CN113150133B - Monoclonal antibodies against SARS-CoV-2 or antigen binding fragments thereof - Google Patents

Monoclonal antibodies against SARS-CoV-2 or antigen binding fragments thereof Download PDF

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CN113150133B
CN113150133B CN202110277206.9A CN202110277206A CN113150133B CN 113150133 B CN113150133 B CN 113150133B CN 202110277206 A CN202110277206 A CN 202110277206A CN 113150133 B CN113150133 B CN 113150133B
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李强
武翠
翁仕强
张晓峰
周宇
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Anyuan Pharmaceutical Technology Shanghai Co ltd
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Abstract

The present invention provides monoclonal antibodies or antigen-binding fragments thereof against SARS-CoV-2. Using N protein of novel coronavirus SARS-CoV-2 as antigen to immunize mouse, obtaining mouse with highest immune serum titer, obtaining hybridoma cell and preserving; the monoclonal antibody or the antigen binding fragment thereof which is secreted by the obtained hybridoma cell and aims at the novel coronavirus N protein antigen can be specifically bound with the N protein of SARS-CoV-2, and further can be prepared into a kit for detecting the existence condition of SARS-CoV-2.

Description

Monoclonal antibodies or antigen-binding fragments thereof against SARS-CoV-2
Technical Field
The invention relates to the technical field of biological medicines, in particular to an antibody aiming at a novel coronavirus, and especially relates to a monoclonal antibody or an antigen binding fragment thereof aiming at the novel coronavirus.
Background
The novel coronavirus (SARS-CoV-2) was shown to be a novel member of the genus beta coronavirus based on the genomic nucleic acid sequence. Like other coronaviruses, SARS-CoV-2 infection causes an acute respiratory infectious disease, i.e., a novel coronavirus pneumonia (COVID-19). The novel coronavirus SARS-CoV-2 is a positive sense RNA virus encoding several major proteins S, M, N and E, RNA dependent RNA polymerase RDRP and ten non-structural proteins. Where the S, M, N and E proteins are used for packaging viral structures, RDRP and a dozen non-structural proteins are used for replication of viral genomic RNA and synthesis of individual protein mRNAs.
The N protein is the most abundant protein in coronaviruses. During virion assembly, the N protein binds to viral RNA and leads to 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. Due to the sequence conservation and strong immunogenicity of the N protein, the N protein is often used as a diagnostic detection tool for coronavirus.
The existing clinical SARS-CoV-2 detection reagent includes enzyme-linked immunosorbent assay for serum antibody detection and RT-PCR assay for virus genetic material detection. Since a window of 1-3 weeks is required for antibody production after infection, SARS-CoV-2 latency is generally 3-7 days, and not more than 14 days at the longest, based on current epidemiological investigations, antibody detection is not significant for early diagnosis. In addition, the current antibody detection has higher false positive rate, and the specificity can not 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 specific for SARS-CoV-2, especially rapid, sensitive, specific antigen diagnostic methods, have irreplaceable significance in COVID-19 diagnosis. At present, there is an urgent need to provide a method and corresponding product for diagnosing CODVI-19 by detecting SARS-CoV-2 antigen in a patient sample at the early stage of infection, thereby satisfying the clinical needs.
Disclosure of Invention
In view of the problems in the prior art, it is an object of the present invention to provide a monoclonal antibody or an antigen-binding fragment thereof against a novel coronavirus. In the invention, SARS-CoV-2N protein antigen is used for immunizing a mouse to obtain the mouse with the highest immune serum titer, and hybridoma cells are obtained and preserved; the monoclonal antibody or antigen binding fragment thereof secreted by the hybridoma cell can specifically bind to the N protein of SARS-CoV-2, and can be further prepared into a kit for detecting the existence condition of SARS-CoV-2.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a monoclonal antibody against SARS-CoV-2 virus, or an antigen-binding fragment thereof, which is any one of the following monoclonal antibodies:
(I) mAb1, which is prepared from a nucleic acid sequence with a preservation number of CCTCC NO: c2020231 hybridoma cell strain # mAb1 secretes; (II) mAb2, consisting of a nucleic acid sequence with a preservation number of CCTCC NO: c2020233 hybridoma cell strain # mAb2 secretion; (III) mAb4, consisting of a sequence with a preservation number of CCTCC NO: c2020234 hybridoma cell line # mAb4 secretion; (IV) mAb5, prepared from a mixture of mAb and mAb, wherein the preservation number is CCTCC NO: c2020235 hybridoma cell line # mAb5 secretion. The monoclonal antibody of the present invention can be produced by the hybridoma of the present invention, and thus can be obtained from a culture solution in which the hybridoma of the present invention is cultured.
The monoclonal antibody of the present invention can be obtained by using the nucleocapsid protein of SARS-CoV-2 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.
Therefore, the monoclonal antibody of the present invention can be obtained using a polypeptide having an amino acid sequence shown by SEQ ID NO. 1 as an immunogen. Also natural mutants of the amino acid sequence of SEQ ID NO. 1. The invention discovers that the specific antibody aiming at the SARS-CoV-2 virus N protein can be used for determining the existence condition or the content of SARS-CoV-2 virus or the corresponding antigen in a sample, and can achieve high sensitivity and stability.
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 also provides a hybridoma cell strain, wherein the hybridoma cell strain secretes the monoclonal antibody or the antigen binding fragment thereof according to the first aspect; the hybridoma cell strain comprises: CCTCC NO: c2020231, CCTCC NO: c2020233, CCTCC NO: c2020234 or CCTCC NO: any one of C2020235.
The biological material preservation information of the present invention is as follows:
1. the preservation number is CCTCC NO: the hybridoma cell strain C2020231 has been registered and deposited 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 # mAb1;
2. the preservation number is CCTCC NO: the hybridoma cell strain C2020233 has been registered and deposited 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 # mAb2.
3. The preservation number is CCTCC NO: the hybridoma cell strain of C2020234 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 # mAb4.
4. The preservation number is CCTCC NO: the hybridoma cell strain of C2020235 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 # mAb5.
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 SARS-CoV-2 virus is administered to an animal such as a mouse by subcutaneous multiple injections 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, according to the method conventionally used in Khler and Milstein (G.Kohler and C.Milstein, nature,1975, 256.
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 fluid or serum, and isolation of the monoclonal antibody of the present invention from the ascites fluid or serum may be a method which is conventional or known in the art.
In a third aspect, the invention also provides the use of the monoclonal antibody or the antigen binding fragment thereof according to the first aspect or the hybridoma cell strain according to the second aspect in the preparation of a SARS-CoV-2 virus 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 provides a SARS-CoV-2 virus detection kit, comprising the monoclonal 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 the preferable technical scheme of the invention, the SARS-CoV-2 virus detection kit also comprises a solid phase carrier, and the monoclonal 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 SARS-CoV-2 virus detection kit also comprises a solid phase carrier and a second antibody, the monoclonal antibody or the antigen binding fragment thereof is used as a first antibody to be bound on the surface of the solid phase carrier, and the second antibody carries a marker to be used together with the first antibody.
The detection kit is a kit for detection by a sandwich method, and in the immunoassay reagent by the sandwich method, one monoclonal antibody may be used as a solid phase antibody and a 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 detected, respectively. Furthermore, any of the solid-phase antibody and the labeled antibody may be selected from 2 or more monoclonal antibodies and used in combination.
In the present invention, the following reagents can 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 addition, the second antibody may be other polyclonal antibodies.
In the context of the present invention, an antibody that is "capable of binding" and "capable of pairing" refers to a combination of antibodies that are directed against different epitopes of the SARS-CoV-2N protein antigen and that do not interfere with or antagonize each other in binding to the N protein antigen.
Preferably, 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 # mAb1, and the second antibody is a monoclonal antibody or an antigen binding fragment thereof secreted by a hybridoma cell strain # mAb 4;
combination II: the first antibody is a monoclonal antibody or an antigen binding fragment thereof secreted by a hybridoma cell strain # mAb1, and the second antibody is a monoclonal antibody or an antigen binding fragment thereof secreted by a hybridoma cell strain # mAb 2;
combination III: the first antibody is a monoclonal antibody or an antigen binding fragment thereof secreted by a hybridoma cell strain # mAb5, and the second antibody is a monoclonal antibody or an antigen binding fragment thereof secreted by a hybridoma cell strain # mAb4.
As a preferred embodiment of the present invention, the marker includes: radioactive isotopologueAny one or a combination of at least two of a hormone, an enzyme substrate, a phosphorescent substance, a fluorescent substance, biotin, or a coloring substance; the radioactive isotope comprises 125 I、 3 H、 14 C or 32 Any one or a combination of at least two of P; the enzyme comprises any one of alkaline phosphatase, horseradish peroxidase, beta-galactosidase, urease or glucose oxidase or the combination of at least two of the alkaline phosphatase, the horseradish peroxidase, the beta-galactosidase and the glucose oxidase; the fluorescent substance comprises any one or 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.
As a preferred technical scheme of the invention, the solid phase carrier comprises any one of or a combination of at least two of a nitrocellulose membrane, a latex particle, a magnetic particle, colloidal gold, a bead, glass, fiber glass, a polymer or a fiber optical sensor.
By using the monoclonal antibody of the present invention, a reagent for immunoassay of SARS-CoV-2 virus can be produced by using the antibody as at least one of the solid-phase antibody and the labeled antibody. The solid phase to which the monoclonal antibody is bound may be any of various solid phases conventionally used in 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 light-emitting 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 a fifth aspect, the present invention provides a method for detecting the presence of SARS-CoV-2 virus or an antigen corresponding thereto in a sample for non-disease diagnostic and therapeutic purposes, said method comprising the steps of:
mixing a sample to be tested with the monoclonal antibody or the antigen binding fragment thereof in the first aspect, incubating, and detecting the existence of SARS-CoV-2 virus 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 various immunoassays described above, 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 and labeled SARS-CoV-2 virus N protein in the detected specimen 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 the antibody or antigen-binding fragment thereof is 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.
Preferably, the sample to be tested is a biological sample and/or an environmental sample, wherein the biological sample comprises any one of plasma, whole blood, mouthwash, throat swab, urine, feces or bronchial perfusion fluid or a combination of at least two of the above. The sample to be used in the immunoassay method is not particularly limited as long as it contains nucleocapsid protein of SARS-CoV-2 virus, and examples thereof include serum, plasma and whole blood derived from human and animals, and in addition, body fluid extracts such as nasal swab (nasal swab), nasal aspirate and pharyngeal swab (pharyngeal swab), respiratory secretions, and cell and tissue homogenates.
The method described in the present invention is preferably 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.
Specifically, the sandwich method comprises the following steps:
(1) Binding a monoclonal antibody or an antigen-binding fragment thereof as in any one of the first aspects 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 control by using a control sample;
(3) After the incubation is completed, washing the solid support, adding a second antibody labeled with a labeling substance, which binds to SARS-CoV-2 virus or its corresponding antigen, and incubating again;
(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 existence and relative amount of SARS-CoV-2 virus in the sample to be tested;
preferably, the sandwich method further comprises: in the step (3), the second antibody is labeled with biotin, and then is bound to enzyme-labeled avidin or streptavidin, and then the substrate for the enzyme is added in the step (4) to develop color.
Preferably, the labeled antigen and the sample to be tested are added at substantially the same time in the above procedure.
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.
Specifically, the competition method comprises the following steps:
(1') binding a predetermined amount of the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect to a solid phase carrier;
(2 ') adding a sample to be tested and a predetermined amount of SARS-CoV-2 virus labeled with a labeling substance or its corresponding antigen to the solid phase carrier obtained in the step (1'), incubating, and performing control with a control sample;
(3') washing said solid phase carrier after the reaction, and detecting a signal value of the marker bound to SARS-CoV-2 virus or the antigen corresponding thereto;
(4') comparing the measured signal value with the signal value of a control sample, and determining the presence and relative amount of SARS-CoV-2 virus in the test sample.
The labeled virus monoclonal antibody can be prepared by binding a monoclonal antibody against SARS-CoV-2 virus to the label. The labeling substance 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, β -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 method of bonding are: 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, a functional group present in the antibody may be used depending on the method, and in addition, a labeled anti-SARS-CoV-2 virus monoclonal antibody can be prepared by introducing a functional group such as thiol, amino, carboxyl, hydroxyl or the like into the antibody according to 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' -Tetramethylbenzidine (TMB), diaminobenzidine (DAB) in combination with hydrogen peroxide for peroxidase; 5-bromo-4-chloro-3-indolyl phosphate (BCIP), p-nitrophenyl phosphate (p-NPP), sodium 5-bromo-4-chloro-3-indolyl phosphate (BCIP. Na) was used for alkaline phosphatase.
(b) Fluorescent substrate: 4-methylumbelliferyl phosphate (4-MUP) for alkaline phosphatase; 4-Methylumbelliferyl phenyl-beta-D-galactoside (4 MUG) 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 "-beta-D-galactopyranosyl) phenyl-1, 2-dioxetane (AMGPD) for beta-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) After the reaction, washing thoroughly 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 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 at least the following beneficial effects:
(1) The monoclonal antibody or the antigen binding fragment thereof is prepared from four hybridoma cell strains CCTCC NO: c2020231, CCTCC NO: c2020233, CCTCC NO: c2020234 or CCTCC NO: c2020235, the monoclonal antibody or the antigen binding fragment thereof can specifically recognize the N protein of SARS-CoV-2 virus, has the affinity up to pM grade, and 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 matched three groups of antibodies (mAb 1/Bio-mAb4, mAb1/Bio-mAb2 and mAb5/Bio-mAb 4), when the concentration range of SARS-CoV-2N protein is 1-200 ng/mL or 1-400 ng/mL, the measured OD value is in linear positive correlation with the concentration of NP, and the lower limit of detection is lower than 12.5pg/mL;
(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 SARS-CoV-2 antigen detection kit provided by the invention can detect the N antigen in the early stage of virus infection of a patient, solves the problem of rapid diagnosis of SARS-CoV-2 virus infectors in clinic, has higher accuracy, and provides a rapid and accurate diagnosis method for clinical detection of the SARS-CoV-2 virus infectors.
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 the measurement of the cross-reactivity of the anti-SARS-CoV-2 monoclonal antibody with SARS virus in example 3.
FIG. 3 is a graph showing the results of the measurement of the cross-reactivity of the anti-SARS-CoV-2 monoclonal antibody with MERS virus in example 3.
FIG. 4 is a graph showing the results of the double antibody sandwich ELISA assay of the capture antibody mAb1 and the labeled antibody Bio-mAb4 of example 5.
FIG. 5 is a graph showing the results of the double antibody sandwich ELISA assay of the capture antibody mAb1 and the labeled antibody Bio-mAb2 of example 5.
FIG. 6 is a graph showing the results of the double antibody sandwich ELISA assay of the capture antibody mAb5 and the labeled antibody Bio-mAb4 in 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 methods of testing, methods of preparation, and methods of preparation disclosed herein 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, molecula clone, ALABORATORY MANUAL, second edition, cold Spring Harbor Laboratory Press,2001; ausubel et al, current PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987and periodic updates; (ii) METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; METHOD IN ENZYMOLOGY, vol.304, chromatin (P.M. Wassarman and A.P.Wolffe, eds.), academic Press, san Diego,1999 and METHOD IN MOLECULAR BIOLOGY, vol.119, chromatin Protocols (P.B. Becker, ed.) Humana Press, totowa,1999 et al.
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) Immunization of animals
Fully emulsifying SARS-CoV-2N protein antigen (Sino Biological, 40588-V08B) with complete 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.
(2) Establishment of indirect ELISA method for determining 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 10mM PBS buffer solution, vacuum drying, vacuum packaging with aluminum film bag, and storing at 4 deg.C for measuring titer of mouse immune serum;
orbital bleeds were performed 10 days after 3 rd immunization, and the collected mouse sera were diluted with 1.5 mass-fraction BSA in 10mM PBS for a series of concentration gradients of 1;
after washing the plate, 100. Mu.L/well of a buffer solution containing 0.05% (w/v) TMB and 0.06% (w/v) dioxygen pH5.0 citric acid was added, and the mixture was protected from light at room temperature for 10 minutes, and 0.2M H was added 2 SO 4 Stopping reaction, reading an absorption value at the position of dual wavelength 450/620nm by an enzyme-labeling instrument at 100 mu L/hole, taking the serum of the mouse before immunization as negative control, and judging the titer of the immune serum by taking the ratio of the measured value to the control value to be more than or equal to 2.0 as positive;
all 4 mice developed a strong specific immune response after the third immunization, with the highest titers of immune sera for mice M21 and M22, slightly lower for M20 mice, and the weakest immune response in M19 mice, the results of which are shown in fig. 1.
(3) Preparation of hybridomas
3 days before fusion, the mice producing the highest antibody titer were boosted once; after 3 days, the mice were sacrificed and their spleens were aseptically removed to make a splenocyte suspension, which was then mixed with 2 × 10 cells at a ratio of 1 8 Several 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);
iscove's medium (containing 10% fetal bovine serum, 100U/mL penicillin, 100. Mu.g/mL streptomycin, 0.1mM hypoxanthine, 0.4. Mu.M aminopterin, and 16. Mu.M thymidine) was used to adjust the number of spleen cells to 5X 10 5 /mL, adding 0.3mL into wells of a 96-well plate, and placing at 37 deg.C, 5% 2 Culturing 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 replated with this selection medium every 3 days until clonal cells formed.
(4) Screening hybridoma secreting monoclonal antibody of SARS-CoV-2 virus N protein
For screening positive clones producing antibodies, the cell culture supernatants were assayed by indirect ELISA as follows:
adding the culture supernatant of the hybridoma cells into an ELISA plate coated with an antigen in advance, wherein each hole is 100 mu L, taking the culture supernatant of the SP2/0 cells 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. No. 115-035-07) at a working concentration of 100. Mu.L/well at 37 ℃ for 30 minutes, washing the plate, adding TMB as a substrate at 100. Mu.L/well, keeping the plate dark at room temperature for 10 minutes, adding 0.2M H at 100. Mu.L/well 2 SO 4 The reaction was stopped and the microplate reader read the absorbance at dual wavelength 450/620 nm. The OD 450/620nm reading of the detected hole is more than twice of the negative control, and the positive result is judged;
(5) Cloning of Positive hybridoma cells
Carrying out subcloning on the screened strong positive cell clone for 2-3 times by adopting a limiting dilution method to obtain 5 hybridoma cell strains which stably secrete antibodies, wherein the 5 hybridoma cell strains are respectively named as # mAb1, # mAb2, # mAb3, # mAb4 and # mAb5; and 4 strains of which were biologically deposited, # mAb1, # mAb2, # mAb4 and # mAb5;
the corresponding preservation number is CCTCC NO: c2020231, CCTCC NO: c2020233, CCTCC NO: c2020234 and CCTCC NO: a hybridoma cell line of C2020235.
(6) Purification of antibodies
The positive hybridoma cell clones # mAb1, # mAb2, # mAb3, # mAb4 and # mAb5 obtained by the above screening were cultured in RPMI 1640 medium supplemented with 10% FCS;
when the cell density reaches about 5X 10 5 Replacing the medium with serum-free medium at individual cells/mL, centrifuging after culturing and collecting the culture supernatant, then purifying the antibody using a Protein G affinity chromatography column, dialyzing the monoclonal antibody eluate against 150mM NaCl, and filter-sterilizing the dialyzed solution through a 0.2 μm filter,a purified antibody sample was obtained.
(7) Labeling of antibodies
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 buffer 2 ) 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 a Biotin derivative NHS-LC-Biotin (Thermo Scientific, cat. No. 21435), and the monoclonal antibodies were labeled with Biotin according to the method described in the specification of the Thermo Scientific EZ-Link Biotin labeling kit to obtain Biotin-labeled monoclonal antibodies Bio-mAb1, bio-mAb2, bio-mAb3, bio-mAb4 and Bio-mAb5.
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:
8-10mg 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 (pH9.6). Dissolving HRP 4mg in 1mL of purified water, and slowly adding 0.1M NaIO 4 0.1mL, stirring at room temperature in the dark for 20 minutes, putting into a dialysis bag, and dialyzing overnight at 4 ℃ with 0.001M acetate buffer solution with pH 4.4;
to HRP was added 0.2M Na 2 CO 3 0.05mL 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 4mg/mL of NaBH was added 4 0.1mL, 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 obtained precipitate was dissolved in 1mL PBS and dialyzed with PBS at 4 ℃ for 48 hours to obtain enzyme-labeled monoclonal antibodies HRP-mAb1, HRP-mAb2, HRP-mAb3, HRP-mAb4 and HRP-mAb5.
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 using the "double antibody sandwich" method.
The 5 monoclonal antibodies mAb1, mAb2, mAb3, mAb4 and mAb5 were coated with each pair of antibodies in a 5X 5 matrix as capture antibodies, which were paired with 5 biotin-labeled monoclonal antibodies Bio-mAb1, bio-mAb2, bio-mAb3, bio-mAb4 and Bio-mAb5, respectively, to rapidly screen pairs of captured and labeled monoclonal antibodies in sandwich ELISA.
By comparing the detection sensitivity, the monoclonal antibody mAb1 is shown as a capture antibody, and can generate stronger signals when being matched with Bio-mAb2 or Bio-mAb4 respectively; in addition, mAb5, as a capture antibody, also gave a strong signal when paired with Bio-mAb 4.
Example 3 functional characterization of anti-N protein monoclonal antibodies
(1) Monoclonal antibody affinity constant determination
The binding affinity constants of the purified murine monoclonal antibodies mAb1, mAb2, mAb4 and mAb5 to the SARS-CoV-2N protein were determined using the 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.
First, biotinylated SARS-CoV-2N protein was immobilized on the surface of an SA sensor, and the above monoclonal antibody against SARS-CoV-2N protein was used as an analyte. The data were processed and fitted with a model incorporating analytical software 1, the fitted data essentially overlapping the experimental data, to yield the association and dissociation rate constants K a And K d By K d Except for K a Obtaining the equilibrium dissociation constant K D (see Table 1).
The result shows that the mAb5 of the mouse has the highest affinity, K D The value is 2.69X 10 -11 M, whereas mAb1, mAb2, mAb4 have comparable affinities, one order of magnitude lower than mAb5.
TABLE 1
Name of antibody K D (M) K a (1/Ms) K d (1/s)
mAb1 3.61E-10 2.18E+05 7.88E-05
mAb2 7.89E-10 1.60E+05 1.26E-04
mAb4 4.076E-10 2.054E+05 8.374E-05
mAb5 2.69E-11 2.79E+05 7.51E-06
(2) Cross-reactivity of monoclonal antibodies with SARS and MERS viruses
The cross-reactivity of the above anti-SARS-CoV-2 monoclonal antibodies mAb1, mAb2, mAb3, mAb4 and mAb5 against the human SARS and MERS coronavirus nucleocapsid proteins 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 in 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, 0.05% Tween20 in PBS), 200. Mu.L/well of PBST/1% skim milk was added and incubated at room temperature for 1h to block.
After removing the blocking solution and washing the plate for 3 times by the PBST buffer solution, the monoclonal antibody with the initial concentration of 10 mug/mL is diluted into gradient concentration according to the proportion of 1. The reaction was removed and after 3 PBST washes the plate, 50 μ L/well of a 1. 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 dual wavelength 450/620 nm.
As shown in FIG. 2, mAb1 and mAb5 of the above four monoclonal antibodies can specifically bind to the SARS virus N protein antigen, and none of the other three antibodies has cross-reactivity with SARS N protein; in addition, none of the five mabs was able to specifically bind MERS virus N protein (see fig. 3).
Therefore, the antibody mAb1 which has cross reaction with SARS N protein is used as capture antibody, and the antibody which does not cross with SARS N protein (such as mAb4 and mAb 2) is used as marker antibody, and the combined use can detect SARS-CoV-2 virus, not only has higher specificity, but also has higher sensitivity, because it is directed against the common epitope of SARS-CoV-2 and SARS N protein, and the common dominant epitope has higher abundance in infected cells or immune serum than other antigen epitopes, thus can reduce virus detection limit.
EXAMPLE 4 establishment of double antibody Sandwich ELISA kit for detecting SARS-CoV-2 Using monoclonal antibody against SARS-CoV-2N protein
Using the monoclonal antibody mAb1 and biotin-labeled monoclonal antibody Bio-mAb2 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, PH7.4 (PBST); the blocking solution is PBST containing 1.5% Bovine Serum Albumin (BSA); the standard and test sample dilutions are PBST containing 1.5% BSA; the biotin-labeled antibody dilution was PBST containing 1.5% BSA; streptavidin-HRP (Thermo Scientific, lot: 21130) was formulated as a working solution using 1.5% BSA in PBST according to the instructions for its formulation.
The detection method comprises the following steps:
1) Antibody mAb1 was diluted to a final concentration of 5. Mu.g/mL with 1 XPBS, 100. Mu.L per well coated with polystyrene microplate, shaken at room temperature for 1 hour at 500rpm/min, washed with washing solution, and patted dry.
2) Add blocking solution 200. Mu.L/well and let stand at room temperature for 1 hour.
3) The SARS-CoV-2N protein standard was diluted to different concentrations as a standard curve (taking the standard diluted with 1.5% BSA in PBST to the following concentrations: 400pg/mL, 200pg/mL, 100pg/mL, 50pg/mL, 25pg/mL, 12.5 pg/mL), 100. Mu.L per well was added to the reaction plate; the test sample was diluted 100-fold and added to different wells as well, and the BSA-PBS solution negative control was 1.5% by volume, left to stand at room temperature for 1 hour, washed 3 times with the washing solution, and patted dry.
4) Biotin-labeled secondary antibody Bio-mAb2 (1.5% bsa-PBS solution by 1: 5000-fold dilution), standing at room temperature for 1 hour, washing with a washing solution 3 times, and patting to dry.
5) Add prepared Streptavidin-HRP solution, 100. Mu.L per well, and let stand at room temperature for 1 hour.
6) Washing solution is washed for 3 times, dried by beating, and then added with the prepared TMB,100 mu L/hole, and developed for 20-30 minutes at room temperature.
7) Addition of 0.2M H 2 SO 4 Stop, 50. Mu.L/well.
8) And reading the absorption value at the position of dual wavelength of 450/620nm 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 measurement of the Performance of ELISA method and kit therefor
The performance of the double antibody sandwich ELISA method and kit for measuring the SARS-CoV-2 virus content, which were established by the best-matched 3 groups of antibodies (mAb 1/Bio-mAb4, mAb1/Bio-mAb2 and mAb5/Bio-mAb 4) screened in example 2, was evaluated.
(1) Detection range
As can be seen from FIGS. 4-6, in which FIG. 4 shows mAb1/Bio-mAb4, FIG. 5 shows mAb1/Bio-mAb2, FIG. 6 shows mAb5/Bio-mAb4, the OD value increases with the increase of the concentration of SARS-CoV-2N protein (NP), and when the concentration of NP to be detected is in the range of 1-200 ng/mL or 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. 2 Not less than 0.995). The lower limit of detection of the best-matched three groups of antibodies screened above was less than 12.5pg/mL.
(2) Specificity of
The reaction of the ELISA kit composed of the three antibodies selected above with several other serum substances was measured, and the reaction specificity was observed. The detection is carried out by a double-antibody sandwich ELISA standard method, the capture antibody is coated at 4mu g/mL, the biotin-labeled antibody (diluted by 1 in 5000) is detected, and the following samples are respectively detected: human serum albumin and bovine serum albumin 100ng/mL, 5 parts of SARS-CoV-2N protein negative serum sample (NP negative serum) diluted by 1. After the reaction was completed, color development was performed, and the results were judged as shown in Table 2.
TABLE 2
Figure BDA0002977127890000211
Remarking: "+" indicates that the sample test result was positive, and "-" indicates that the sample test result was negative.
(3) Sensitivity of the probe
The detection of the reference/standard was performed according to the established experimental method described previously. The detection limit is at least 12.5pg/mL, and the conditions are that the OD value is more than 3 times SD of the blank control OD value, and the OD value is more than 2 times the blank control OD value. The method of the invention has good sensitivity.
(4) Repeatability of
The ELISA kit consisting of the three groups of antibody pairs is subjected to repeated measurement. Using mAb1/Bio-mAb4 as an example, the results show that: setting 8 multiple holes for one sample, and repeatedly detecting for 4 times, wherein CV values of 8 results in the same plate at the same time are all less than 4%; different people operate at different times, the detection is repeated for 4 times, the CV value of the average value of each result is 1.89%, other three groups of antibodies have good reproducibility to the ELISA kit formed, and the repeatability of the kit for measuring the SARS-CoV-2N protein according to the method of the invention is better.
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 monoclonal 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 SARS-CoV-2 virus.
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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cttcctcaag gaacaacatt gccaaaaggc ttctacgcag aagggagcag aggcggcagt 540
caagcctctt ctcgttcctc atcacgtagt cgcaacagtt caagaaattc aactccaggc 600
agcagtaggg gaacttctcc tgctagaatg gctggcaatg gcggtgatgc tgctcttgct 660
ttgctgctgc ttgacagatt gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa 720
caacaaggcc aaactgtcac taagaaatct gctgctgagg cttctaagaa gcctcggcaa 780
aaacgtactg ccactaaagc atacaatgta acacaagctt tcggcagacg tggtccagaa 840
caaacccaag gaaattttgg ggaccaggaa ctaatcagac aaggaactga ttacaaacat 900
tggccgcaaa ttgcacaatt tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt 960
ggcatggaag tcacaccttc gggaacgtgg ttgacctaca caggtgccat caaattggat 1020
gacaaagatc caaatttcaa agatcaagtc attttgctga ataagcatat tgacgcatac 1080
aaaacattcc caccaacaga gcctaaaaag gacaaaaaga agaaggctga tgaaactcaa 1140
gccttaccgc agagacagaa gaaacagcaa actgtgactc ttcttcctgc tgcagatttg 1200
gatgatttct ccaaacaatt gcaacaatcc atgagcagtg ctgactcaac tcaggcctaa 1260

Claims (5)

1. A SARS-CoV-2 virus detection kit, wherein the SARS-CoV-2 virus detection kit comprises:
a first antibody bound to a solid support; and
a second antibody carrying a label in combination with the first antibody;
the first antibody and the second antibody are selected from any one of the following combinations:
combination I: the first antibody is a polypeptide with the preservation number of CCTCC NO: the monoclonal antibody secreted by the hybridoma cell strain # mAb1 of C2020231, wherein the second antibody is a monoclonal antibody expressed by a nucleic acid sequence with the preservation number of CCTCC NO: a monoclonal antibody secreted by a hybridoma cell strain # mAb4 of C2020234;
combination II: the first antibody is a polypeptide with the preservation number of CCTCC NO: the monoclonal antibody secreted by the hybridoma cell strain # mAb1 of C2020231, wherein the second antibody is a monoclonal antibody with the preservation number of CCTCC NO: a monoclonal antibody secreted by a hybridoma cell strain # mAb2 of C2020233;
combination III: the first antibody is a polypeptide with the preservation number of CCTCC NO: the monoclonal antibody secreted by the hybridoma cell strain # mAb5 of C2020235, wherein the second antibody is a monoclonal antibody expressed by a nucleic acid sequence with the preservation number of CCTCC NO: the monoclonal antibody secreted by the hybridoma cell strain # mAb4 of C2020234.
2. The SARS-CoV-2 virus detection kit of claim 1, wherein the marker comprises: any one of a radioisotope, an enzyme substrate, a phosphorescent substance, a fluorescent substance, or biotin;
the radioactive isotope is 125 I、 3 H、 14 C or 32 Any one of P;
the enzyme is any one of alkaline phosphatase, horseradish peroxidase, beta-galactosidase, urease or glucose oxidase;
the fluorescent substance is any one of fluorescein dye, rhodamine dye, rare earth elements or rare earth element compound.
3. A SARS-CoV-2 virus detection kit according to claim 2, wherein the solid support comprises any one of or a combination of at least two of nitrocellulose membrane, latex particles, magnetic particles, colloidal gold, glass, fiber glass or fiber optic sensors.
4. A method for detecting the presence of SARS-CoV-2 virus or an N protein antigen thereof in a sample for non-disease diagnostic and therapeutic purposes, said method comprising the steps of:
mixing the sample to be tested with the first antibody and the second antibody as claimed in claim 1, incubating, detecting the existence of SARS-CoV-2 virus or its N protein antigen,
the second antibody is combined with the first antibody, and the first antibody and the second antibody are selected from any one of the following combinations:
combination I: the first antibody is a polypeptide with the preservation number of CCTCC NO: the monoclonal antibody secreted by the hybridoma cell strain # mAb1 of C2020231, wherein the second antibody is a monoclonal antibody expressed by a nucleic acid sequence with the preservation number of CCTCC NO: a monoclonal antibody secreted by a hybridoma cell strain # mAb4 of C2020234;
combination II: the first antibody is a polypeptide with the preservation number of CCTCC NO: the monoclonal antibody secreted by the hybridoma cell strain # mAb1 of C2020231, wherein the second antibody is a monoclonal antibody with the preservation number of CCTCC NO: a monoclonal antibody secreted by the hybridoma cell strain # mAb2 of C2020233;
combination III: the first antibody is a polypeptide with the preservation number of CCTCC NO: the monoclonal antibody secreted by the hybridoma cell strain # mAb5 of C2020235, wherein the second antibody is a monoclonal antibody expressed by a nucleic acid sequence with the preservation number of CCTCC NO: and (3) a monoclonal antibody secreted by the hybridoma cell strain # mAb4 of C2020234.
5. The method according to claim 4, characterized in that the sample to be tested is a biological and/or environmental sample.
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