional or known in the art. The method of inoculation, collection of ascites or serum, and isolation of the monoclonal antibody of the present invention from ascites or serum may be a method which is conventional or known in the art.

In a third aspect, the invention provides an application of the antibody or the antigen-binding fragment thereof according to the first aspect or the hybridoma cell strain according to the second aspect in preparing a novel coronavirus detection kit.

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

In a fourth aspect, the present invention also provides a method for detecting the presence of a novel coronavirus, or a corresponding antigen thereof, in a sample for non-disease diagnostic and therapeutic purposes, said method comprising the steps of:

mixing a sample to be tested with the antibody or antigen-binding fragment thereof according to the first aspect, incubating, and detecting the presence of the novel coronavirus or the corresponding antigen thereof.

The invention provides an immunoassay method for detecting or determining the existence of SARS-CoV-2 virus or an antigen thereof in a biological sample and/or an environmental sample or for quantification by using the above antibody; the method comprises incubating a biological sample and/or an environmental sample to be tested with the anti-SARS-CoV-2 virus N protein monoclonal antibody or antigen binding fragment thereof of the present invention to form an antigen-antibody complex, and performing qualitative detection and quantitative determination on the formed binding complex, wherein the presence or amount of the complex indicates the presence or amount of SARS-CoV-2 virus.

The immunoassay method is known per se, and any known immunoassay method can be used, and need not be described in detail in this specification. That is, there are a sandwich method, a competition method, an aggregation method, a western blot method and the like if classification is performed in a measurement format, and there are a fluorescence method, an enzymatic method, a radiation method, a biotin method and the like if classification is performed with a label used, and these methods can be used. Diagnosis can also be made by immunohistological staining. When a labeled antibody is used in the immunoassay method, the method of labeling the antibody is known per se, and any known method can be used.

The immunoassay method of the present invention is carried out using at least one monoclonal antibody or an antigen-binding fragment thereof secreted from the hybridoma cell line. Immunoassays for detecting antigens are well known in the art. The monoclonal antibody or antigen-binding fragment thereof according to the present invention can be used in the above immunoassay method independently of the label used (e.g., enzyme, fluorescence, etc.) and independently of the detection mode (e.g., fluorescence immunoassay, enzyme-linked immunosorbent assay, chemiluminescence assay, etc.) or assay principle (e.g., sandwich method, competition method, etc.).

The above immunoassay methods, including enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, western blot, immunochromatography, latex agglutination assay, etc.; furthermore, the above-mentioned immunoassays can be used for measuring a target antigen in a biological sample and/or an environmental sample by a competitive method or a sandwich method using an antigen or an antibody labeled with a labeling substance.

Among the above-mentioned various immunoassays, enzyme immunoassay, fluorescence immunoassay and chemiluminescence immunoassay are preferable. The competitive method is based on the quantitative competitive binding reaction of SARS-CoV-2 virus in the detected specimen and labeled SARS-CoV-2 virus N protein and the monoclonal antibody or antigen binding fragment thereof; that is, the present invention includes qualitative or quantitative determination of the presence or amount of SARS-CoV-2 virus in a biological component by measuring color, fluorescence, time-resolved fluorescence, chemiluminescence, electrochemical fluorescence, or radioactivity. The sandwich method is a method in which an antibody or an antigen-binding fragment of the present invention is immobilized as a first antibody to a solid phase, reacted with a biological sample and/or an environmental sample to be measured, rinsed, reacted with a second antibody, and then rinsed, followed by measurement of the second antibody bound to the solid phase. The second antibody can be labeled with an enzyme, a fluorescent substance, a radioactive substance, biotin, or the like, and the second antibody bound to the solid phase can be measured. In the agglutination method, the antibody or antigen-binding fragment thereof of the present invention is immobilized on particles such as latex, and reacted with a sample to measure the absorbance. By measuring a plurality of standards of known concentrations by the above method, preparing a standard curve based on the relationship between the amount of the measured marker and the content of the standard, and comparing the measurement result of the test sample of unknown concentration with the standard curve, the SARS-CoV-2 virus antigen in the test sample can be quantified.

When SARS-CoV-2 immunoassay is carried out by the above competition method and sandwich method, the solid phase needs to be sufficiently washed to measure the activity of binding to the label.

When the label is a radioisotope, the measurement is performed using a well counter or a liquid scintillation counter. For example, when the label is an enzyme, a substrate is added and the enzyme activity is measured colorimetrically or by fluorescence after development. When the labeling substance is a fluorescent substance, a phosphorescent substance, or a coloring substance, the measurement can be performed by a method known in the art, respectively.

By measuring a plurality of standards of known concentrations by the above method, preparing a standard curve based on the relationship between the amount of the measured marker and the content of the standard, and comparing the measurement result of the test sample of unknown concentration with the standard curve, the SARS-CoV-2 virus antigen in the test sample can be quantified.

As a preferred embodiment of the present invention, the sample to be tested includes a biological sample and/or an environmental sample.

The sample to be supplied to the immunoassay method is not particularly limited as long as it contains the nucleocapsid protein of SARS-CoV-2 virus, and examples thereof include serum, plasma and whole blood derived from human and animals, and in addition thereto, body fluid extracts such as nasal swab (nasal swab), nasal aspirate and pharyngeal swab (pharyngeal swab), airway secretions, and cell and tissue homogenates.

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

As a preferred embodiment of the present invention, the method includes a sandwich method or a competition method. In brief, the sandwich method is a method in which an antibody or an antigen-binding fragment of the present invention is immobilized as a first antibody to a solid phase, reacted with a sample to be tested, washed, then reacted with a second antibody, and washed, and then the second antibody bound to the solid phase is measured. The second antibody can be labeled with an enzyme, a fluorescent substance, a radioactive substance, biotin, or the like, and the second antibody bound to the solid phase can be measured.

Wherein, the sandwich method comprises the following steps:

(1) binding an antibody or antigen-binding fragment thereof according to the first aspect as a first antibody to a solid support;

(2) mixing the solid phase carrier obtained in the step (1) with a sample to be detected, incubating, and performing comparison by using a comparison sample;

(3) after the incubation is completed, washing the solid phase carrier, adding a second antibody labeled with a labeling substance, and incubating again, wherein the second antibody is combined with the novel coronavirus or the corresponding antigen thereof;

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

(5) comparing the measured signal value with the signal value of a control sample to determine the presence and relative amount of the novel coronavirus in the test sample.

Preferably, the sandwich method further comprises: labeling the second antibody in the step (3) by using biotin, and then combining the labeled second antibody with enzyme-labeled avidin or streptavidin;

then, a substrate for the enzyme is added in step (4) to develop color.

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

after the reaction, fully washing the solid phase, adding a second antibody which is marked by a proper amount of a marker and can be combined with N protein of SARS-CoV-2 virus, and incubating again; after the reaction, washing the solid phase sufficiently and detecting a signal value of the label bound to the second antibody by an appropriate method; the measured signal value is compared to a signal value of a control sample of a predetermined amount measured in parallel to determine the presence and relative amount of SARS-CoV-2 virus in the sample.

Further, the competition method includes the steps of:

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

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

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

(4') comparing the measured signal value with the signal value of a control sample to determine the presence and relative amount of the novel coronavirus in the test sample.

The labeled virus monoclonal antibody can be prepared by binding a monoclonal antibody against SARS-CoV-2 virus to a label. The label may be an enzyme, a colloidal metal particle, a colored latex particle, a fluorescent latex particle, a luminescent substance, a fluorescent substance, or the like. The enzyme may be various enzymes used in Enzyme Immunoassay (EIA), such as alkaline phosphatase, peroxidase, beta-D-galactosidase, etc.; as the colloidal metal particles, for example, colloidal gold particles, colloidal selenium particles, and the like can be used.

Among them, the method of binding the marker to the monoclonal antibody against SARS-CoV-2 virus can be a known method of generating a covalent bond or a non-covalent bond. Examples of the bonding method include: glutaraldehyde method, periodic acid method, maleimide method, pyridyl disulfide method, and method using various crosslinking agents (for example, refer to "protein nucleic acid enzyme", 1985, appendix 31, pages 37-45).

Among these, in the binding method using a crosslinking agent, for example, N-succinimidyl-4-maleimidobutyric acid (GMBS), N-succinimidyl-6-maleimidocaproic acid, N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid, and the like can be used as the crosslinking agent. In the method of covalent bonding, depending on the use of a functional group present in the antibody, a labeled monoclonal antibody against SARS-CoV-2 virus can be produced by introducing a functional group such as thiol, amino, carboxyl, hydroxyl or the like into the antibody by a conventional method, and then binding the functional group to the label by the above-mentioned binding method. The non-covalent bonding method may be a physical adsorption method.

As the substrate, various chromogenic substrates, fluorescent substrates, luminescent substrates, etc., which correspond to the enzyme of the label and are represented as follows, can be used. For example:

(a) chromogenic substrate: 2, 2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS), 3', 5,5 ' -Tetramethylbenzidine (TMB), Diaminobenzidine (DAB) in combination with hydrogen peroxide for peroxidases; 5-bromo-4-chloro-3-indolyl phosphate (BCIP), p-nitrophenyl phosphate (p-NPP), sodium 5-bromo-4-chloro-3-indolyl phosphate (BCIP. Na) are used for alkaline phosphatase.

(b) Fluorescent substrate: 4-methylumbelliferyl phosphate (4-MUP) for alkaline phosphatase; 4-Methylumbelliferyl phenyl-beta-D-galactoside (4MUG) was used for beta-D-galactosidase.

(c) Luminescent substrate: 3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxetane.2sodium salt (AMPPD) for alkaline phosphatase; 3- (2 '-spiroadamantane) -4-methoxy-4- (3' - β -D-galactopyranosyl) phenyl-1, 2-dioxetane (AMGPD) for β -D-galactosidase; luminol, isoluminol obtained in combination with hydrogen peroxide is used in peroxidases.

In a preferred embodiment of the present invention, a double antibody sandwich ELISA detection kit is provided, and a method for detecting whether SARS-CoV-2 virus exists in a sample by using the kit is provided, which comprises:

(1) providing a monoclonal antibody or an antigen-binding fragment thereof (primary antibody or solid phase antibody) that binds to the N protein of SARS-CoV-2 virus and coating a solid phase carrier with said primary antibody;

(2) adding a sample to be detected and a control sample (or standard substance) to the coated solid phase carrier in the step (1) and incubating under proper conditions;

(3) washing thoroughly after the reaction to remove any unbound sample, and adding an appropriate amount of biotin-labeled monoclonal antibody or antigen-binding fragment thereof (secondary antibody or labeled antibody) that binds to another epitope of the N protein of SARS-CoV-2 virus and incubating again;

(4) fully washing after reaction to remove any unbound second antibody, binding the biotinylated second antibody with enzyme-labeled avidin or streptavidin, then adding an enzyme substrate for color development, and then measuring a corresponding light absorption value by using an enzyme-labeling instrument;

(5) the measured absorbance is compared to the absorbance of a known quantity of 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, and further, the sample is blood plasma, blood serum and whole blood; the solid phase carrier is a microtiter plate; the avidin or streptavidin is marked by horseradish peroxidase; the enzyme substrate is TMB.

In a fifth aspect, the present invention also provides a novel coronavirus detection kit comprising the antibody or antigen-binding fragment thereof according to the first aspect.

By assaying various biological samples and/or environmental samples derived from a human or an animal using the monoclonal antibody against the nucleoprotein of SARS-CoV-2 virus of the present invention, diagnosis of SARS-CoV-2 virus infection can be carried out. The nucleocapsid protein of SARS-CoV-2 virus in various body fluids, cells, tissues and the like from humans or animals and/or environmental samples can be directly determined by immunochemical or immunohistochemical methods using the monoclonal antibody of the present invention.

As a preferred technical scheme, the novel coronavirus detection kit further comprises a solid phase carrier, and the antibody or the antigen-binding fragment thereof is embedded in the solid phase carrier.

The kit is a kit for detection by a competitive method, and the immunoassay reagent may be prepared as a predetermined amount of virus antigen labeled with, for example, an enzyme, colloidal metal particles, colored latex particles, a luminescent substance, a fluorescent substance, a radioactive substance, or the like. The reagent can be used to conduct an immunoassay by, for example, subjecting a sample containing a certain amount of the monoclonal antibody of the present invention, the labeled viral antigen described above and the antigen to be assayed to a competitive reaction, and quantifying the amount of the labeled viral antigen bound or unbound to the antibody relative to the amount of the antigen in the sample to be assayed.

Or, the novel coronavirus detection kit further comprises a solid-phase carrier and a second antibody, wherein the monoclonal antibody or the antigen binding fragment thereof is bound on the solid-phase carrier as a first antibody, and the second antibody carries a marker to be combined with the first antibody.

The detection kit is a kit for detection by the sandwich method, and in the immunoassay reagent by the sandwich method, one monoclonal antibody may be used as the solid phase antibody and the labeled antibody (for example, when the antigen is a polymer), but it is usually preferable to use 2 or more antibodies that can recognize two different epitopes of the antigen to be measured, respectively. Further, any of the solid-phase antibody and the labeled antibody may be used in combination selected from 2 or more kinds of monoclonal antibodies.

In the present invention, the following reagents may be used: for example, two kinds of the monoclonal antibodies of the present invention are prepared, one of which is the labeled antibody described above, and the other of which 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 (second antibody) is reacted with the antigen captured by the solid-phase antibody, whereby the presence or activity of the label bound to the insoluble carrier is detected, whereby immunoassay can be performed. Similarly, immunoassay can be performed by reacting a sample containing an antigen to be measured with a solid-phase antibody, subsequently reacting a labeled antibody (second antibody) with the antigen captured on the solid-phase antibody, and determining the presence or activity of the label bound to the insoluble carrier, that is, quantifying the amount of the antigen to be measured by the amount of the labeled antibody.

Preferably, the second antibody is selected from any one of the antibodies of the present invention that is capable of being conjugated to the first antibody, i.e., a monoclonal antibody or antigen-binding fragment thereof that is capable of being used in pair. In the context of the present invention, an antibody that is "capable of being matched" and "capable of being used in a pair" refers to a combination of antibodies that are directed against different epitopes in the N protein antigen of a novel coronavirus and that do not interfere with or antagonize each other in binding to the N protein antigen.

In addition, the second antibody may be other polyclonal antibodies.

In a preferred embodiment 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 an antigen binding fragment thereof secreted by a hybridoma cell strain # mAb7, and the second antibody is a monoclonal antibody or an antigen binding fragment thereof secreted by a hybridoma cell strain # mAb 6;

combination II: the first antibody is a monoclonal antibody or an antigen binding fragment thereof secreted by a hybridoma cell strain # mAb7, and the second antibody is a monoclonal antibody or an antigen binding fragment thereof secreted by a hybridoma cell strain # mAb 8;

combination III: the first antibody is a monoclonal antibody or an antigen-binding fragment thereof secreted by a hybridoma cell strain # mAb7, and the second antibody is a monoclonal antibody or an antigen-binding fragment thereof secreted by a hybridoma cell strain # mAb 9.

As a preferred embodiment of the present invention, the marker includes: any one or a combination of at least two of a radioisotope, an enzyme substrate, a phosphorescent substance, a fluorescent substance, biotin, or a coloring substance.

Preferably, the radioisotope comprises¹²⁵I、³H、¹⁴C or³²Any one or a combination of at least two of P.

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

Preferably, the fluorescent substance includes any one of a fluorescein derivative, a rhodamine derivative, a rare earth element or a rare earth element complex or a combination of at least two thereof.

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

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

By using the monoclonal antibody of the present invention, a reagent for measuring SARS-CoV-2 virus immunity can be produced by using the antibody as at least one of a solid-phase antibody and a labeled antibody.

The solid phase to which the monoclonal antibody is bound can be any of various solid phases used in conventional immunoassays, and examples thereof include: ELISA plate, latex, gelatin particle, magnetic particle, polystyrene, glass and other various solid phase beads, liquid-transmissible matrix and other insoluble carrier. The labeled antibody can be prepared by labeling an antibody with an enzyme, colloidal metal particles, colored latex particles, a luminescent substance, a fluorescent substance, a radioactive substance, or the like. By combining these reagents such as the solid-phase antibody and/or the labeled antibody, a reagent used in enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, or the like can be prepared. These assay reagents are reagents for assaying a target antigen in a sample by a sandwich method or a competitive binding assay.

In addition, another aspect of the present invention provides the use of the above immunoassay reagent for the diagnosis of a disease caused by SARS-CoV-2 virus infection.

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

Compared with the prior art, the invention has the beneficial effects that:

(1) the monoclonal antibody or the antigen binding fragment thereof is prepared from four hybridoma cell strains CCTCC NO: c2020236, CCTCC NO: c2020237, CCTCC NO: c2020238 or CCTCC NO: c2020239, the monoclonal antibody or the antigen binding fragment thereof can specifically recognize the N protein of SARS-CoV-2 virus, the affinity is up to pM grade, and the monoclonal antibody has very high sensitivity and specificity;

(2) the invention also provides a SARS-CoV-2 virus detection kit, the detection kit can utilize various immunoassay methods to detect the existence condition of SARS-CoV-2 virus, especially utilizes a sandwich method and a competition method to detect the SARS-CoV-2 virus detection kit, because the monoclonal antibody or the antigen binding fragment thereof used by the detection kit has high affinity and specificity, the obtained detection result is more accurate; the invention also provides a double-antibody sandwich ELISA detection kit which is constructed by optimally paired three groups of antibodies (mAb 7/Bio-mAb6, mAb7/Bio-mAb8 and mAb7/Bio-mAb 9), the detection limit of the kit is 12.5 pg/mL, and the kit has good repeatability;

(3) the double-antibody sandwich ELISA immunoassay method constructed in the invention is a very sensitive virus detection technology, has higher specificity than other serological methods when detecting most viruses, and has high ELISA sensitivity and simple and convenient operation, and the development of matched instruments and equipment ensures that the operation procedure is normalized and automated, thereby further improving the stability. Therefore, the novel coronavirus antigen detection kit provided by the invention can detect the nucleocapsid protein in the early stage of virus infection of a patient, solves the problem of rapid clinical diagnosis of a patient infected with the novel coronavirus, has high accuracy, and provides a rapid and accurate diagnosis method for clinical detection of the disease.

Drawings

FIG. 1 is a graph showing the results of measurement of the titer of the mouse immune sera in example 1.

FIG. 2 is a graph showing the results of measurement of the cross-reactivity of the antibody against the novel coronavirus nucleocapsid protein and SARS virus in example 3.

FIG. 3 is a graph showing the results of the cross-reactivity assay of the antibody against the novel coronavirus nucleocapsid protein and MERS virus in example 3.

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

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

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

Detailed Description

The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature and may be found, for example, in: 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; metals IN ENZYMOLOGY, Academic Press, San Diego, Wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, Academic Press, San Diego, 1998; related documents including 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, etc.

Example 1 preparation of hybridoma cell lines and monoclonal antibodies

The embodiment is used for obtaining hybridoma cell strains and preparing monoclonal antibodies, and comprises the following specific steps:

1.1 animal immunization

Fully emulsifying SARS-CoV-2N protein antigen (Sino Biological, 40588-V08B) with Freund's adjuvant, and immunizing male Balb/C mouse (Shanghai Leike laboratory animals Co., Ltd.) with multi-point immunization at a dose of 50 μ g/mouse, wherein the immunization period is once in three weeks;

on day 10 after the 3 rd immunization, blood was taken through the eye sockets and the degree of immune response of the mice was monitored by testing the serum antibody titer according to the indirect ELISA method.

1.2 immune serum titer determination

Establishing an indirect ELISA method for measuring the immune serum titer: coating a polystyrene micro 96-well plate with 0.1 mu g/mL recombinant SARS-CoV-2N protein, 100 mu L/well, and staying overnight at 4 ℃; the next day, adding blocking solution with mass fraction of 1% bovine serum albumin (BSA, Sigma), 0.3 mg/hole, standing overnight at 4 deg.C, treating the coated lath with 10% sucrose-containing 10 mM PBS buffer solution, vacuum drying, vacuum packaging with aluminum film bag, and storing at 4 deg.C for measuring titer of mouse immune serum;

collecting blood in orbit 10 days after 3 rd immunization, diluting the collected mouse serum with 10 mM PBS containing 1.5% BSA by 1:100 times to obtain a series of concentration gradients, adding the diluted serum into a 96-well plate, 100 uL/well, and washing the plate at 37 ℃ for 30 minutes by using 10 mM PBS containing 0.1% Tween-20 washing solution for 3 times, and adding horseradish peroxidase (HRP) labeled goat anti-mouse IgG (Jackson Laboratory Inc, cat # 115-035-07), 100 uL/well, and 37 ℃ for 30 minutes by 1:5000 times;

after washing the plate, 100. mu.L/well of a buffer containing 0.05% (w/v) TMB and 0.06% (w/v) dioxygen pH5.0 citric acid was added, and the plate was protected from light at room temperature for 10 minutes, and 0.2M H was added₂SO₄Stop the reaction, 100. mu.L/well, microplate reader at dual wavelength 450/6Reading an absorption value at 20 nm, taking the serum of the mouse before immunization as a negative control, and taking the ratio of the measured value to the control value to be more than or equal to 2.0 as positive to judge the titer of the immune serum;

four mice all produced stronger specific immune responses after the third immunization, the reaction results are shown in figure 1, and the titers of immune sera of the mice M51, M52, M53 and M54 are all higher.

1.3 hybridoma preparation

3 days before fusion, the mice producing the highest antibody titer were boosted once; 3 days later, the mice were sacrificed, the spleens of the mice were aseptically taken out to prepare a splenocyte suspension, which was then mixed with 2X 10 cells at a ratio of 1:1⁸Several mouse myeloma Sp2/0 cell lines at logarithmic growth phase were mixed and then fused in a solution containing 50% polyethylene glycol (molecular weight 1450) and 5% dimethyl sulfoxide (DMSO);

iscove's medium (containing 10% fetal calf serum, 100U/mL penicillin, 100. mu.g/mL streptomycin, 0.1 mM hypoxanthine, 0.4. mu.M aminopterin and 16. mu.M thymidine) was used to adjust the number of spleen cells to 5X 10⁵ 0.3 mL/mL, added to wells of a 96-well plate and placed at 37 ℃ in 5% CO₂Culturing in an incubator;

after 1 day, 100. mu.L of selection medium containing hypoxanthine, aminopterin-thymidine (HAT, Sigma) was added to each well, and the cultures were then replated with this selection medium every 3 days until clonal cells were formed.

1.4 screening of hybridoma cells secreting novel coronavirus N protein antibody

For screening positive clones producing antibodies, the cell culture supernatants were assayed by indirect ELISA as follows:

adding the hybridoma cell culture supernatant into an ELISA plate coated with an antigen in advance, wherein each hole is 100 mu L, taking SP2/0 cell culture supernatant as a negative control, taking immune multi-antiserum as a positive control, and keeping the temperature at room temperature for one hour; PBST wash 3 times; adding HRP labeled goat anti-mouse IgG antibody (Jackson Laboratory Inc, cat # 115-035-07) at a working concentration of 100. mu.L/well at 37 ℃ for 30 minutes, washing the plate, adding TMB substrate at 100. mu.L/well, shielding from light at room temperature for 10 minutes, adding 0 to 100. mu.L/well.2M H₂SO₄The reaction was stopped and the microplate reader read the absorbance at two wavelengths of 450/620 nm. The measured hole OD 450/620 nm reading is more than twice of the negative control, and the result is judged to be positive;

1.5 cloning of Positive hybridoma cells

Carrying out subcloning on the screened strong positive cell clones for 2-3 times by adopting a limiting dilution method to obtain 6 strains of hybridoma cell strains which stably secrete antibodies, wherein the 6 strains are respectively named as # mAb6, # mAb7, # mAb8, # mAb9, # mAb10 and # mAb11, and biological preservation is carried out on 4 strains of the hybridoma cell strains, and the # mAb6, # mAb7, # mAb8 and # mAb9 respectively correspond to the strains with the preservation numbers of CCTCC NO: c2020236, CCTCC NO: c2020237, CCTCC NO: c2020238 and CCTCC NO: a hybridoma cell line of C2020239.

1.6 purification of antibodies

The positive hybridoma 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 reaches about 5X 10⁵At individual cells/mL, the medium was replaced with serum-free medium. After the culture, centrifugation was performed and the culture supernatant was collected, and then the antibody was purified using a Protein G affinity chromatography column, the monoclonal antibody eluate was dialyzed against 150 mM NaCl, and the dialyzed solution was filter-sterilized through a 0.2 μm filter to obtain a purified antibody sample.

1.7 antibody labeling

Labeling with biotin: N-N-hydroxysuccinimide ester (NHS) is the most common biotin labeling reagent, and NHS-activated biotin is capable of reacting with a primary amine group (-NH) in alkaline buffer₂) Reacting to form stable amido bond. Proteins (e.g., antibodies) typically have many primary amine groups and thus can serve as targets for biotin labeling;

this example was labeled with Biotin derivative NHS-LC-Biotin (Thermo Scientific, cat # 21435), and the monoclonal antibodies were biotinylated as described in the Thermo Scientific EZ-Link Biotin labeling kit protocol to obtain Biotin-labeled antibodies Bio-mAb6, Bio-mAb7, Bio-mAb8, Bio-mAb9, Bio-mAb10 and Bio-mAb 11.

The monoclonal antibodies of the invention may also be labeled using other methods known in the art, such as with HRP (horseradish peroxidase), 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 ℃ against 0.01M carbonate buffer solution of pH 9.6. Dissolving HRP 4 mg in 1mL of pure water, and slowly adding 0.1M NaIO₄0.1mL, stirred at room temperature for 20 minutes in the dark, put into a dialysis bag, dialyzed overnight at 4 ℃ with 0.001M acetate buffer solution with pH 4.4;

to HRP was added 0.2M Na₂CO₃0.05mL of NaBH was added to adjust the pH to 9.6, the mixture was mixed with antibody, stirred for 2 hours in the dark, and 4 mg/mL of NaBH was added₄0.1mL, left to stand at 4 ℃ for 2 hours, and dialyzed with PBS at 4 ℃ overnight.

Adding saturated ammonium sulfate with the same volume, stirring for 30 minutes, standing at 4 ℃ for 2 hours, centrifuging at 3000rpm for 20 minutes, and discarding the supernatant. The resulting precipitate was then repeated with 50% saturated ammonium sulfate. The resulting precipitate was dissolved in 1mL PBS and dialyzed against PBS at 4 ℃ for 48 hours to obtain enzyme-labeled monoclonal antibodies HRP-mAb6, HRP-mAb7, HRP-mAb8, HRP-mAb9, HRP-mAb10 and HRP-mAb 11.

Example 2 double antibody sandwich ELISA screening for best-paired monoclonal antibodies

Monoclonal hybridoma cells produce antibodies directed against only one epitope of the antigen, and the best paired solid phase antibody and labeled antibody are screened by the double antibody sandwich method.

The 6X 6 matrix is adopted for antibody pairwise pairing screening, the 6 monoclonal antibodies are respectively coated to be used as capture antibodies, and the 6 monoclonal antibodies are respectively paired with 6 biotin-labeled monoclonal antibodies Bio-mAb6, Bio-mAb7, Bio-mAb8, Bio-mAb9, Bio-mAb10 and Bio-mAb11, so as to rapidly screen the capture and labeled monoclonal antibody pairs in the sandwich ELISA.

By comparing the sensitivity of the assay, it was shown that monoclonal antibody mAb7 or mAb11 as capture antibody produced stronger signals when paired with Bio-mAb6, Bio-mAb8, Bio-mAb9 and Bio-mAb10, respectively.

Example 3 functional characterization of anti-N protein monoclonal antibodies

3.1 monoclonal antibody affinity constant determination

The binding affinity constants of the purified murine monoclonal antibodies mAb6, mAb7, mAb8, mAb9 and the novel coronavirus N protein were determined using biofilm interference technique (BLI). The assay was performed using the ForteBio Octet RED & QK platform from PALL, methods according to the instructions for the platform.

Firstly, biotinylated SARS-CoV-2N protein is fixed on the surface of SA sensor, and the monoclonal antibody for resisting SARS-CoV-2N protein is used as analyte. The data was processed and analyzed with analysis software 1:1 fitting the combined model, and basically overlapping fitting data with experimental data to obtain a combination and dissociation rate constantK _aAndK _d by usingK _d Removing deviceK _aObtaining the equilibrium dissociation constant K _D (see Table 1).

The results showed that the affinity of murine mAb7 was highest, whereas the affinity of mAb6, mAb8 and mAb9 was comparable, K _D All values were below the pM level.

TABLE 1

Name of antibody	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

The cross-reactivity of the above anti-SARS-CoV-2 monoclonal antibodies mAb6, mAb7, mAb8, mAb9, mAb10 and mAb11 to the nucleocapsid protein of human SARS and MERS coronavirus was also examined in this example.

SARS virus N protein (Beijing Yi Qiao Shen Biotechnology Co., Ltd., cat # 40143-V08B) or MERS virus N protein (Beijing Yi Qiao Shen Biotechnology Co., Ltd., cat # 40068-V08B) was diluted to 0.1. mu.g/mL with PBS buffer, added to a 96-well plate at a volume of 100. mu.L/well, and left at 4 ℃ for 20 hours. The 96-well plate was aspirated off PBS buffer, and after washing the plate 1 time with PBST (pH 7.4, PBS containing 0.05% Tween 20) buffer, 200. mu.L/well of PBST/1% skim milk was added and incubated at room temperature for 1 h to block.

After removing the blocking solution and washing the plate for 3 times by PBST buffer solution, diluting the monoclonal antibody with the initial concentration of 10 mu g/mL according to the proportion of 1:4 into gradient concentration, adding the monoclonal antibody into the microplate, incubating for 1.5 h at room temperature, wherein the concentration of the monoclonal antibody is 100 mu L/well. The reaction was removed and after 3 PBST washes, a 1:5000 dilution of a secondary HRP-labeled goat anti-mouse IgG Fc antibody (Jackson Laboratory Inc, cat # 115-035-07) was added at 50. mu.L/well and incubated at room temperature for 1 hour. After PBST washing the plate for 3 times, adding 100 mu L/hole TMB, incubating at room temperature, and adding 50 mu L/hole 0.2M sulfuric acid to terminate the reaction; the microplate reader reads the absorbance at a dual wavelength of 450/620 nm.

As shown in FIG. 2, all of the 6 monoclonal antibodies were able to specifically bind to the N protein antigen of SARS virus. In addition, none of the 6 mabs specifically bound the MERS virus N protein (see fig. 3).

EXAMPLE 4 establishment of double antibody Sandwich ELISA kit for detecting SARS-CoV-2 antigen Using monoclonal antibody against SARS-CoV-2N protein

The antibody mAb7 provided by the invention and the monoclonal antibody Bio-mAb8 labeled by biotin are taken as examples to establish a double-antibody sandwich ELISA immunoassay method for SARS-CoV-2 virus.

The washing solution is PBS-Tween20, PH7.4 (PBST); the blocking solution is PBST containing 1.5% Bovine Serum Albumin (BSA); the standard substance and the diluent of the sample to be detected are PBST containing 1.5% BSA; the biotin-labeled antibody diluent is PBST containing 1.5% BSA; Streptavidin-HRP (Thermo Scientific, Lot: 21130) was formulated into working solution using PBST with 1.5% BSA according to the protocol of the protocol.

The detection method comprises the following steps:

(1) diluting the antibody mAb7 with 1 × PBS to a final concentration of 5 μ g/mL, coating polystyrene microplate with 100 μ L per well, shaking for 1 hr at room temperature, 500 rpm, washing with washing solution, and patting dry;

(2) adding confining liquid, standing at 200 muL/hole for 1 hour at room temperature.

(3) The SARS-CoV-2N protein standard was diluted to different concentrations as a standard curve (the standard was diluted with 1.5% BSA in PBST to concentrations of 400 pg/mL, 200 pg/mL, 100 pg/mL, 50 pg/mL, 25 pg/mL, 12.5 pg/mL) and 100. mu.L per well was added to the reaction plate; diluting the sample to be detected by 100 times, adding the diluted sample into different wells, carrying out negative control by using 1.5% BSA-PBS solution, standing the sample at room temperature for 1 hour, washing the sample for 3 times by using a washing solution, and drying the sample by patting the sample.

(4) Biotin-labeled secondary antibody Bio-mAb8 (diluted with 1.5% BSA-PBS at a ratio of 1: 5000) was added to each well at 100. mu.L, and the mixture was allowed to stand at room temperature for 1 hour, washed 3 times with washing solution, and patted dry.

(5) Adding the prepared Streptavidin-HRP solution, standing for 1 hour at room temperature, wherein each well is 100 muL.

(6) Washing with the washing liquid for 3 times, drying, adding the prepared TMB at 100 muL/hole, and developing at room temperature for 20-30 minutes.

(7) Adding 0.2M H₂SO₄And stopping, 50 muL/hole.

(8) And reading an absorption value at the position of double wavelength 450/620 nm by the microplate reader, and drawing a standard curve according to the OD value of the measured sample and the concentration of the standard substance to obtain a linear regression equation. Substituting into OD value of the sample to be detected to obtain the content of SARS-CoV-2N protein in the sample.

Example 5 ELISA method and Performance of the kit

We evaluated the performance of the double antibody sandwich ELISA method and kit for determining SARS-CoV-2 virus content, which was established from the best-paired 3 groups of antibodies selected in example 2, mAb7/Bio-mAb6, mAb7/Bio-mAb8, and mAb7/Bio-mAb 9.

5.1 detection Range

As can be seen from FIGS. 4-6, in which FIG. 4 is mAb7/Bio-mAb6, FIG. 5 is mAb7/Bio-mAb8, FIG. 6 is mAb7/Bio-mAb9, as the concentration of SARS-CoV-2N protein (NP) increases, the OD value also increases, and when the concentration of NP is measured in the range of 1-400 ng/mL, the OD value measured by the established sandwich method is linearly and positively correlated with the concentration of NP (R.sub.²Not less than 0.988). The lower limit of detection of the best-matched antibodies in all three groups screened above was as low as 12.5 pg/mL.

5.2 specificity

The reaction of the three antibodies selected above with the ELISA kit composed of the antibodies and several other serum substances was measured, and the reaction specificity was observed. Coating capture antibody 4 mug/mL, human serum albumin and bovine serum albumin 100 ng/mL, SARS-CoV-2N protein negative serum sample (NP negative serum) 5 parts diluted by 1:100, NP positive control pure product 100 ng/mL, biotin labeling antibody (diluted by 1: 5000), determining result after developing, see Table 2.

TABLE 2

Sample (I)	Human albumin	Bovine albumin	NP negative serum (5 parts)	Positive control (NP pure)
					mAb7/Bio-mAb6	-	-	- - - - -	+
mAb7/Bio-mAb8	-	-	- - - - -	+
					mAb7/Bio-mAb9	-	-	- - - - -	+

Remarking: "+" indicates that the sample test result was positive, and "-" indicates that the sample test result was negative.

5.3 sensitivity

The detection of the reference/standard was performed according to the established experimental method described previously. The detection limit is 12.5 pg/mL, and the conditions are that the OD value is more than 3 times of the blank OD value SD, and the OD value is more than 2 times of the blank OD value. The method of the invention has good sensitivity.

5.4 repeatability

The detection is repeated for a sample for 4 times, the CV values of 8 results in the same plate at the same time are all less than 4%, and the CV value of the average value of 4 results operated by different people at different times is 1.63%, so that the kit for detecting the novel coronavirus N protein has better repeatability.

The double antibody sandwich enzyme-linked immunosorbent assay can be used for detecting SARS-CoV-2N protein antigen in serum or plasma samples of human or animals, and is used for early diagnosis of SARS-CoV-2 infection, so as to achieve the purposes of early detection, early isolation and prevention of spread. The antibody of the present invention is used in labeling fluorescence and then immunofluorescence to detect virus antigen in biological sample and/or environment sample infected with SARS-CoV-2, and may be also used in identifying new type coronavirus.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

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aaaacattcc caccaacaga gcctaaaaag gacaaaaaga agaaggctga tgaaactcaa 1140

gccttaccgc agagacagaa gaaacagcaa actgtgactc ttcttcctgc tgcagatttg 1200

gatgatttct ccaaacaatt gcaacaatcc atgagcagtg ctgactcaac tcaggcctaa 1260