WO2022193980A1 - Anticorps ou fragment de liaison à l'antigène de celui-ci pour protéine nucléocapsidique du nouveau coronavirus, et son utilisation - Google Patents

Anticorps ou fragment de liaison à l'antigène de celui-ci pour protéine nucléocapsidique du nouveau coronavirus, et son utilisation Download PDF

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WO2022193980A1
WO2022193980A1 PCT/CN2022/079557 CN2022079557W WO2022193980A1 WO 2022193980 A1 WO2022193980 A1 WO 2022193980A1 CN 2022079557 W CN2022079557 W CN 2022079557W WO 2022193980 A1 WO2022193980 A1 WO 2022193980A1
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antibody
antigen
novel coronavirus
binding fragment
sample
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Chinese (zh)
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李强
武翠
翁仕强
张晓峰
周宇
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安源医药科技（上海）有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G01N33/546—Synthetic resin as water suspendable particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses

Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to an antibody against a novel coronavirus, in particular to an antibody against a novel coronavirus nucleocapsid protein or an antigen-binding fragment thereof and an application thereof.
  • the novel coronavirus belongs to the genus Betacoronavirus, and both the MERS virus known to cause Middle East Respiratory Syndrome and the SARS virus known to cause Severe Acute Respiratory Syndrome belong to this genus.
  • the acute respiratory infectious disease caused by the novel coronavirus is novel coronavirus pneumonia (COVID-19). Its clinical manifestations are mainly fever, fatigue, and dry cough. The upper respiratory tract symptoms such as nasal congestion and runny nose are rare, and hypoxia and hypoxia state may occur. .
  • the existing clinical novel coronavirus detection reagents include enzyme-linked immunosorbent assay for serum antibody detection and RT-PCR method for viral genetic material detection. Since the production of antibodies after infection requires a window period of 1-3 weeks, based on the current epidemiological investigation, the incubation period of the new coronavirus is generally 3-7 days, and the longest is not more than 14 days, so antibody detection is not meaningful for early diagnosis. In addition, the current antibody detection has a high false positive rate, and the specificity cannot meet the needs.
  • Nucleic acid detection has high specificity and sensitivity, but this detection method has high technical requirements, is prone to false negatives, requires special handling of specimens, and requires professional equipment such as PCR amplifiers and gel electrophoresis. It takes a long time to test and requires professional technicians to operate and judge the test results. It cannot be used for early preliminary screening in communities, grass-roots hospitals, airports, customs and even families. Therefore, the antigen diagnosis method for the new coronavirus, especially the rapid, sensitive and specific antigen diagnosis method, has irreplaceable significance in the diagnosis of COVID-19.
  • nucleocapsid protein binds to the viral RNA and leads to the formation of the helical nucleocapsid.
  • Nucleocapsid protein is a highly immunogenic phosphoprotein involved in viral genome replication and regulation of cellular signaling pathways. Nucleocapsid protein is also the most abundant protein in coronaviruses. Due to the sequence conservation and strong immunogenicity of nucleocapsid proteins, nucleocapsid proteins are often used as diagnostic tools for coronaviruses.
  • the purpose of the present invention is to provide an antibody or an antigen-binding fragment thereof against the nucleocapsid protein of the novel coronavirus and its application.
  • the novel coronavirus nucleocapsid protein as an antigen to immunize mice, obtain a mouse with the highest immune serum titer, and obtain hybridoma cells and store them; the antibodies or their antigen-binding fragments secreted by the obtained hybridoma cells can specifically bind to the nucleocapsid capsid protein, which can then be made into a kit for the detection of the new coronavirus.
  • the present invention provides an antibody against a novel coronavirus nucleocapsid protein or an antigen-binding fragment thereof, the antibody is selected from any one of mAb6 antibody, mAb7 antibody, mAb8 antibody and mAb9 antibody;
  • the mAb6 antibody is secreted by the hybridoma cell line #mAb6 with the deposit number CCTCC NO: C2020236, the mAb7 antibody is secreted by the hybridoma cell line #mAb7 with the deposit number CCTCC NO: C2020237, and the mAb8 antibody is secreted by the deposit The hybridoma cell line #mAb8 with the number CCTCC NO: C2020238 is secreted, and the mAb9 antibody is secreted by the hybridoma cell line #mAb9 with the deposit number CCTCC NO: C2020239.
  • the specific antibody against the SARS-CoV-2 virus N protein can be used to determine the presence or content of the SARS-CoV-2 virus or its corresponding antigen in a sample, and can achieve high sensitivity and stability sex.
  • the monoclonal antibody of the present invention can be obtained using the nucleocapsid protein of the novel coronavirus as an immunogen.
  • the amino acid sequence of nucleocapsid protein is well known, and the amino acid sequence is shown in SEQ ID NO:1.
  • the base sequence of the nucleic acid encoding the amino acid sequence shown in SEQ ID NO:1 is shown in SEQ ID NO:2.
  • the monoclonal antibody of the present invention can be obtained using a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 as an immunogen. It can also be a natural mutant of the amino acid sequence of SEQ ID NO:1.
  • antibody fragments referred to as "antigen-binding fragments" in the present invention
  • antigen-binding fragments having binding properties to the corresponding antigens such as Fab fragments or F(ab')2 fragments
  • the antigen-binding fragment of the present invention can also be used in the same manner as the monoclonal antibody of the present invention, and is included in the scope of the present invention.
  • the present invention provides a hybridoma cell line that secretes the antibody or its antigen-binding fragment according to the first aspect, wherein the hybridoma cell line has a deposit number of CCTCC NO: C2020236, CCTCC NO: C2020237, CCTCC Either NO: C2020238 or CCTCC NO: C2020239.
  • the biological material preservation information of the present invention is as follows:
  • hybridoma cell line with the deposit number CCTCC NO: C2020236 has been registered and deposited in the China Collection of Type Cultures (CCTCC) (Address: Wuhan University, Wuhan, China) on November 26, 2020, and the classification name is: Hybridoma cell line #mAb6.
  • CTCC China Collection of Type Cultures
  • the hybridoma cell line with the deposit number CCTCC NO: C2020237 has been registered and deposited in the China Center for Type Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China) on November 26, 2020, and the classification name is: Hybridoma cell line #mAb7.
  • CTCC China Center for Type Culture Collection
  • hybridoma cell line with the deposit number CCTCC NO: C2020238 has been registered and deposited in the China Center for Type Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China) on November 26, 2020, and the classification name is: Hybridoma cell line #mAb8.
  • CTCC China Center for Type Culture Collection
  • hybridoma cell line with the deposit number CCTCC NO: C2020239 has been registered and deposited in the China Center for Type Culture Collection (CCTCC) (Address: Wuhan University, Wuhan, China) on November 26, 2020, and the classification name is: Hybridoma cell line #mAb9.
  • CTCC China Center for Type Culture Collection
  • the hybridoma of the present invention can be produced by a cell fusion technique known in the art: immunizing an animal with an immunogen by a conventional method, cell fusion of an anti-nucleocapsid protein antibody-producing cell obtained from the animal and a tumor cell, thereby obtaining hybridomas to produce the above antibodies.
  • Animals immunized to produce hybridomas of the present invention are not particularly limited and include, but are not limited to, goats, sheep, guinea pigs, mice, rats and rabbits. Preferred among them are mice.
  • the above-mentioned hybridoma can be obtained, for example, by subcutaneously administering the nucleocapsid protein of the novel coronavirus together with Freund's complete adjuvant to animals such as mice by subcutaneous multiple injections at intervals of 2 to 3 weeks. immunity.
  • an antibody-producing cell derived from a spleen or the like obtained from an immunized animal is fused with a tumor cell, and the tumor cell is a myeloma cell selected from an immortalized cell line such as a myeloma cell line. cultured tumor cells.
  • the above-mentioned fusion method can be, for example, according to the conventional method of Khler and Milstein (G. Kohler and C. Milstein, Nature, 1975, 256: 495-497), using the polyethylene glycol method, or the Sendai virus method or the like.
  • the monoclonal antibody of the present invention can be produced by the hybridoma of the present invention.
  • the method for producing the monoclonal antibody of the present invention from the hybridoma of the present invention can be conventional or known methods in the art, for example, it can be obtained from the tissue culture medium for culturing the hybridoma of the present invention, or it can be inoculated into mice Proliferated in vivo, isolated from collected ascites fluid or serum.
  • the culturing of the hybridomas of the present invention can be carried out according to conventional or known methods in the art.
  • the methods of inoculating, collecting ascites fluid or serum, and isolating the monoclonal antibody of the present invention from ascites fluid or serum may be conventional or known methods in the art.
  • the present invention provides the application of the antibody or antigen-binding fragment thereof described in the first aspect or the hybridoma cell line described in the second aspect in the preparation of a novel coronavirus detection kit.
  • the application field of the monoclonal antibody or the antigen-binding fragment thereof in the present invention is not particularly limited, and can especially be applied to immunoassays for judging the infection status of the SARS-CoV-2 virus.
  • the present invention also provides a non-disease diagnosis and treatment purpose, a method for detecting the presence of a novel coronavirus or its corresponding antigen in a sample, the method comprising the following steps:
  • the sample to be tested is mixed with the antibody or its antigen-binding fragment as described in the first aspect, incubated, and the presence of the novel coronavirus or its corresponding antigen is detected.
  • the present invention provides an immunoassay method for detecting or quantifying the presence or quantification of SARS-CoV-2 virus or its antigen in biological samples and/or environmental samples by using the above-mentioned antibodies; the method comprises combining the biological samples to be tested and /or the environmental sample is incubated with the anti-SARS-CoV-2 virus N protein monoclonal antibody of the present invention or its antigen-binding fragment to form an antigen-antibody complex, and qualitative detection and quantitative determination of the formed binding complex are performed.
  • the presence or amount of SARS-CoV-2 virus is indicative of the presence or amount.
  • the immunoassay method itself is well-known, and any well-known immunoassay method can be used, which need not be repeated in this specification. That is, if it is classified by the measurement format, there are sandwich methods, competition methods, aggregation methods, Western blotting methods, etc., and if they are classified by the labels used, there are fluorescence methods, enzymatic methods, radioactive methods, biotin methods, etc. can be used. Diagnosis can also be made by immunohistostaining. When a labeled antibody is used in the immunoassay method, the method for labeling the antibody itself is known, and any known method can be used.
  • the immunoassay of the present invention is performed using at least one monoclonal antibody or antigen-binding fragment thereof secreted by the above-mentioned hybridoma cell line.
  • Immunoassays for detecting antigens are well known in the art.
  • the monoclonal antibodies or antigen-binding fragments thereof according to the present invention may be independent of the label used (eg, enzyme, fluorescence, etc.) and independent of the detection mode (eg, fluorescence immunoassay, enzyme-linked immunosorbent assay, or chemiluminescence assay). method, etc.) or assay principles (eg sandwich method, competition method, etc.) are used in the above immunoassay methods.
  • the above immunoassays include enzyme immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescence immunoassays, Western blotting, immunochromatography, latex agglutination assays, etc.;
  • the method uses a marker-labeled antigen or antibody to determine the target antigen in a biological sample and/or an environmental sample.
  • the competitive method is based on the detection of SARS-CoV-2 virus in the specimen and a known amount of labeled SARS-CoV-2 virus N protein and the monoclonal antibody of the present invention or its antigen-binding fragment for quantitative competitive binding reaction; namely, the present invention Including qualitative or quantitative determination of the presence or content of SARS-CoV-2 virus in biological components using assay color, fluorescence, time-resolved fluorescence, chemiluminescence, electrochemiluminescence, or radioactivity.
  • the sandwich method is to immobilize the antibody or antigen-binding fragment of the present invention as a first antibody as a solid phase, react with the biological sample to be tested and/or an environmental sample, rinse it, and then react it with the second antibody, and measure the difference after rinsing.
  • Solid phase bound secondary antibody The second antibody bound to the solid phase can be measured by labeling the second antibody with an enzyme, a fluorescent substance, a radioactive substance, biotin, or the like.
  • the antibody or antigen-binding fragment thereof of the present invention is immobilized on particles such as latex, reacted with a sample, and the absorbance is measured.
  • a plurality of standard substances of known concentration are measured, and a standard curve is prepared according to the relationship between the measured labeled amount and the content of the standard substance. Quantification of SARS-CoV-2 viral antigens in samples.
  • the solid phase needs to be washed sufficiently to measure the activity of binding to the label.
  • the label is a radioisotope
  • the measurement is performed with a pore counter or a liquid scintillation counter.
  • the label is an enzyme
  • the substrate is added and the enzyme activity is measured colorimetrically or fluorometrically after color development.
  • the label is a fluorescent substance, a phosphorescent substance, or a coloring substance, it can be measured by methods known in the art, respectively.
  • the samples to be tested include biological samples and/or environmental samples.
  • the sample to be supplied to the above-mentioned immunoassay is not particularly limited as long as it contains the nucleocapsid protein of the SARS-CoV-2 virus.
  • it can be derived from human and animal serum, plasma, whole blood, and others.
  • Nasal swab nasal swab
  • nasal aspiration a nasal swab
  • throat swab a nasal swab
  • other body fluid extracts derived from human and animal serum, plasma, whole blood, and others.
  • the biological sample includes any one or a combination of at least two of plasma, whole blood, mouthwash, throat swab, urine, feces or bronchial perfusate.
  • the method includes a sandwich method or a competition method.
  • the sandwich method is to fix the antibody or antigen-binding fragment of the present invention as a first antibody on a solid phase, react with the sample to be tested, rinse it, and then react it with a second antibody, and measure the solid phase after rinsing. conjugated secondary antibody.
  • the second antibody bound to the solid phase can be measured by labeling the second antibody with an enzyme, a fluorescent substance, a radioactive substance, biotin, or the like.
  • the sandwich method comprises the steps:
  • step (2) mixing the solid-phase carrier obtained in step (1) with the sample to be tested, incubating, and comparing with the control sample;
  • the sandwich method further comprises: in step (3), the second antibody is first labeled with biotin, and then combined with the enzyme-labeled avidin or streptavidin;
  • step (4) the substrate of the enzyme is added to develop color.
  • the above sandwich method is based on the fact that the monoclonal antibody or its antigen-binding fragment of the present invention as a capture antibody (or solid-phase antibody) and the labeled antibody that can be used in combination can specifically bind to the SARS-CoV-2 virus in the sample to be tested, The amount of SARS-CoV-2 virus in the sample was determined by quantifying the labeled antibody.
  • the sandwich method binds the specific monoclonal antibody against the SARS-CoV-2 virus N protein or its antigen-binding fragment of the present invention to a solid-phase carrier to form a solid-phase antibody (also called capture antibody or primary antibody) , and then add the sample to be tested and the control sample to the coated solid phase carrier and incubate for a long enough time under appropriate conditions;
  • a solid-phase carrier also called capture antibody or primary antibody
  • the solid phase is thoroughly washed, and an appropriate amount of labeled secondary antibody that can bind to the SARS-CoV-2 virus N protein is added and incubated again; Detecting the signal value of the label bound to the secondary antibody; comparing the measured signal value with the signal value of a predetermined amount of control samples measured in parallel to determine the presence of SARS-CoV-2 virus in the sample and its relative quantity.
  • the competition law includes the following steps:
  • step (1') adding the sample to be tested and a predetermined amount of the marker-labeled novel coronavirus or its corresponding antigen to the solid-phase carrier obtained in step (1'), incubating, and comparing with the control sample;
  • Labeled virus monoclonal antibodies can be prepared by binding an anti-SARS-CoV-2 virus monoclonal antibody to a label.
  • Labels can be enzymes, colloidal metal particles, colored latex particles, fluorescent latex particles, luminescent substances, fluorescent substances, and the like.
  • the enzyme can be various enzymes used in enzyme-linked immunoassay (EIA), such as alkaline phosphatase, peroxidase, ⁇ -D-galactosidase, etc.; colloidal metal particles such as colloidal gold particles, colloidal Selenium particles, etc.
  • EIA enzyme-linked immunoassay
  • the binding method of the marker and the anti-SARS-CoV-2 virus monoclonal antibody can use a known method for generating covalent bonds or non-covalent bonds.
  • the binding methods include: glutaraldehyde method, periodate method, maleimide method, pyridyl ⁇ disulfide method, methods using various cross-linking agents, etc. (for example, refer to "Protein Nucleic Acid Enzymes", 1985, Separate volume No. 31, pages 37-45).
  • N-succinimidyl-4-maleimidobutyric acid N-succinimidyl-6-maleimide
  • Aminocaproic acid N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, etc.
  • the functional groups present in the antibody can be used depending on the use of functional groups.
  • functional groups such as thiol group, amino group, carboxyl group, and hydroxyl group can be introduced into the antibody according to conventional methods, and then the above-mentioned binding method can be used.
  • the functional group is combined with the label, thereby preparing a labeled anti-SARS-CoV-2 virus monoclonal antibody.
  • chromogenic substrates As the substrate, various chromogenic substrates, fluorescent substrates, luminescent substrates, and the like can be used corresponding to the enzymes of the labels and shown below.
  • fluorescent substrates As the substrate, various chromogenic substrates, fluorescent substrates, luminescent substrates, and the like can be used corresponding to the enzymes of the labels and shown below.
  • luminescent substrates As the substrate, various chromogenic substrates, fluorescent substrates, luminescent substrates, and the like can be used corresponding to the enzymes of the labels and shown below. E.g:
  • Chromogenic substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3′,5, 5'-tetramethylbenzidine (TMB), diaminobenzidine (DAB) for peroxidase; 5-bromo-4-chloro-3-indolyl phosphate (BCIP), p-nitrophenyl phosphate (p-NPP), 5-bromo-4-chloro-3-indolyl sodium phosphate (BCIP ⁇ Na) was used for alkaline phosphatase.
  • Fluorescent substrates 4-methylumbelliferyl phenyl phosphate (4-MUP) for alkaline phosphatase; 4-methylumbelliferyl phenyl- ⁇ -D-galactoside (4MUG) for in ⁇ -D-galactosidase.
  • 4-MUP 4-methylumbelliferyl phenyl phosphate
  • 4-MUG 4-methylumbelliferyl phenyl- ⁇ -D-galactoside
  • Luminescent substrate 3-(2′-Spiroadamantane)-4-methoxy-4-(3′-phosphoryloxy)phenyl-1,2-dioxetane ⁇ 2 Sodium salt (AMPPD) for alkaline phosphatase; 3-(2'-spiroadamantane)-4-methoxy-4-(3'- ⁇ -D-galactopyranosyl)phenyl-1, 2-Dioxetane (AMGPD) was used for ⁇ -D-galactosidase; luminol, isoluminol obtained in combination with hydrogen peroxide were used for peroxidase.
  • AMPPD AMPPD
  • AMGPD 2-Dioxetane
  • a double-antibody sandwich ELISA detection kit is provided, and a method for using the kit to detect the presence of SARS-CoV-2 virus in a sample is provided, including:
  • a monoclonal antibody or its antigen-binding fragment that can bind to the N protein of SARS-CoV-2 virus and coat the solid-phase carrier with the first antibody;
  • step (1) adding the sample to be tested and the control sample (or standard) to the solid phase carrier coated in step (1) and incubating under appropriate conditions;
  • the biotinylated secondary antibody is combined with the enzyme-labeled avidin or streptavidin, and then the enzyme substrate is added for color development, and then Use a microplate reader to determine the corresponding absorbance value;
  • the measured absorbance value is compared with the absorbance of the known quantity standard measured in parallel to determine the presence and relative amount of SARS-CoV-2 virus in the sample.
  • the sample to be tested is a biological sample
  • the sample is plasma, serum and whole blood
  • the solid phase carrier is a microtiter plate
  • the avidin or streptavidin is expressed in horseradish Peroxidase label
  • the enzyme substrate is TMB.
  • the present invention also provides a novel coronavirus detection kit, the novel coronavirus detection kit includes the antibody or its antigen-binding fragment as described in the first aspect.
  • Diagnosis of SARS-CoV-2 virus infection can be performed by assaying various biological samples and/or environmental samples from humans or animals using the monoclonal antibodies of the present invention against the nucleoprotein of SARS-CoV-2 virus.
  • the monoclonal antibody of the present invention the nucleocoat of SARS-CoV-2 virus in various body fluids, cells, tissues, etc. and/or environmental samples from humans or animals can be directly determined by immunochemical methods or immunohistochemical methods. shell protein.
  • the novel coronavirus detection kit further includes a solid-phase carrier, and the antibody or its antigen-binding fragment is embedded in the solid-phase carrier.
  • the kit is a kit for detection using a competitive method, wherein the immunoassay reagent can be prepared, for example, into a certain amount of viral antigens labeled with enzymes, colloidal metal particles, colored latex particles, luminescent substances, fluorescent substances, radioactive substances, and the like.
  • a competitive reaction can be carried out with, for example, a sample containing a certain amount of the monoclonal antibody of the present invention, the above-mentioned labeled viral antigen and the antigen to be determined, and the amount of the labeled viral antigen bound or unbound to the antibody is relative.
  • Immunoassays are performed by measuring the amount of antigen in a sample for quantification.
  • the novel coronavirus detection kit further includes a solid-phase carrier and a second antibody, the monoclonal antibody or its antigen-binding fragment is bound to the solid-phase carrier as a first antibody, and the second antibody carries a label with primary antibody combination.
  • This detection kit is a kit for detection by a sandwich method.
  • a monoclonal antibody can be used as a solid-phase antibody and a labeled antibody (for example, when the antigen is a polymer), but it is usually preferred Two or more antibodies are used that can recognize two different epitopes of the antigen to be assayed, respectively.
  • any solid-phase antibody and labeled antibody can be selected and used in combination from two or more monoclonal antibodies.
  • the following reagents can be used.
  • two types of monoclonal antibodies of the present invention can be prepared, one of which is the labeled antibody and the other is a solid-phase antibody bound to a solid-phase carrier.
  • a sample containing an antigen to be measured is reacted with the solid-phase antibody, and then a labeled antibody (secondary antibody) is reacted with the antigen captured on the solid-phase antibody to detect the presence of the label bound to the insoluble carrier. or activity, immunoassays can be performed.
  • a sample containing the antigen to be assayed is reacted with a solid-phase antibody, and then a labeled antibody (secondary antibody) is reacted with the antigen captured on the solid-phase antibody, and the presence of the label bound to the insoluble carrier or the Activity, ie, quantification of the amount of antigen to be assayed by the amount of labeled antibody, allows immunoassays to be performed.
  • the second antibody is selected from any one of the antibodies of the present invention that can cooperate with the first antibody, that is, a monoclonal antibody or an antigen-binding fragment thereof that can be paired for use.
  • antibodies that "can be matched" and “can be used in pairs” mean that the antibodies are directed against different epitopes in the N protein antigen of the novel coronavirus, and are different from the N protein antigen. Antibody combinations that do not interfere or antagonize each other in their binding.
  • the second antibody can also be other polyclonal antibody.
  • the first antibody and the second antibody are selected from any one of the following combinations:
  • Combination I the first antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb7, and the second antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb6;
  • the first antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb7
  • the second antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb8;
  • Combination III the first antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb7, and the second antibody is a monoclonal antibody or its antigen-binding fragment secreted by hybridoma cell line #mAb9.
  • the label includes any one or a combination of at least two of radioisotopes, enzymes, enzyme substrates, phosphorescent substances, fluorescent substances, biotin or coloring substances.
  • the radioisotope includes any one or a combination of at least two of125I , 3H , 14C or32P .
  • the enzyme comprises any one or a combination of at least two of alkaline phosphatase, horseradish peroxidase, beta-galactosidase, urease or glucose oxidase.
  • the fluorescent substance includes any one or a combination of at least two of fluorescein derivatives, rhodamine derivatives, rare earth elements or rare earth element complexes.
  • the phosphorescent substance comprises acridine ester and/or isoluminol.
  • the solid phase carrier comprises: any one or at least two of nitrocellulose membrane, latex particles, magnetic particles, colloidal gold, glass, fiberglass, polymer or fiber optical sensor combination.
  • the antibody can be used as at least one of a solid-phase antibody and a labeled antibody to prepare a SARS-CoV-2 virus immunoassay reagent.
  • solid phases used in conventional immunoassays can be used as the solid phase bound to the above-mentioned monoclonal antibodies, such as ELISA plates, latex, gelatin particles, magnetic particles, polystyrene, glass and other solid phase beads. Insoluble carriers such as liquid bases, etc.
  • labeled antibodies can be prepared by labeling antibodies with enzymes, colloidal metal particles, colored latex particles, luminescent substances, fluorescent substances, radioactive substances, and the like.
  • reagents for use in enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, and the like can be prepared.
  • These assay reagents are reagents for measuring the target antigen in a sample by a sandwich method or a competitive binding assay.
  • Another aspect of the present invention provides the application of the above immunoassay reagent in diagnosing diseases caused by SARS-CoV-2 virus infection.
  • the disease is novel coronavirus pneumonia (COVID-19).
  • the monoclonal antibody of the present invention or its antigen-binding fragment is secreted by four hybridoma cell lines CCTCC NO: C2020236, CCTCC NO: C2020237, CCTCC NO: C2020238 or CCTCC NO: C2020239, the monoclonal antibody or its antigen
  • the binding fragment can specifically recognize the N protein of SARS-CoV-2 virus with an affinity up to pM level, with very high sensitivity and specificity;
  • the present invention also provides a SARS-CoV-2 virus detection kit, which can detect the presence of SARS-CoV-2 virus by using a variety of immunoassays, especially the sandwich method and the competition method.
  • the detection kit for SARS-CoV-2 virus because the monoclonal antibody or its antigen-binding fragment used has high affinity and specificity, the obtained detection result is relatively accurate; wherein, the present invention also provides a double-antibody sandwich ELISA detection
  • the kit is constructed with the best paired three groups of antibodies (mAb7/Bio-mAb6, mAb7/Bio-mAb8 and mAb7/Bio-mAb9), the detection limit is 12.5pg/mL, and its repeatability is good;
  • the double-antibody sandwich ELISA immunoassay method constructed in the present invention is a very sensitive virus detection technology, which is more specific than other serological methods when detecting most viruses, and has high ELISA sensitivity and easy operation.
  • the development of supporting instruments and equipment standardizes and automates operating procedures, thereby further improving stability. Therefore, the novel coronavirus antigen detection kit provided by the present invention can detect the nucleocapsid protein in the early stage of infection of the patient, solves the problem of rapid clinical diagnosis of the novel coronavirus infection, and has high accuracy, It provides a rapid and accurate diagnostic method for clinical detection of the disease.
  • FIG. 1 is a graph showing the results of measuring the titer of mouse immune serum in Example 1.
  • FIG. 1 is a graph showing the results of measuring the titer of mouse immune serum in Example 1.
  • Fig. 2 is the cross-reactivity determination result curve diagram of the antibody against the new coronavirus nucleocapsid protein and SARS virus in embodiment 3.
  • FIG. 3 is a graph showing the results of cross-reactivity determination between the antibody against the new coronavirus nucleocapsid protein and MERS virus in Example 3.
  • FIG. 4 is a graph showing the results of the double-antibody sandwich ELISA test of the capture antibody mAb7 and the labeled antibody Bio-mAb6 in Example 5.
  • FIG. 4 is a graph showing the results of the double-antibody sandwich ELISA test of the capture antibody mAb7 and the labeled antibody Bio-mAb6 in Example 5.
  • FIG. 5 is a graph showing the results of the double-antibody sandwich ELISA test of the capture antibody mAb7 and the labeled antibody Bio-mAb8 in Example 5.
  • FIG. 5 is a graph showing the results of the double-antibody sandwich ELISA test of the capture antibody mAb7 and the labeled antibody Bio-mAb8 in Example 5.
  • FIG. 6 is a graph showing the results of the double-antibody sandwich ELISA test of the capture antibody mAb7 and the labeled antibody Bio-mAb9 in Example 5.
  • FIG. 6 is a graph showing the results of the double-antibody sandwich ELISA test of the capture antibody mAb7 and the labeled antibody Bio-mAb9 in Example 5.
  • This embodiment is used to obtain hybridoma cell lines and prepare monoclonal antibodies, and the specific steps are as follows:
  • SARS-CoV-2 N protein antigen (Sino Biological, 40588-V08B) was fully emulsified with complete Freund's adjuvant, male Balb/C mice (Shanghai Slack Laboratory Animal Co., Ltd.) were immunized by multi-point immunization, 50 ⁇ g / only, the immunization cycle is once every three weeks;
  • 96-well polystyrene microplates were coated with 0.1 ⁇ g/mL recombinant SARS-CoV-2 N protein, 100 ⁇ L/well, overnight at 4°C; the next day, the mass fraction was 1% Blocking solution of bovine serum albumin (BSA, Sigma), 0.3 mg/well, overnight at 4°C, the coated strips were treated with 10 mM PBS buffer containing 10% sucrose the next day, vacuum-dried and then vacuum-packed in aluminum film bags Store at 4°C for the determination of mouse immune serum titer;
  • BSA bovine serum albumin
  • Orbital blood was collected 10 days after the third immunization, and the collected mouse serum was diluted with 1:100 in 10mM PBS containing 1.5% BSA for a series of concentration gradients, and then added to a 96-well plate, 100 ⁇ L/well, 30 minutes at 37°C, wash the plate 3 times with 10mM PBS containing 0.1% Tween-20 washing solution, add a 1:5000-fold dilution of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (Jackson Laboratory Inc, Cat. No. 115- 035-07), 100 ⁇ L/well, 30 minutes at 37°C;
  • HRP horseradish peroxidase
  • the microplate reader After washing the plate as above, add citrate buffer containing 0.05% (w/v) TMB and 0.06% (w/v) hydrogen peroxide pH5.0, 100 ⁇ L/well, and protect from light at room temperature for 10 minutes, and stop by adding 0.2M H 2 SO 4 Reaction, 100 ⁇ L/well, the microplate reader reads the absorption value at the dual wavelengths of 450/620 nm, with the mouse serum before immunization as the negative control, and the ratio of the measured value to the control value ⁇ 2.0 is positive to judge the titer of the immune serum ;
  • mice M51, M52, M53 and M54 were all higher.
  • mice with the highest antibody titer were boosted once; three days later, the mice were sacrificed, and the mouse spleen was taken under aseptic conditions to prepare a spleen cell suspension, which was then mixed with 2 in a ratio of 1:1.
  • ⁇ 10 8 mouse myeloma Sp2/0 cell lines in logarithmic growth phase were mixed and then fused in a solution containing 50% polyethylene glycol (molecular weight 1450) and 5% dimethyl sulfoxide (DMSO) ;
  • Iscove's medium containing 10% fetal bovine serum, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 0.1 mM hypoxanthine, 0.4 ⁇ M aminopterin, and 16 ⁇ M thymidine
  • Iscove's medium containing 10% fetal bovine serum, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 0.1 mM hypoxanthine, 0.4 ⁇ M aminopterin, and 16 ⁇ M thymidine
  • screening medium containing hypoxanthine and aminopterin-thymidine (HAT, Sigma) to each well, and then use this screening medium to change the culture medium every 3 days until the colony. cell formation.
  • HAT hypoxanthine and aminopterin-thymidine
  • the hybridoma cell culture supernatant was added to the ELISA plate pre-coated with antigen, 100 ⁇ L/well, the SP2/0 cell culture supernatant was used as a negative control, and the immune polyantibody serum was used as a positive control, one hour at room temperature; PBST washed 3 times; Add working concentration of HRP-labeled goat anti-mouse IgG antibody (Jackson Laboratory Inc, Cat. No. 115-035-07), 100 ⁇ L/well, 30 minutes at 37°C, wash the plate as above, add substrate TMB, 100 ⁇ L/well, avoid at room temperature.
  • HRP-labeled goat anti-mouse IgG antibody Jackson Laboratory Inc, Cat. No. 115-035-07
  • the screened strong positive cell clones were subcloned 2 to 3 times by the limiting dilution method, and a total of 6 hybridoma cell lines that stably secreted antibodies were obtained, named #mAb6, #mAb7, #mAb8, #mAb9, # mAb10 and #mAb11, and 4 of them were bio-deposited, #mAb6, #mAb7, #mAb8 and #mAb9 correspond to the deposit numbers CCTCC NO: C2020236, CCTCC NO: C2020237, CCTCC NO: C2020238 and CCTCC NO: C2020239 of hybridoma cell lines.
  • the positive hybridoma cell clones #mAb6, #mAb7, #mAb8, #mAb9, #mAb10 and #mAb11 obtained from the above screening were cultured in RPMI 1640 medium supplemented with 10% FCS.
  • the medium was replaced with serum-free medium. After incubation, centrifuge and collect the culture supernatant, then use Protein G affinity chromatography column to purify the antibody, dialyze the monoclonal antibody eluate with 150mM NaCl, and filter sterilize the dialyzed solution through a 0.2 ⁇ m filter to obtain purified Antibody samples.
  • Biotin labeling NN-hydroxysuccinimide ester (NHS) is the most common biotin labeling reagent, NHS-activated biotin can react with primary amine groups (-NH 2 ) in alkaline buffer to form stable amide bond. Proteins (such as antibodies) often have many primary amine groups and thus can be targeted for biotin labeling;
  • biotin derivative NHS-LC-Biotin (Thermo Scientific, Item No. 21435) was used for antibody labeling, and the monoclonal antibody was labeled with biotin according to the method described in the manual of Thermo Scientific EZ-Link Biotin Labeling Kit to obtain Biotinylated antibodies Bio-mAb6, Bio-mAb7, Bio-mAb8, Bio-mAb9, Bio-mAb10 and Bio-mAb11.
  • the monoclonal antibody of the present invention can also be labeled by other methods known in the art, such as HRP (horseradish peroxidase) labeling, and the specific method is as follows:
  • Monoclonal hybridoma cells produce antibodies against only one epitope of the antigen, and the "double antibody sandwich method" is used to screen the best paired solid-phase antibody and labeled antibody.
  • a 6 ⁇ 6 matrix was used for pairwise screening of antibodies, and the above 6 monoclonal antibodies were coated as capture antibodies, respectively, and the 6 biotin-labeled monoclonal antibodies Bio-mAb6, Bio-mAb7, Bio-mAb8, Bio-mAb9, Bio-mAb10, and Bio-mAb11 were paired for rapid screening of captured and labeled monoclonal antibody pairs in sandwich ELISA.
  • the binding affinity constants of purified mouse monoclonal antibodies mAb6, mAb7, mAb8, mAb9 and the N protein of 2019-nCoV were determined by biofilm interferometry (BLI).
  • the assay was performed using the ForteBio Octet RED&QK platform of PALL Company, and the method was referred to the instruction manual of the platform.
  • the biotinylated SARS-CoV-2 N protein was immobilized on the surface of the SA sensor, and the aforementioned monoclonal antibody against SARS-CoV-2 N protein was used as the analyte.
  • the data were processed and fitted with a 1:1 binding model of the analysis software.
  • the fitted data basically overlapped with the experimental data to obtain the association and dissociation rate constants Ka and K d . Divide Ka by K d to obtain the equilibrium dissociation constant K D (see Table 1).
  • mice mAb mAb7 had the highest affinity, while the affinity of mAb6, mAb8 and mAb9 were comparable, and the K D values were all lower than the pM level.
  • Example 4 Establishment of a double-antibody sandwich ELISA kit for detecting SARS-CoV-2 antigen with anti-SARS-CoV-2 N protein monoclonal antibody
  • the washing solution is PBS-Tween20, pH 7.4 (PBST); the blocking solution is PBST containing 1.5% bovine serum albumin (BSA); the standard and test sample dilution solution is PBST containing 1.5% BSA; biotin-labeled antibody
  • the diluent is PBST containing 1.5% BSA; Streptavidin-HRP (Thermo Scientific, Lot: 21130) is prepared into a working solution with PBST containing 1.5% BSA according to the method of its matching instructions.
  • the detection method is as follows:
  • the microplate reader reads the absorption value at the dual wavelengths of 450/620nm, draws a standard curve according to the measured OD value of the sample and the concentration of the standard substance, and obtains a linear regression equation.
  • the content of SARS-CoV-2 N protein in the sample can be obtained by substituting the OD value of the tested sample.
  • Figure 4 in which Figure 4 is mAb7/Bio-mAb6, Figure 5 is mAb7/Bio-mAb8, Figure 6 is mAb7/Bio-mAb9, with the increase of SARS-CoV-2 N protein (NP) concentration, The OD value also increased.
  • NP concentration range was 1-400 ng/mL
  • the OD value measured by the established sandwich method was linearly positively correlated with the NP concentration (R 2 ⁇ 0.988).
  • the detection limits of the three best paired antibodies screened above were all as low as 12.5 pg/mL.
  • the reaction of the ELISA kit composed of the three antibody pairs screened above with several other serum substances was measured, and the specificity of the reaction was observed.
  • the detection of the reference substance/standard substance is carried out.
  • the detection limit was 12.5pg/mL, and the condition satisfies the OD value>3 times SD of the OD value of the blank control, and the OD value>2 times the OD value of the blank control. It shows that the method of the present invention has good sensitivity.
  • the kit has good reproducibility for the determination of the N protein of 2019-nCoV.
  • the above double-antibody sandwich enzyme-linked immunosorbent assay can be used to detect the SARS-CoV-2 N protein antigen in human or animal serum or plasma samples for early diagnosis of SARS-CoV-2 infection to achieve early detection, early isolation, and avoidance of purpose of dissemination.
  • the immunofluorescence method is used to detect viral antigens in biological samples and/or environmental samples infected with SARS-CoV-2, which can also be used to identify the existence of novel coronaviruses.

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Abstract

La présente invention concerne un anticorps ou un fragment de liaison à l'antigène de celui-ci pour une protéine nucléocapsidique du nouveau coronavirus, et son utilisation. L'anticorps est choisi parmi l'un quelconque d'un anticorps mAb6, un anticorps mAb7, un anticorps mAb8 et un anticorps mAb9 ; et l'anticorps est sécrété par des cellules d'hybridome ayant les numéros d'accession CCTCC NO : C2020236, CCTCC NO : C2020237, CCTCC NO : C2020238 ou CCTCC NO : C2020239. La présence d'un nouveau coronavirus ou d'un antigène de celui-ci dans un échantillon environnemental et/ou un échantillon biologique peut être détectée à l'aide de l'anticorps. De plus, la présente invention concerne également un kit de détection du nouveau coronavirus préparé à l'aide de l'anticorps, capable de détecter la protéine nucléocapsidique dans l'étape précoce d'infection, ce qui permet de fournir un moyen pour effectuer la détection clinique rapide et précise d'un nouveau coronavirus.
PCT/CN2022/079557 2021-03-15 2022-03-07 Anticorps ou fragment de liaison à l'antigène de celui-ci pour protéine nucléocapsidique du nouveau coronavirus, et son utilisation WO2022193980A1 (fr)

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CN114409767A (zh) * 2021-12-08 2022-04-29 广东菲鹏生物有限公司 一种鉴别新冠突变型抗原的抗体、试剂及方法
CN114574449B (zh) * 2022-04-14 2023-07-18 青岛硕景生物科技有限公司 一株分泌抗新型冠状病毒n蛋白单克隆抗体的杂交瘤细胞株及其应用
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