WO2023036152A1 - 一种SARS-Cov-2检测方法和试剂盒 - Google Patents

一种SARS-Cov-2检测方法和试剂盒 Download PDF

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WO2023036152A1
WO2023036152A1 PCT/CN2022/117381 CN2022117381W WO2023036152A1 WO 2023036152 A1 WO2023036152 A1 WO 2023036152A1 CN 2022117381 W CN2022117381 W CN 2022117381W WO 2023036152 A1 WO2023036152 A1 WO 2023036152A1
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antibody
seq
amino acid
antibodies
acid sequence
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PCT/CN2022/117381
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English (en)
French (fr)
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卢盛萍
李婷
刘丽萍
刘春艳
易嘉乐
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广东唯实生物技术有限公司
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Publication of WO2023036152A1 publication Critical patent/WO2023036152A1/zh

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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
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • 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
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the field of protein detection. Specifically, the present disclosure relates to detection methods and kits for SARS-Cov-2.
  • the main detection methods for the diagnosis of new coronary pneumonia include nucleic acid detection and antigen-antibody immunoassay; the nucleic acid detection method is mainly fluorescent RT-PCR technology, and the antigen-antibody immunoassay method is divided into detection of virus antibodies and detection of virus antigens. detection.
  • the antibody detection reagent is to detect the IgM or IgG antibody that is stimulated by the human body after the virus enters the human body in the serum.
  • the IgM antibody appears earlier and the IgG antibody appears later.
  • Viral antigen detection is mainly to detect some proteins on the surface of the virus such as S protein. How to find specific antigenic epitope peptides is crucial for the detection and development of antibody-based COVID-19 detection methods.
  • Nucleic acid detection has the characteristics of early diagnosis, high sensitivity and specificity, and is the "gold standard" for the diagnosis of new coronary pneumonia.
  • RNA extraction and testing equipment due to the high requirements for sample collection, RNA extraction and testing equipment, false negatives are prone to occur. Therefore, there is an urgent need for fast immunological detection methods as a supplement to nucleic acid detection.
  • Studies have shown that the collaborative detection of new crown total antibody and nucleic acid can significantly improve the sensitivity of early diagnosis of new crown virus infection, and can effectively make up for the shortcomings of false negative nucleic acid detection that easily lead to missed diagnosis.
  • the structural genome of SARS-Cov-2 is 29883bp, and the genome includes 14 ORF boxes.
  • the larger protein molecules include the following protein regions, N: nucleocapsid protein (referred to as N protein); S: Spike protein; M: membrane protein; HE: hemagglutinin esterase; E: envelope protein.
  • N protein nucleocapsid protein
  • S Spike protein
  • M membrane protein
  • HE hemagglutinin esterase
  • E envelope protein.
  • the N protein is also the most abundant viral structural protein in the infection process of the new coronavirus. After infection, human cells will produce strong immune reactivity, and the gene sequence is relatively conservative and stable. Compared with the S protein, the N protein has fewer mutations over time.
  • the present disclosure provides an antibody combination, comprising a first group of antibodies and a second group of antibodies, the first group of antibodies comprising antibody 1 selected from binding to the N protein amino acid fragment 44-180 of SARS-Cov-2, and the second group of antibodies
  • the group of antibodies includes antibody 2 selected from the amino acid fragment 44-180 of the N protein binding to SARS-Cov-2, and the antibody 1 and the antibody 2 can be the same antibody or different antibodies.
  • the first group of antibodies further includes at least one of antibody 3-antibody 6.
  • the second group of antibodies further includes at least one of antibody 3-antibody 6.
  • the first group of antibodies further includes antibody 4 or antibody 5
  • the second group of antibodies further includes antibody 6.
  • Antibody 3 binds to N protein amino acid fragment 50-107;
  • Antibody 4 binds to N protein amino acid fragment 1-43;
  • Antibody 5 binds to N protein amino acid fragment 248-361;
  • Antibody 6 binds amino acid fragment 74-105 of N protein.
  • the antibody has a reactive titer higher than 10 5 .
  • the first set of antibodies is used for labeling, and the second set of antibodies is used for coating; or the second set of antibodies is used for labeling, and the first set of antibodies is used for coating.
  • the antibody for labeling is labeled with a detectable label, such as metal particles, such as colloidal gold, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, radioactive labels, enzyme labels, such as radioactive Isotope, Fluorophore, Rhodamine, Luciferase, Luciferin, Horseradish Peroxidase, Alkaline Phosphatase, ⁇ -Galactosidase, Glucoamylase, Lysozyme, Carbohydrate Oxidase, Glucose Oxidase , galactose oxidase, glucose-6-phosphate dehydrogenase, biotin/avidin, spin labeling, phage labeling, e.g. acridinium ester labeling, e.g. addition of fluorescent labels via linkers such as biotin-avidin such as Acridine ester labeling.
  • a detectable label such as metal particles, such as
  • the antibody used for coating is attached to a solid phase, such as magnetic particles, latex particles, ELISA plates, microtiter plates, nitrocellulose membranes or microfluidic chips.
  • a solid phase such as magnetic particles, latex particles, ELISA plates, microtiter plates, nitrocellulose membranes or microfluidic chips.
  • the heavy chain complementarity determining region VH-CDR1 of the antibody 1 includes the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 13, and the heavy chain complementarity determining region VH-CDR2 includes the amino acid sequence shown in SEQ ID NO: 8.
  • the heavy chain complementarity determining region VH-CDR3 includes the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 14; the light chain complementarity determining region VL-CDR1 of the antibody 1 includes SEQ ID NO: 10
  • the light chain complementarity determining region VL-CDR2 includes the amino acid sequence shown in SEQ ID NO:11
  • the light chain complementarity determining region VL-CDR3 includes the amino acid sequence shown in SEQ ID NO:12.
  • the antibody 1 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16 and the antibody 1 light chain variable region includes the amino acid sequence shown in SEQ ID NO: 17 sequence.
  • the Antibody 1 heavy chain includes the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 19, and the Antibody 1 light chain includes the amino acid sequence shown in SEQ ID NO: 20.
  • the antibody 1 binds to an epitope 1 that is associated with a heavy chain variable region comprising SEQ ID NO: 15 or SEQ ID NO: 16 and a light chain variable region of SEQ ID NO: 17 or an antibody that binds to the same epitope as an antibody comprising a heavy chain of SEQ ID NO: 18 or SEQ ID NO: 19 and a light chain of SEQ ID NO: 20.
  • the heavy chain complementarity determining region VH-CDR1 of the antibody 2 includes the amino acid sequence shown in SEQ ID NO:21 or SEQ ID NO:27
  • the heavy chain complementarity determining region VH-CDR2 includes the amino acid sequence shown in SEQ ID NO:22.
  • the heavy chain complementarity determining region VH-CDR3 includes the amino acid sequence shown in SEQ ID NO:23; the light chain complementarity determining region VL-CDR1 of the antibody 2 includes SEQ ID NO:24 or SEQ ID NO:28
  • the light chain complementarity determining region VL-CDR2 includes the amino acid sequence shown in SEQ ID NO:25
  • the light chain complementarity determining region VL-CDR3 includes the amino acid sequence shown in SEQ ID NO:26.
  • the antibody 2 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 30, and the antibody 2 light chain variable region includes SEQ ID NO: 31 or SEQ ID NO : Amino acid sequence shown in 32.
  • the antibody 2 heavy chain includes the amino acid sequence shown in SEQ ID NO:33 or SEQ ID NO:34
  • the antibody 2 light chain includes the amino acid sequence shown in SEQ ID NO:35 or SEQ ID NO:36 sequence.
  • the antibody 2 binds to an epitope 2 that is associated with a heavy chain variable region comprising SEQ ID NO:29 or SEQ ID NO:30 and SEQ ID NO:31 or SEQ ID NO:32
  • the antibody of the light chain variable region of or binds to the same epitope as the antibody comprising the heavy chain of SEQ ID NO:33 or SEQ ID NO:34 and the light chain of SEQ ID NO:35 or SEQ ID NO:36.
  • the heavy chain complementarity determining region VH-CDR1 of the antibody 3 includes the amino acid sequence shown in SEQ ID NO:37, and the heavy chain complementarity determining region VH-CDR2 includes SEQ ID NO:38 or SEQ ID NO:43
  • the amino acid sequence shown, the heavy chain complementarity determining region VH-CDR3 includes the amino acid sequence shown in SEQ ID NO:39;
  • the light chain complementarity determining region VL-CDR1 of the antibody 3 includes the amino acid sequence shown in SEQ ID NO:40,
  • the light chain complementarity determining region VL-CDR2 includes the amino acid sequence shown in SEQ ID NO:41 or SEQ ID NO:44, and the light chain complementarity determining region VL-CDR3 includes the amino acid sequence shown in SEQ ID NO:42.
  • the antibody 3 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46, and the antibody 3 light chain variable region includes SEQ ID NO: 47 or SEQ ID NO : the amino acid sequence shown in 48.
  • the antibody 3 heavy chain includes the amino acid sequence shown in SEQ ID NO:49 or SEQ ID NO:50
  • the antibody 3 light chain includes the amino acid sequence shown in SEQ ID NO:51 or SEQ ID NO:52 sequence.
  • the antibody 3 binds to an epitope 3 that is associated with a heavy chain variable region comprising SEQ ID NO:45 or SEQ ID NO:46 and SEQ ID NO:47 or SEQ ID NO:48
  • the antibody of the light chain variable region of or binds to the same epitope as the antibody comprising the heavy chain of SEQ ID NO:49 or SEQ ID NO:50 and the light chain of SEQ ID NO:51 or SEQ ID NO:52.
  • the antibody 1 or the antibody 2 binds to a conformation-dependent epitope of the N protein of SARS-Cov-2.
  • the antibody 1 or the antibody 2 does not bind to any polypeptide consisting of 6 consecutive amino acids of the N protein of SARS-Cov-2.
  • the present disclosure also provides a SARS-Cov-2 antigen detection kit, including the above-mentioned antibody combination.
  • the present disclosure also provides a SARS-Cov-2 antigen detection kit, which is used to detect SARS-Cov-2 N protein in a sample from a subject, including the above-mentioned antibody combination.
  • the kit is an immunochromatographic test strip, an enzyme-linked immunosorbent reagent or a chemiluminescence reagent.
  • the sample includes healthy or pathological biological tissues, cells or body fluids, such as saliva, nasopharyngeal swabs.
  • the present disclosure provides a SARS-Cov-2 immunochromatographic test strip
  • the immunochromatographic test strip includes a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and an absorbent pad, and the nitrocellulose T lines and C lines are arranged on the plain film, the second group of antibodies is arranged on the binding pad, and the first group of antibodies is coated on the T lines; or the first group of antibodies is arranged on the binding pad, and the T lines are coated with was given a second set of antibodies.
  • the present disclosure also provides the application of the antibody combination in the preparation of a kit for detecting SARS-Cov-2.
  • the present disclosure also provides the use of the above-mentioned antibody combination, or the above-mentioned kit, or the immunochromatographic test strip for detecting SARS-Cov-2.
  • the present disclosure also provides a method for detecting SARS-Cov-2, comprising:
  • the present disclosure also provides a method for diagnosing a subject infected with SARS-Cov-2 or a disease related to SARS-Cov-2 infection, comprising:
  • the diseases associated with SARS-Cov-2 infection include respiratory symptoms, fever, cough, shortness of breath, dyspnea, pneumonia, severe acute respiratory syndrome, and renal failure.
  • antibody is used in the broadest sense, which may include full-length monoclonal antibodies, polyclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, so long as they exhibit the desired biological activity.
  • Antibody fragment includes a portion of a full-length antibody, optionally an antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab') 2 , Fd, Fv, dAb, complementarity determining region (CDR) fragments, single chain antibodies (eg, scFv), diabodies or domain antibodies.
  • CCD complementarity determining region
  • a complete or complete antibody comprises two heavy chains and two light chains; each heavy chain contains a variable region (VH) and constant region (CH); each light chain contains a variable region (VL) and a constant region (CL); the antibody has a "Y" shape, and the stem of Y is composed of two consists of the second and third constant regions of the heavy chain; each arm of Y includes the variable and first constant regions of a single heavy chain combined with the variable and constant regions of a single light chain; the light The variable region of the chain and the variable region of the heavy chain are responsible for antigen binding; the variable regions in both chains usually contain three hypervariable regions, called complementarity determining regions.
  • constant region refers to a relatively stable region near the C-terminal amino acid sequence in the light chain and heavy chain of an antibody molecule.
  • variable region refers to the region in the light chain and heavy chain of an antibody molecule, where the amino acid sequence near the N-terminus varies greatly.
  • aa refers to the amino acid at position ... of a peptide.
  • the term "subject” refers to a vertebrate, optionally a mammal, optionally a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro. It should be understood that the terminology used herein is for the purpose of describing the context of the disclosure such as the embodiments and examples only, and is not intended to limit the scope of the disclosure.
  • genotype refers to a hybrid cell resulting from the fusion of genotype-identical cells known as a homokaryon.
  • Some embodiments of the present disclosure provide an antibody combination, comprising a first group of antibodies and a second group of antibodies, the first group of antibodies comprising antibody 1 selected from binding to the amino acid fragment of the N protein at positions 44-180 of SARS-Cov-2, and the first group of antibodies
  • the second group of antibodies includes antibody 2 selected from the 44th-180th N protein amino acid sheet that binds SARS-Cov-2, and antibody 1 and antibody 2 can be the same antibody or different antibodies;
  • the first set of antibodies may also include at least one of Antibody 3, Antibody 4, Antibody 5, or Antibody 6.
  • the second set of antibodies may also include at least one of Antibody 3, Antibody 4, Antibody 5, or Antibody 6.
  • the first set of antibodies or the second set of antibodies further includes Antibody 3.
  • the first set of antibodies further includes Antibody 4 or Antibody 5
  • the second set of antibodies further includes Antibody 6.
  • Antibody 3 binds to an amino acid fragment of the N protein at positions 50-107;
  • Antibody 4 binds to the N protein amino acid fragment at positions 1-43;
  • Antibody 5 binds to the N protein amino acid fragment at positions 248-361;
  • Antibody 6 binds the N protein amino acid fragment at positions 74-105.
  • the kit of the present disclosure includes a SARS-Cov-2 detection reagent card (test strip).
  • SARS-Cov-2 detection reagent card test strip
  • colloidal gold lateral flow chromatography technology can be used to label antibodies on detectable markers such as colloidal gold, and the principle of double-antibody sandwich method can be used to detect whether a subject is infected with the new coronavirus SARS-Cov-2.
  • the methods and kits disclosed herein increase the detection rate and can overcome the problem of missed detection of mutant strains.
  • the present disclosure provides a detection method and a kit, which can improve the detection rate and reduce the Risk of missed detection.
  • the present disclosure may be performed by or include reagents for performing colloidal gold immunochromatography.
  • immunochromatography can be achieved by a double-antibody sandwich method with a test strip.
  • the reagents and devices for the double-antibody sandwich method are not particularly limited, and any appropriate reagents and/or devices can be used.
  • an immunochromatographic test strip can include a sample pad, a conjugate pad, an NC membrane, and an absorbent pad.
  • the colloidal gold-antibody complex with multiple epitopes can be coated on the binding pad; two lines can be coated on the NC membrane, such as T line and C line respectively; T lines can be mixed and coated Antibodies with multiple epitopes can capture the colloidal gold-antibody complex on the binding pad to form a double-antibody sandwich complex, thereby immobilizing the colloidal gold on the T line; the C line can be coated to capture free colloidal gold- Antibody to antibody complex.
  • the sample to be tested is added to the sample port of the detection reagent card, and under the action of the lateral capillary, the sample to be tested first passes through the binding pad, and then undergoes specific immunity with different colloidal gold-antibody complexes on the binding pad.
  • each combined to form an antigen-antibody colloidal gold complex which was fixed in the T line.
  • the free colloidal gold-antibody complex passes through the C line, it can specifically immunocombined with the C line antibody, and thus be fixed in the C line.
  • the color intensity of the control band (C) is inversely proportional to that of the test band (T), allowing correction for individual sample differences.
  • the present disclosure can use a multi-epitope antibody to recognize multiple epitopes on an antigen, reduce the risk of missed detection, and increase the detection rate.
  • the present disclosure can use the combination of antigen epitopes to cover a large area, use multi-epitope antibody pairing to recognize multiple epitopes on the antigen, reduce the risk of missed detection, and increase the detection rate.
  • the labeled antibody of the present disclosure can be mixed with the 44-180aa, 50-107aa or 74-105aa antibody epitope of the N protein of SARS-Cov-2, and the coated antibody can be 44-180aa, 1 Antibody epitopes of -43aa or 248-361aa were mixed.
  • N protein epitopes or fragments is carried out according to the corresponding position of SEQ ID NO:1.
  • the present disclosure provides a detection method and kit for SARS-Cov-2.
  • the disclosed kit has improved sensitivity, increased detection rate, and overcomes the problem of missed detection of mutant strains.
  • the first panel of antibodies and/or the second panel of antibodies of the present disclosure can be monoclonal antibodies.
  • the antibodies of the present disclosure can be prepared using methods known in the art.
  • antibodies of the present disclosure can be prepared by immunizing an animal with an antigen comprising an amino acid fragment described herein.
  • an antigen comprising an amino acid fragment described herein.
  • larger antigenic compounds including but not limited to BSA (bovine serum albumin), ovalbumin, KLH (keyhole limpet hemocyanin), etc.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • the immunogenic portion can include various proteins or polypeptides, which can function as immunogen carriers. These types of polypeptides include albumin, serum protein, globulin, crystallin, lipoprotein and/or fragments thereof.
  • synthetic polypeptides can be used.
  • the immunogenic moiety or carrier may also be a particle or microparticle.
  • Immunogenic particles can be organic or inorganic, porous or non-porous. Immunogenic particles can be biological materials such as cells and microorganisms, and can also contain organic and inorganic polymers, liposomes, latex, phospholipid vesicles, liposomes, cationic liposomes, anionic liposomes, lipoproteins, lipid polymer etc. Accordingly, the epitope peptides described in the present disclosure can be used to generate antibodies with affinity for the N protein.
  • the effects of the antibodies of the disclosure can be tested using any suitable in vitro assay, cell-based assay, in vivo assay, animal model, etc.
  • the assay may include, for example, ELISA (Enzyme-Linked Immunosorbent Assay enzyme-linked immunosorbent assay), FACS (Fluorescence-Activated Cell Sorting fluorescence-activated cell sorting) binding assay, Biacore (Biomolecule Interaction Analysis core technique ), competitive binding assays, etc.
  • an antibody of the present disclosure (or an antigen-binding fragment thereof) can have an EC50 value for binding to an antigen, eg, in ELISA or FACS, of eg 1 ⁇ M-1 pM, eg 1 nM-1 pM, eg 100 pM-1 pM.
  • the antibody of the present disclosure is used to detect the N protein antigen with a reaction titer higher than 10 5 , including for example higher than 10 6 , for example higher than 10 7 .
  • the first group of antibodies and/or the second group of antibodies in the kit can be used as a coating antibody (or called a capture antibody) and a labeling antibody, respectively, for example, the first group of antibodies is a coating antibody, and the second group of antibodies is a coating antibody.
  • the second set of antibodies is labeled antibodies, or the first set of antibodies is labeled antibodies and the second set of antibodies is coated antibodies.
  • the first group of antibodies is labeled antibodies
  • the second group of antibodies is coated antibodies (or called capture antibodies).
  • the coating antibody is bound to a solid phase.
  • the coating antibody can be used to coat a solid support.
  • the solid phase support is not particularly limited, and it can be, for example, magnetic particles, latex particles, microtiter plates, nitrocellulose membranes or microfluidic chips, and optionally the kit includes or is prepared to Chromatographic test strips or test cards.
  • the labeled antibody is labeled with a detectable label, eg, with a fluorescent label, eg, via a linker such as biotin-avidin with a fluorescent label.
  • Some embodiments of the present disclosure provide a SARS-Cov-2 immunochromatographic test strip
  • the immunochromatographic test strip includes a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and an absorbent pad, and the nitrocellulose membrane T lines and C lines are provided, and the above-mentioned second group of antibodies is provided on the binding pad, wherein the above-mentioned first group of antibodies is coated on the T line; or the above-mentioned first group of antibodies is provided on the binding pad.
  • the detectable labels used in the present disclosure are not particularly limited.
  • the labels may include, but are not limited to, metal particles such as colloidal gold, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels, as well as indirect labels such as enzymes or ligands, For example, indirect detection through enzymatic reactions or molecular interactions.
  • exemplary labels include, but are not limited to, radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase (HRP), alkaline phosphatase, ⁇ - Galactosidase, glucoamylase, lysozyme, carbohydrate oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin labeling , phage markers, and more.
  • HRP horseradish peroxidase
  • alkaline phosphatase e.g., ⁇ - Galactosidase
  • glucoamylase glucoamylase
  • lysozyme carbohydrate oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphat
  • the heavy chain complementarity determining region VH-CDR1 of Antibody 1 includes the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 13, and the heavy chain complementarity determining region VH-CDR2 includes the amino acid sequence shown in SEQ ID NO: 8
  • the amino acid sequence shown, the heavy chain complementarity determining region VH-CDR3 includes the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:14;
  • the light chain complementarity determining region VL-CDR1 of antibody 1 includes the amino acid sequence shown in SEQ ID NO:10
  • the amino acid sequence of the light chain complementarity determining region VL-CDR2 includes the amino acid sequence shown in SEQ ID NO:11, and the light chain complementarity determining region VL-CDR3 includes the amino acid sequence shown in SEQ ID NO:12.
  • variable region of the heavy chain of Antibody 1 includes the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16 and the variable region of the light chain of Antibody 1 includes the amino acid sequence shown in SEQ ID NO: 17.
  • the heavy chain of Antibody 1 includes the amino acid sequence shown in SEQ ID NO:18 or SEQ ID NO:19, and the light chain of Antibody 1 includes the amino acid sequence shown in SEQ ID NO:20.
  • Antibody 1 binds Epitope 1 that binds to an antibody comprising a heavy chain variable region of SEQ ID NO: 15 or SEQ ID NO: 16 and a light chain variable region of SEQ ID NO: 17 Or the same epitope as the antibody that comprises the heavy chain of SEQ ID NO: 18 or SEQ ID NO: 19 and the light chain of SEQ ID NO: 20 binds, Antibody 1 is identical to SARS-Cov- shown in SEQ ID NO: 1 Binding to the amino acid region between 44 and 180 in the N protein amino acid sequence of 2.
  • the heavy chain complementarity determining region VH-CDR1 of antibody 2 includes the amino acid sequence shown in SEQ ID NO:21 or SEQ ID NO:27, and the heavy chain complementarity determining region VH-CDR2 includes the amino acid sequence shown in SEQ ID NO:22
  • the light chain complementarity determining region VL-CDR1 of antibody 2 includes the amino acid sequence shown in SEQ ID NO:24 or SEQ ID NO:28
  • the amino acid sequence of the light chain complementarity determining region VL-CDR2 includes the amino acid sequence shown in SEQ ID NO:25, and the light chain complementarity determining region VL-CDR3 includes the amino acid sequence shown in SEQ ID NO:26.
  • variable region of the heavy chain of Antibody 2 comprises the amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:30, and the variable region of the light chain of Antibody 2 comprises SEQ ID NO:31 or SEQ ID NO:32 Amino acid sequence shown.
  • the heavy chain of Antibody 2 includes the amino acid sequence shown in SEQ ID NO:33 or SEQ ID NO:34, and the light chain of Antibody 2 includes the amino acid sequence shown in SEQ ID NO:35 or SEQ ID NO:36.
  • Antibody 2 binds to Epitope 2 that binds to a heavy chain variable region comprising SEQ ID NO:29 or SEQ ID NO:30 and a light chain variable region of SEQ ID NO:31 or SEQ ID NO:32
  • the antibody of chain variable region or with the antibody that comprises the heavy chain of SEQ ID NO:33 or SEQ ID NO:34 and the antibody of the light chain of SEQ ID NO:35 or SEQ ID NO:36 binds the same epitope, antibody 2 and In the N protein amino acid sequence of SARS-Cov-2 shown in SEQ ID NO: 1, the amino acid region between 44 and 180 is combined.
  • the heavy chain complementarity determining region VH-CDR1 of antibody 3 comprises the amino acid sequence shown in SEQ ID NO:37
  • the heavy chain complementarity determining region VH-CDR2 comprises SEQ ID NO:38 or SEQ ID NO:43
  • the amino acid sequence shown in the heavy chain complementarity determining region VH-CDR3 includes the amino acid sequence shown in SEQ ID NO:39
  • the light chain complementarity determining region VL-CDR1 of antibody 3 includes the amino acid sequence shown in SEQ ID NO:40
  • the complementarity determining region VL-CDR2 includes the amino acid sequence shown in SEQ ID NO:41 or SEQ ID NO:44
  • the light chain complementarity determining region VL-CDR3 includes the amino acid sequence shown in SEQ ID NO:42.
  • the antibody 3 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46, and the antibody 3 light chain variable region includes SEQ ID NO: 47 or SEQ ID NO: 48 Amino acid sequence shown.
  • the heavy chain of Antibody 3 includes the amino acid sequence shown in SEQ ID NO:49 or SEQ ID NO:50, and the light chain of Antibody 3 includes the amino acid sequence shown in SEQ ID NO:51 or SEQ ID NO:52.
  • Antibody 3 binds to Epitope 3 that binds to a heavy chain variable region comprising SEQ ID NO:45 or SEQ ID NO:46 and a light chain variable region of SEQ ID NO:47 or SEQ ID NO:48
  • the antibody of the chain variable region or the same epitope as the antibody binding to the heavy chain of SEQ ID NO:49 or SEQ ID NO:50 and the light chain of SEQ ID NO:51 or SEQ ID NO:52, Antibody 3 and SEQ ID NO:52 Binding to the amino acid region between positions 50 and 107 in the N protein amino acid sequence of SARS-Cov-2 shown in ID NO:1.
  • Antibody 1 or Antibody 2 binds a conformation-dependent epitope of the N protein of SARS-Cov-2.
  • the conformational epitope consists of discrete portions of the amino acid sequence of the antigen.
  • Antibody 1 and Antibody 2 are analyzed by PepScan, and the synthetic peptide consists of 6 consecutive amino acids, each of which is shifted by 1 amino acid, eg: 44-49, 45-50 and so on, up to 175-180. Analyze the binding state of the synthetic peptide and the antibody, specifically, use the HRP-labeled secondary antibody to detect the binding state of the synthetic peptide and the antibody. The detection results showed that both antibody 1 and antibody 2 were conformational epitopes.
  • kits of the present disclosure include reagents suitable for performing an immunoassay.
  • the kits of the present disclosure can be used to perform immunoassays, such as ELISA, indirect immunofluorescence assay IFA, radioimmunoassay RIA, and other non-enzyme-linked antibody binding assays or methods.
  • the N protein antibody can be coated on a solid phase such as magnetic beads, the N protein antigen in the sample can be captured, and then the labeled antibody can be used to bind the antigen bound to the magnetic beads again , read the result after adding the luminescent substrate.
  • the N protein antibodies of the present disclosure can be used to coat a solid phase such as magnetic beads or as a second set of antibodies for labeling.
  • the antibody or antigen-binding fragment thereof is immobilized on a surface, eg, a solid support, eg, a plastic plate, an ELISA plate.
  • a sample from a subject is contacted with the solid support, and then contacted with a detectably labeled antibody indicator to develop a color.
  • blocking agents such as bovine serum albumin, milk powder solution, gelatin, PVP, and Superblock can be used to block non-specific sites, thereby reducing the background caused by non-specific binding.
  • diluents such as BSA and phosphate buffered saline (PBS)/Tween can be used to help reduce non-specific background.
  • a sample from a subject may include biological tissues, cells or body fluids in a healthy or pathological state, such as saliva, nasopharyngeal swab.
  • the present disclosure provides a combination of a first set of antibodies and a second set of antibodies for detecting N protein, and their use in preparing a kit for detecting SARS-Cov-2.
  • the present disclosure provides combinations of immunogenic polypeptides comprising the amino acid fragment epitopes described herein, and their use for the preparation of antibodies for the detection of N protein.
  • the present disclosure provides a method for preparing an antibody for detecting the N protein, the method comprising immunizing animals with immunogenic polypeptides comprising the amino acid fragment epitopes described herein, thereby preparing an antibody for detecting the N protein, such as a monoclonal Antibodies or polyclonal antibodies.
  • Monoclonal or polyclonal antibodies can be produced by methods known in the art.
  • the epitopes identified in this disclosure (which can be artificially synthesized by, for example, chemical methods) can be linked to appropriate carrier proteins and used to immunize animals to prepare antibodies such as monoclonal antibodies.
  • suitable carrier proteins are known to those skilled in the art, such as KLH and BSA, among others.
  • a kit of the present disclosure may comprise the first set of antibodies and the second set of antibodies described above.
  • the present disclosure provides the use of an immunogenic polypeptide comprising an amino acid fragment epitope described herein in the preparation of a reagent or kit for detecting SARS-Cov-2.
  • the method may comprise contacting a sample with at least one protein N antibody or fragment thereof coated on a solid phase to form an immune complex; detecting the presence of the complex to determine the presence of protein N in the sample. The presence.
  • the method may comprise contacting the sample with at least one protein N antibody or fragment thereof coated on a solid phase to form an immune complex; adding to the resulting complex a first antibody linked to a detectable label; diN protein antibody; and detecting the signal generated to determine the presence and/or amount of N protein in said sample.
  • the present disclosure provides kits for use in the methods, comprising 1) a container comprising at least one N protein antibody coated on a solid phase.
  • the kit optionally comprises a second N protein antibody linked to a detectable label.
  • the at least one protein N antibody is a monoclonal antibody to protein N.
  • the present disclosure provides a kit comprising magnetic beads or test strips.
  • kits of the present disclosure may include reagents suitable for mechanochemiluminescent detection.
  • the kit of the present disclosure can use a chemiluminescent automatic instrument to detect N protein quickly and accurately with high throughput, shorten the detection time, and quickly obtain the test result.
  • the present disclosure includes antibodies labeled on magnetic beads or test strips.
  • the kit of the present disclosure can use magnetic beads or test strips as the solid phase, directly coat antibodies on the magnetic beads or test strips, and use the principle of double-antibody sandwich method to detect N protein to improve the detection rate. Rate.
  • Some embodiments of the present disclosure also provide the use of the above-mentioned antibody combination, or the above-mentioned kit, or the immunochromatographic test strip for detecting SARS-Cov-2.
  • Some embodiments of the present disclosure also provide a method for detecting SARS-Cov-2, comprising:
  • Some embodiments of the present disclosure also provide a method for diagnosing a subject infected with SARS-Cov-2 or a disease related to SARS-Cov-2 infection, comprising:
  • diseases associated with SARS-Cov-2 infection include respiratory symptoms, fever, cough, shortness of breath, dyspnea, pneumonia, severe acute respiratory syndrome, renal failure.
  • the antibody combination or prepared detection kit or immunochromatographic test strip provided by the present disclosure has higher sensitivity and detection rate when detecting SARS-Cov-2, and can effectively improve the problem of missed detection of mutant strains.
  • mice of the same strain as myeloma cells Take BALB/c mice of the same strain as myeloma cells at the age of 8-12 weeks, mix well with N protein antigen containing 100 ⁇ g of protein per mouse and the same amount of complete Freund's adjuvant, inject into the abdominal cavity of the mice, and For 2 weeks, 100 ⁇ g/mouse N protein antigen was fully mixed with the same amount of Freund's incomplete adjuvant, and injected into the abdominal cavity of mice several times to boost immunization. After the detection of mouse serum (indirect ELISA method), those whose titer is above 1:2000 can be used for fusion, and 3 days before fusion, the mice are given intraperitoneal booster immunization again, with a dose of 50 ⁇ g/mouse.
  • N protein antigen sequence SEQ ID NO:1.
  • BALB/c mouse peritoneal macrophages were used as feeder cells.
  • the BALB/c mice were killed by pulling the neck, soaked in 75% alcohol, and cut the abdominal skin with scissors under aseptic operation in an ultra-clean bench to expose the peritoneum. Wash repeatedly, recover the washing solution, centrifuge at 1000rpm for 5 minutes, save the precipitate, resuspend in RPMI 1640 screening culture medium (in RPMI 1640 complete culture medium containing HAT), adjust the cell concentration to 1 ⁇ 105 cells/mL, and add to 96 wells plate, 150 ⁇ L/well, and culture overnight at 37°C, 5% CO 2 .
  • the spleen was taken out under aseptic conditions, placed in a plate, washed once with RPMI 1640 basal culture medium, placed on a nylon mesh in a small beaker, ground and filtered to make a cell suspension. Centrifuge, discard the supernatant, resuspend in RPMI 1640 basal culture medium, repeat this three times, and count.
  • Immunized mouse splenocytes and mouse myeloma cells after PEG treatment, form a mixture of various cell components, including unfused myeloma cells and immune splenocytes; myeloma cell synkaryocytes and immune splenocytes symkaryon, and heterokaryon in myeloma cells and immune spleen cells. Only the latter form hybridoma cells. For this reason, unfused cells and allofused synkaryons must be removed from the various cell mixtures, and true hybrid cells must be selected. Therefore, on the 1st, 3rd, 5th, and 7th days after cell fusion, use the above-mentioned HAT medium for culture;
  • Hybridoma cells were cloned by limiting dilution. After culture, a single cell can proliferate into homologous cell clones; a total of 40 cell lines stably secreting anti-N protein monoclonal antibodies were obtained through reactivity screening (see Table 1 below for details), and their titers were all in the range of 10 5 - Between 10 and 7 .
  • microtiter plates (Nunc, Maxisorb) were coated with 100 ⁇ l/well of coating buffer containing 2.5 ⁇ g/ml N protein (as antigen) while stirring (coating buffer, catalog number 0726 559, Scil Diagnostics, GmbH) were coated for 1 hour. Post-coating treatments were performed by incubation in PBS buffer and 1% monoclonal antibody for 30 minutes. Subsequently, wash with wash buffer. 100 ⁇ l/well antibody samples were incubated for 1 hour at room temperature with agitation. Then wash 2 more times with washing solution.
  • the cell lines in the above table are from Feipeng Biological Co., Ltd. after screening and preservation, and are obtained through reactive screening. Those skilled in the art can use this method or similar ELISA method or immunofluorescence
  • the above-mentioned cell lines with similar functions can also be screened by techniques such as immunoblotting and immunoblotting, and the selected cell lines are cell lines with good reactivity and stable secretion of anti-N protein monoclonal antibodies.
  • the preparation method and selection of the above-mentioned cell lines do not limit the scope of the present disclosure in any way, but are only exemplary illustrations.
  • N protein short peptide antigens were used to coat the microwells, and PBS+20% NBS was used as the diluent, and the monoclonal antibody was diluted to a concentration of 1 ⁇ g/ml as the primary antibody, and goat anti-mouse IgG-HRP was used as the secondary antibody.
  • Antibody responses to different antigens were used to determine the epitope of the monoclonal antibody. According to statistics, the above 41 antibodies are respectively directed against the following segments of the N protein antigen (see Table 2 below for details):
  • the segment sequence of N protein antigen is as follows:
  • the 1-43 fragment sequence (i.e. the 1-43 N protein amino acid fragment of SARS-Cov-2) is shown in SEQ ID NO: 2:
  • the 44-180 fragment sequence (i.e. the 44-180 N protein amino acid fragment of SARS-Cov-2) is shown in SEQ ID NO: 3:
  • the 248-361 fragment sequence (i.e. the 248-361 N protein amino acid fragment of SARS-Cov-2) is shown in SEQ ID NO:4:
  • the 50-107 fragment sequence (i.e. the 50-107 N protein amino acid fragment of SARS-Cov-2) is shown in SEQ ID NO:5:
  • the 74-105 fragment sequence (i.e. the 74-105 N protein amino acid fragment of SARS-Cov-2) is shown in SEQ ID NO: 6:
  • Anti-N protein antibody epitope pairing The antibody with the highest antibody titer against different epitopes is used as the paired combination, and the ELISA double-antibody sandwich detection method is used, with 100 positive nucleic acid test samples as the test sample, and the detection rate is ⁇ 95%. For better matching results, see Table 3 for details.
  • the detection rates of pairing 6-8 and pairing 12-14 are both higher than 95%.
  • the factor for the improvement of the detection rate is that at least the binding 44-180aa (16D8) segment is used in both coating and labeling antibodies.
  • pairing 6-8 and pairing 12-14 can effectively detect the D103Y and P13L+R203K+G204R+G214C mutant strains at a concentration range of 0.2ng/ml, and some pairing combinations can also be detected at a concentration as low as 50pg/ml. The results can be checked out.
  • the above pairing can still have a high detection rate after replacing other strains of antibodies.
  • the detection rates of pairing 15 to pairing 34 were all higher than 95%.
  • the reason for the high detection rate was that both the coated antibody and the labeled antibody in the above pairing scheme included antibodies that bind to the 44-180aa segment.
  • the above data also prove that only 3 kinds of antibodies need to be selected in the above scheme, and the technical effect of the combination of 4 kinds of antibodies can be achieved.
  • Antibodies 9F5, 4B5, and 3A7 in the above pairing scheme 15 are taken as examples; 9F5 is Antibody 1, 4B5 is Antibody 2, and 3A7 is Antibody 3.
  • the heavy chain, light chain, and the number in the CDR correspond to the sequence number on SEQ ID NO:, for example, 7 corresponds to SEQ ID NO:7.
  • the above antibodies were used to replace the corresponding antibodies in the original pair 15, and the detection rates were above 96%.
  • Whether an antibody recognizes the same epitope as other antibodies can be confirmed by competition between the two for the epitope. Competition between antibodies can be assessed by competition binding assays such as: ELISA, fluorescence energy transfer assay, or fluorescence microassay techniques. After identification, the 9F5, 4B5, 3A7 corresponding mutant antibodies had the same binding epitope as the unmutated antibody.
  • the immunochromatographic test strip includes a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and a water-absorbing pad, the nitrocellulose membrane is provided with T lines and C lines, and the binding pad is provided with the above-mentioned A second set of antibodies, wherein the T-lines are coated with the first set of antibodies described above.
  • the preparation of the immunochromatographic test strips includes the following steps: firstly fix the nitrocellulose membrane in the middle of the bottom plate, and press one end of the treated colloidal gold binding pad (spraying the second group of antibodies labeled with colloidal gold on the binding pad) on the nitrocellulose
  • the end of the detection T line of the plain film (the T line is sprayed with the first group of antibodies), with an overlap of 1mm.
  • One end of the treated sample pad is pressed against the other end of the colloidal gold bonding pad, with an overlap of 2mm.
  • Stick the absorbent pad on the bottom plate one end of which is pressed against the end of the quality control line C of the nitrocellulose membrane, with an overlap of 2mm. Cut the assembled test strip test piece into 4mm width with a strip cutter, put it into the groove of the plastic buckle base, and press the plastic cover tightly.
  • the disclosure provides a detection method and kit for SARS-Cov-2.
  • the method and detection kit of the disclosure can improve the detection sensitivity and detection rate, especially for the detection of new crown mutant strains, which can effectively improve the problem of missed detection of mutant strains , has excellent practical performance, and can be widely used in the field of novel coronavirus detection.

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Abstract

一种SARS-Cov-2检测方法和试剂盒，所述SARS-Cov-2检测试剂盒包括用于检测来自受试者样品中SARS-Cov-2的第一组和第二组抗体，第一组抗体包括选自结合SARS-Cov-2的N蛋白氨基酸片段44-180的抗体1，第二组抗体包括选自结合SARS-Cov-2的N蛋白氨基酸片段44-180的抗体2，抗体1和抗体2可以是相同的抗体也可以是不同的抗体。所述检测方法和试剂盒可以提高检测灵敏度和检出率，尤其可以检出新冠变异株。

Description

一种SARS-Cov-2检测方法和试剂盒
相关申请的交叉引用
本公开要求于2021年09月10日提交中国专利局的申请号为CN202111064565.2、名称为“一种SARS-Cov-2检测方法和试剂盒”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及蛋白检测领域。具体而言，本公开涉及SARS-Cov-2的检测方法和试剂盒。
背景技术
自2019年以来，全球爆发了新型冠状病毒(严重急性呼吸综合征冠状病毒2，SARS-Cov-2)感染引起的肺炎，但目前尚无针对新型冠状病毒引起的肺炎的特异性治疗药物，给全球健康带来巨大的威胁。目前确诊新冠肺炎的主要检测方法有核酸检测和基于抗原-抗体免疫检测方法；核酸检测方法主要为荧光RT-PCR技术，抗原-抗体免疫检测的方法分为针对病毒抗体的检测和针对病毒抗原的检测。抗体检测试剂是检测血清中由病毒进入人体后刺激人体产生的IgM或IgG抗体，IgM抗体出现较早，IgG抗体出现较晚。病毒抗原检测主要是检测病毒表面的一些蛋白质如S蛋白。如何找到特异性的抗原表位肽对于检测开发基于抗体的新冠检测方法至关重要。
核酸检测具有早期诊断、灵敏度和特异性高等特点，是确诊新冠肺炎的“金标准”。但是受样本采集、RNA提取及检测设备条件要求较高的影响，容易出现假阴性，因此急需快捷的免疫学检测方法作为核酸检测的补充。有研究表明，新冠总抗体和核酸协同检测可显著提高新冠病毒感染早期诊断的灵敏度，可以有效弥补核酸检测假阴性容易造成漏诊的缺点。
SARS-Cov-2的结构基因组29883bp，基因组中包括了14个ORF框，较大的蛋白分子包括以下蛋白的区域，N：核衣壳蛋白(简称N蛋白)；S：Spike蛋白；M：膜蛋白；HE：血凝素酯酶；E：包膜蛋白。N蛋白也是新冠病毒感染过程中最丰富的病毒结构蛋白，感染后人细胞会产生强烈的免疫反应性，且基因序列相对保守稳定，与S蛋白比较，N蛋白上随着时间推移产生的突变较少，因为其良好的稳定性，国内外诊断(IVD)公司开发的以N蛋白做为主要靶标的抗原检测试剂盒，用于病例筛查和疫情监控；但由于全球疫情的快速传播，流行的突变株上的N蛋白上也出现了位点突变，作为新冠抗原检测试剂盒的主靶标，N蛋白上发生的变异可能对试剂盒灵敏度造成检出率影响，因此，补充靶标位点，而非变异株的区段位点，目前看流行性突变株的位点突变，试剂盒主靶标位点均不受影响，且还能补充一部分灵敏度，提高病毒的检出率，这对全球疫情筛查和诊断尤为重要。
发明内容
本公开提供一种抗体组合，包含第一组抗体和第二组抗体，所述第一组抗体包括选自结合SARS-Cov-2的N蛋白氨基酸片段44-180的抗体1，所述第二组抗体包括选自结合SARS-Cov-2的N蛋白氨基酸片段44-180的抗体2，所述抗体1和所述抗体2可以是相同的抗体也可以是不同的抗体。
可选地，所述第一组抗体还包括抗体3-抗体6中的至少一种。
可选地，所述第二组抗体还包括抗体3-抗体6中的至少一种。
可选地，所述第一组抗体还包括抗体4或抗体5，第二组抗体还包括抗体6。
抗体3：结合N蛋白氨基酸片段50-107；
抗体4：结合N蛋白氨基酸片段1-43；
抗体5：结合N蛋白氨基酸片段248-361；
抗体6：结合N蛋白氨基酸片段74-105。
可选地，所述抗体的反应效价高于10 5。
可选地，所述第一组抗体用于标记，第二组抗体用于包被；或第二组抗体用于标记，第一组抗体用于包被。
可选地，所述用于标记的抗体用可检测标记物标记，例如金属粒子，如胶体金，荧光标记，发色团标记，电子致密标记，化学发光标记，放射性标记，酶标记，例如放射性同位素，荧光团，罗丹明，荧光素酶，荧光素，辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶菌酶，糖类氧化酶，葡萄糖氧化酶，半乳糖氧化酶，葡萄糖-6-磷酸脱氢酶，生物素/抗生物素蛋白，自旋标记，噬菌体标记，例如吖啶酯标记，例如通过接头如生物素-亲和素添加荧光标记如吖啶酯标记。
可选地，所述用于包被的抗体连接至固相，例如磁性颗粒，胶乳粒子、ELISA板、微量滴定板、硝酸纤维素膜或微流控芯片。
可选地，所述抗体1的重链互补决定区VH-CDR1包括SEQ ID NO:7或SEQ ID NO:13所示的氨基酸序列，重链互补决定区VH-CDR2包括SEQ ID NO:8所示的氨基酸序列，重链互补决定区VH-CDR3包括SEQ ID NO:9或SEQ ID NO:14所示的氨基酸序列；所述抗体1的轻链互补决定区VL-CDR1包括SEQ ID NO:10所示的氨基酸序列，轻链互补决定区VL-CDR2包括SEQ ID NO:11所示的氨基酸序列，轻链互补决定区VL-CDR3包括SEQ ID NO:12所示的氨基酸序列。
可选地，所述抗体1重链可变区包括SEQ ID NO:15或SEQ ID NO:16所示的氨基酸序列，所述抗体1轻链可变区包括SEQ ID NO:17所示的氨基酸序列。
可选地，所述抗体1重链包括SEQ ID NO:18或SEQ ID NO:19所示的氨基酸序列，所述抗体1轻链包括SEQ ID NO:20所示的氨基酸序列。
可选地，所述抗体1与表位1结合，所述表位1与包含SEQ ID NO:15或SEQ ID NO:16的重链可变区以及SEQ ID NO:17的轻链可变区的抗体或者与包含SEQ ID NO:18或SEQ ID NO:19的重链以及SEQ ID NO:20的轻链的抗体所结合的表位相同。
可选地，所述抗体2的重链互补决定区VH-CDR1包括SEQ ID NO:21或SEQ ID NO:27所示的氨基酸序列，重链互补决定区VH-CDR2包括SEQ ID NO:22所示的氨基酸序列，重链互补决定区VH-CDR3包括SEQ ID NO:23所示的氨基酸序列；所述抗体2的轻链互补决定区VL-CDR1包括SEQ ID NO:24或SEQ ID NO:28所示的氨基酸序列，轻链互补决定区VL-CDR2包括SEQ ID NO:25所示的氨基酸序列，轻链互补决定区VL-CDR3包括SEQ ID NO:26所示的氨基酸序列。
可选地，所述抗体2重链可变区包括SEQ ID NO:29或SEQ ID NO:30所示的氨基酸序列，所述抗体2轻链可变区包括SEQ ID NO:31或SEQ ID NO:32所示的氨基酸序列。
可选地，所述抗体2重链包括SEQ ID NO:33或SEQ ID NO:34所示的氨基酸序列，所述抗体2轻 链包括SEQ ID NO:35或SEQ ID NO:36所示的氨基酸序列。
可选地，所述抗体2与表位2结合，所述表位2与包含SEQ ID NO:29或SEQ ID NO:30的重链可变区以及SEQ ID NO:31或SEQ ID NO:32的轻链可变区的抗体或者与包含SEQ ID NO:33或SEQ ID NO:34的重链以及SEQ ID NO:35或SEQ ID NO:36的轻链的抗体所结合的表位相同。
可选地，所述抗体3的重链互补决定区VH-CDR1包括SEQ ID NO:37所示的氨基酸序列，重链互补决定区VH-CDR2包括SEQ ID NO:38或SEQ ID NO:43所示的氨基酸序列，重链互补决定区VH-CDR3包括SEQ ID NO:39所示的氨基酸序列；所述抗体3的轻链互补决定区VL-CDR1包括SEQ ID NO:40所示的氨基酸序列，轻链互补决定区VL-CDR2包括SEQ ID NO:41或SEQ ID NO:44所示的氨基酸序列，轻链互补决定区VL-CDR3包括SEQ ID NO:42所示的氨基酸序列。
可选地，所述抗体3重链可变区包括SEQ ID NO:45或SEQ ID NO:46所示的氨基酸序列，所述抗体3轻链可变区包括SEQ ID NO:47或SEQ ID NO:48所示的氨基酸序列。
可选地，所述抗体3重链包括SEQ ID NO:49或SEQ ID NO:50所示的氨基酸序列，所述抗体3轻链包括SEQ ID NO:51或SEQ ID NO:52所示的氨基酸序列。
可选地，所述抗体3与表位3结合，所述表位3与包含SEQ ID NO:45或SEQ ID NO:46的重链可变区以及SEQ ID NO:47或SEQ ID NO:48的轻链可变区的抗体或者与包含SEQ ID NO:49或SEQ ID NO:50的重链以及SEQ ID NO:51或SEQ ID NO:52轻链的抗体所结合的表位相同。
可选地，所述抗体1或所述抗体2结合SARS-Cov-2的N蛋白的构象依赖性表位。
可选地，所述抗体1或所述抗体2不结合由SARS-Cov-2的N蛋白的6个连续氨基酸组成的任何多肽。本公开还提供一种SARS-Cov-2抗原检测试剂盒，包括上述的抗体组合。
本公开还提供一种SARS-Cov-2抗原检测试剂盒，其用于检测来自受试者样品中的SARS-Cov-2 N蛋白，包括上述的抗体组合。
可选地，所述试剂盒是免疫层析试纸条、酶联免疫试剂或化学发光试剂。
可选地，所述样品包括健康或病理状态的生物组织、细胞或体液，例如唾液、鼻咽拭子。
可选地，本公开提供一种SARS-Cov-2免疫层析试纸条，所述免疫层析试纸条包括底板、样品垫、结合垫、硝酸纤维素膜和吸水垫，所述硝酸纤维素膜上设置有T线和C线，所述结合垫上设置有第二组抗体，其中T线上包被有第一组抗体；或所述结合垫上设置有第一组抗体，T线上包被有第二组抗体。
本公开还提供了抗体组合在制备检测SARS-Cov-2的试剂盒中的应用。
本公开还提供上述抗体组合，或上述的试剂盒，或所述疫层析试纸条，用于检测SARS-Cov-2的用途。
本公开还提供一种检测SARS-Cov-2的方法，包括：
A)在足以发生结合反应的条件下，使上述抗体组合，或上述试剂盒，或所述疫层析试纸条与样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
本公开还提供一种诊断受试者在感染SARS-Cov-2或与SARS-Cov-2感染相关疾病中的方法，包括：
A)在足以发生结合反应的条件下，使上述抗体组合，或上述试剂盒，或所述疫层析试纸条与来自所述受试者的样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
可选地，所述与SARS-Cov-2感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、呼吸困难、肺炎、严重急性呼吸综合征、肾衰竭。
具体实施方式
术语定义
如本文所用，术语“抗体”在最广义上使用，其可以包括全长单克隆抗体，多克隆抗体，双特异性或多特异性抗体，嵌合抗体，以及抗体片段，只要他们展示所需的生物学活性。“抗体片段”包括全长抗体的部分，可选地其抗原结合区或可变区。抗体片段的实例包括Fab，Fab'，F(ab') 2，Fd，Fv，dAb，互补决定区(CDR)片段，单链抗体(例如，scFv)，双价抗体或结构域抗体。
如本文所使用，术语“互补决定区(complementarity determining region，简称CRD)”是指：一个完整或完全的抗体包含两条重链及两条轻链；每条重链含有可变区(VH)及恒定区(CH)；每条轻链含有一可变区(VL)及一恒定区(CL)；抗体具有“Y”形状，Y的茎部由透过双硫键结合在一起的两条重链的第二及第三恒定区所组成；Y的每个臂部包括与一单个轻链的可变区及恒定区结合的一单个重链的可变区及第一恒定区；该轻链的可变区及重链的可变区负责抗原结合；两条链中的可变区通常包含三个高度可变区，称为互补决定区。
如本文所使用，术语“恒定区”是指抗体分子的轻链和重链中靠近C端氨基酸序列相对稳定的区域。
如本文所使用，术语“可变区”是指抗体分子的轻链和重链中靠近N端氨基酸序列变化较大的区域。
如本文所使用，“aa”表示肽的第……位的氨基酸。
如本文所用，术语“受试者”是指脊椎动物，可选地为哺乳动物，可选地为人类。哺乳动物包括但不限于鼠类、猿类、人类、家畜、竞技动物和宠物。在体内获得的或在体外培养的生物实体的组织、细胞及其子代也包括在内。应当理解本文所用术语仅用于描述本公开上下文诸如实施方式和实施例的目的，并不旨在限制本公开的范围。
如本文所述，术语“共核体”是指基因型相同的细胞融合成的杂交细胞称为同核体。
本公开一些实施方式提供一种抗体组合，包含第一组抗体和第二组抗体，第一组抗体包括选自结合SARS-Cov-2的第44-180位N蛋白氨基酸片段的抗体1，第二组抗体包括选自结合SARS-Cov-2的第44-180位N蛋白氨基酸片的抗体2，抗体1和抗体2可以是相同的抗体或是不同的抗体；
在一些实施方式中，第一组抗体还可以包括抗体3、抗体4、抗体5或抗体6中的至少一种。
在一些实施方式中，第二组抗体还可以包括抗体3、抗体4、抗体5或抗体6中的至少一种。
在一些实施方式中，第一组抗体或第二组抗体还包括抗体3。
在一些实施方式中，第一组抗体还包括抗体4或抗体5，第二组抗体还包括抗体6。
在一些实施方式中，抗体3结合第50-107位的N蛋白氨基酸片段；
抗体4结合第1-43位的N蛋白氨基酸片段；
抗体5结合第248-361位的N蛋白氨基酸片段；
抗体6结合第74-105位的N蛋白氨基酸片段。
在一些实施方式中，本公开的试剂盒包括SARS-Cov-2检测试剂卡(试纸条)。在一些实施方式中，可以利用胶体金侧向层析技术，将抗体标记在可检测标记如胶体金上，利用双抗夹心法原理检测受试者是否感染新型冠状病毒SARS-Cov-2。在一些实施方式中，本公开的方法和试剂盒提高检出率，能够克服突变株漏检问题。
在一些实施方式中，针对现有大部分技术存在着检出率低，不能快速检测是否感染新型冠状病毒等技术问题，本公开提供一种检测方法和试剂盒，能够实现提高检出率，降低漏检的风险。
在一些实施方式中，本公开可以通过胶体金免疫层析进行或包括进行胶体金免疫层析的试剂。在一些实施方式中，免疫层析可以通过试纸条由双抗体夹心法实现。在一些实施方式中，进行双抗体夹心法的试剂和装置没有特别限制，可以采用任何适当的试剂和/或装置。例如，免疫层析试纸条可以包括样品垫、结合垫、NC膜和吸水垫。在一些实施方式中，结合垫上可以包被有多个表位的胶体金-抗体复合物；NC膜上可以包被两条线，例如分别是T线和C线；T线上可以混合包被多个表位的抗体，能捕捉来自结合垫上的胶体金-抗体复合物形成双抗体夹心的复合物，从而将胶体金固定在T线上；C线上可以包被能够捕捉游离的胶体金-抗体复合物的抗体。在一些实施方式中，待测样本加入到检测试剂卡的加样口中，在侧向毛细管作用下，待检样本先经过结合垫，与结合垫上的不同胶体金-抗体复合物分别发生特异的免疫结合，各自结合形成抗原-抗体胶体金复合物，从而被固定在T线中。游离的胶体金-抗体复合物经过C线时，能够与C线抗体发生特异的免疫结合，从而被固定在C线中。对照带(C)与测试带(T)的显色强度成反比，从而可以校正样本个体差异。
在一些实施方式中，本公开能够利用多表位抗体识别抗原上的多个表位，降低漏检的风险，提高检出率。
在一些实施方式中，本公开可以通过抗原表位的组合，覆盖范围大，利用多表位抗体配对识别抗原上的多个表位，降低漏检的风险，提高检出率。
在一些实施方式中，本公开的标记抗体可以采用SARS-Cov-2的N蛋白的44-180aa、50-107aa或74-105aa抗体表位进行混标，包被抗体可以采用44-180aa、1-43aa或248-361aa的抗体表位进行混标。或者，包被与标记互换也是可行的。
在本文中，如无相反说明，N蛋白表位或片段的编号根据SEQ ID NO:1相应位置进行。
在一些实施方式中，本公开提供了一种SARS-Cov-2的检测方法和试剂盒。本公开的试剂盒具备改善的灵敏度、提高检出率、克服突变株漏检问题。
在一些实施方式中，本公开的第一组抗体和/或第二组抗体可以是单克隆抗体。
在一些实施方式中，可以采用本领域已知的方法制备本公开的抗体，例如第一组抗体和/或第二组抗体。
在一些实施方式中，可以以包含本文描述的氨基酸片段的抗原免疫动物制备本公开的抗体，例如第一组抗体和/或第二组抗体。为了增加免疫原性，可以将较大的抗原化合物(包括但不限于BSA(牛血清白蛋白)、卵清蛋白、KLH(钥孔血蓝蛋白)等)与表位肽偶联。免疫原部分可以包括多种蛋白质或多肽，其可以起免疫原载体的作用。这些类型的多肽包括白蛋白、血清蛋白、球蛋白、晶状体蛋白、脂蛋白和/或其片段。
在一些实施方式中，可以使用合成多肽。免疫原部分或载体还可以是粒子或微粒。免疫原粒子可以 是有机的或无机的，多孔的或无孔的。免疫原粒子可以是生物材料如细胞和微生物，还可以包含有机和无机聚合物、脂质体、胶乳、磷脂囊泡、脂质体、阳离子脂质体、阴离子脂质体、脂蛋白、脂质聚合物等。因此，本公开所述的表位肽可以用于产生具有对N蛋白亲和力的抗体。
在一些实施方式中，可以使用任何适当的体外测定、基于细胞的测定、体内测定、动物模型等检测本公开抗体的效果如结合活性和/或交叉反应性。在一些实施方式中，所述测定可以包括例如ELISA(Enzyme-Linked Immunosorbent Assay酶联免疫吸附剂测定)，FACS(Fluorescence-Activated Cell Sorting荧光激活细胞分选)结合测定，Biacore(Biomolecule Interaction Analysis core technique)，竞争性结合测定等。在一些实施方式中，例如在ELISA或FACS中本公开的抗体(或其抗原结合片段)与抗原结合的EC50值可以为例如1μM-1pM，例如1nM-1pM，例如100pM-1pM。在一些实施方式中，本公开抗体用于检测N蛋白抗原的反应效价高于10 5，包括例如高于10 6，例如高于10 7。
在一些实施方式中，试剂盒中的第一组抗体和/或第二组抗体可以分别用作包被抗体(或称为捕获抗体)和标记抗体，例如第一组抗体为包被抗体，第二组抗体为标记抗体，或者第一组抗体为标记抗体，第二组抗体为包被抗体。可选地，在一些实施方式中，第一组抗体为标记抗体，第二组抗体为包被抗体(或称为捕获抗体)。
在一些实施方式中，所述包被抗体结合至固相。在一些实施方式中，所述包被抗体可以用于包被固相支持物。在一些实施方式中，固相支持物没有特别限制，其可以是例如磁性颗粒，胶乳粒子、微量滴定板、硝酸纤维素膜或微流控芯片，任选地所述试剂盒包括或制备成免疫层析试纸条或检测卡。在一些实施方式中，所述标记抗体用可检测标记物标记，例如用荧光标记物标记，例如通过接头如生物素-亲和素与荧光标记物标记。
本公开的一些实施方式，提供一种SARS-Cov-2免疫层析试纸条，免疫层析试纸条包括底板、样品垫、结合垫、硝酸纤维素膜和吸水垫，硝酸纤维素膜上设置有T线和C线，结合垫上设置有上文所述的第二组抗体，其中T线上包被有上文所述的第一组抗体；或结合垫上设置有上文所述的第一组抗体，T线上包被有上文所述的第二组抗体。
在一些实施方式中，本公开中使用的可检测标记没有特别限制。在一些实施方式中，所述标记可以包括但不限于金属粒子，如胶体金，荧光标记，发色团标记，电子致密标记，化学发光标记，和放射性标记，以及间接标记如酶或配体，例如，通过酶促反应或分子相互作用来间接检测。在一些实施方式中，示例性标记包括但不限于放射性同位素，荧光团，罗丹明及其衍生物，荧光素酶，荧光素，辣根过氧化物酶(HRP)，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶菌酶，糖类氧化酶，例如，葡萄糖氧化酶，半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，生物素/抗生物素蛋白，自旋标记，噬菌体标记等等。
在一些实施方式中，抗体1的重链互补决定区VH-CDR1包括SEQ ID NO:7或SEQ ID NO:13所示的氨基酸序列，重链互补决定区VH-CDR2包括SEQ ID NO:8所示的氨基酸序列，重链互补决定区VH-CDR3包括SEQ ID NO:9或SEQ ID NO:14所示的氨基酸序列；抗体1的轻链互补决定区VL-CDR1包括SEQ ID NO:10所示的氨基酸序列，轻链互补决定区VL-CDR2包括SEQ ID NO:11所示的氨基酸序列，轻链互补决定区VL-CDR3包括SEQ ID NO:12所示的氨基酸序列。
在一些实施方式中，抗体1重链可变区包括SEQ ID NO:15或SEQ ID NO:16所示的氨基酸序列，抗体1轻链可变区包括SEQ ID NO:17所示的氨基酸序列。
在一些实施方式中，抗体1重链包括SEQ ID NO:18或SEQ ID NO:19所示的氨基酸序列，抗体1轻链包括SEQ ID NO:20所示的氨基酸序列。
在一些实施方式中，抗体1与表位1结合，表位1与包含SEQ ID NO:15或SEQ ID NO:16的重链可变区以及SEQ ID NO:17的轻链可变区的抗体或者与包含SEQ ID NO:18或SEQ ID NO:19的重链以及SEQ ID NO:20的轻链的抗体所结合的表位相同，抗体1与SEQ ID NO:1所示的SARS-Cov-2的N蛋白氨基酸序列中44至180位之中的氨基酸区域结合。
Figure PCTCN2022117381-appb-000001
Figure PCTCN2022117381-appb-000002
在一些实施方式中，抗体2的重链互补决定区VH-CDR1包括SEQ ID NO:21或SEQ ID NO:27所示的氨基酸序列，重链互补决定区VH-CDR2包括SEQ ID NO:22所示的氨基酸序列，重链互补决定区VH-CDR3包括SEQ ID NO:23所示的氨基酸序列；抗体2的轻链互补决定区VL-CDR1包括SEQ ID NO:24或SEQ ID NO:28所示的氨基酸序列，轻链互补决定区VL-CDR2包括SEQ ID NO:25所示的氨基酸序列，轻链互补决定区VL-CDR3包括SEQ ID NO:26所示的氨基酸序列。
在一些实施方式中，抗体2重链可变区包括SEQ ID NO:29或SEQ ID NO:30所示的氨基酸序列，抗体2轻链可变区包括SEQ ID NO:31或SEQ ID NO:32所示的氨基酸序列。
在一些实施方式中，抗体2重链包括SEQ ID NO:33或SEQ ID NO:34所示的氨基酸序列，抗体2轻链包括SEQ ID NO:35或SEQ ID NO:36所示的氨基酸序列。
在一些实施方式中，抗体2与表位2结合，表位2与包含SEQ ID NO:29或SEQ ID NO:30的重链可变区以及SEQ ID NO:31或SEQ ID NO:32的轻链可变区的抗体或者与包含SEQ ID NO:33或SEQ ID NO:34的重链以及SEQ ID NO:35或SEQ ID NO:36的轻链的抗体所结合的表位相同，抗体2与SEQ ID NO:1所示的SARS-Cov-2的N蛋白氨基酸序列中44至180位之中的氨基酸区域结合。
Figure PCTCN2022117381-appb-000003
Figure PCTCN2022117381-appb-000004
在一些实施方式中，抗体3的重链互补决定区VH-CDR1包括SEQ ID NO:37所示的氨基酸序列，重链互补决定区VH-CDR2包括SEQ ID NO:38或SEQ ID NO:43所示的氨基酸序列，重链互补决定区VH-CDR3包括SEQ ID NO:39所示的氨基酸序列；抗体3的轻链互补决定区VL-CDR1包括SEQ ID NO:40所示的氨基酸序列，轻链互补决定区VL-CDR2包括SEQ ID NO:41或SEQ ID NO:44所示的氨基酸序列，轻链互补决定区VL-CDR3包括SEQ ID NO:42所示的氨基酸序列。
在一些实施方式中，抗体3重链可变区包括SEQ ID NO:45或SEQ ID NO:46所示的氨基酸序列， 抗体3轻链可变区包括SEQ ID NO:47或SEQ ID NO:48所示的氨基酸序列。
在一些实施方式中，抗体3重链包括SEQ ID NO:49或SEQ ID NO:50所示的氨基酸序列，抗体3轻链包括SEQ ID NO:51或SEQ ID NO:52所示的氨基酸序列。
在一些实施方式中，抗体3与表位3结合，表位3与包含SEQ ID NO:45或SEQ ID NO:46的重链可变区以及SEQ ID NO:47或SEQ ID NO:48的轻链可变区的抗体或者与包含SEQ ID NO:49或SEQ ID NO:50的重链以及SEQ ID NO:51或SEQ ID NO:52轻链的抗体所结合的表位相同，抗体3与SEQ ID NO:1所示的SARS-Cov-2的N蛋白氨基酸序列中50至107位之中的氨基酸区域结合。
Figure PCTCN2022117381-appb-000005
Figure PCTCN2022117381-appb-000006
在一些实施方式中，抗体1或抗体2结合SARS-Cov-2的N蛋白的构象依赖性表位。
在一些实施方案中，所述构象表位由抗原氨基酸序列的不连续部分组成。
在一些实施方案中，抗体1和抗体2通过PepScan分析，合成肽由6个连续氨基酸组成，其各自移位1个氨基酸，例如：44-49、45-50依此类推，直到175-180。分析合成肽与抗体的结合状态，具体为采用HRP标记的二抗检测合成肽与抗体的结合情况。通过检测结果显示抗体1和抗体2均为构象表位。
在一些实施方式中，抗体1或抗体2不结合由SARS-Cov-2的N蛋白的6个连续氨基酸组成的任何多肽。在一些实施方式中，本公开的试剂盒包括适合进行免疫测定的试剂。在一些实施方式中，本公开的试剂盒可以用于进行免疫测定，例如ELISA，间接免疫荧光测定IFA，放射免疫测定RIA以及其它非酶联抗体结合试验或方法。
在一些实施方式中，例如在化学发光实验方案中，可以将N蛋白抗体包被固相如磁珠，捕获样品中的N蛋白抗原，然后用标记的抗体与结合在磁珠上的抗原再次结合，添加发光底物后读取结果。在一些实施方式中，本公开的N蛋白抗体可以用于包被固相如磁珠或用作标记的第二组抗体。在一些实施方式中，例如ELISA实验方案中，抗体或其抗原结合片段固定到一个表面上，例如固相支持物上，例如塑料板、ELISA板。来自受试者的样品与所述固相支持物接触，然后接触带有可检测标记的抗体指示剂显色。在一些实施方式中，可以采用封闭剂如牛血清白蛋白、奶粉溶液、明胶、PVP、Superblock封闭非特异性位点，因此減少非特异性结合造成的背景。在一些实施方式中，可以采用稀释剂，如应用BSA和磷酸缓冲盐液(PBS)/吐温稀释，有助于减少非特异性背景。
在本文中，来自受试者的样品可以包括健康或病理状态的生物组织、细胞或体液，例如唾液、鼻咽拭子。
在一些实施方式中，本公开提供用于检测N蛋白的第一组抗体和第二组抗体的组合，以及提供它们在制备检测SARS-Cov-2的试剂盒中的用途。在一些实施方式中，本公开提供包含本文描述的氨基酸片段表位的免疫原性多肽的组合，以及它们用于制备检测N蛋白的抗体的用途。在一些实施方式中，本公开提供制备检测N蛋白的抗体的方法，所述方法包括将包含本文描述的氨基酸片段表位的免疫原性多肽分别免疫动物，从而制备检测N蛋白的抗体如单克隆抗体或多克隆抗体。单克隆抗体或多克隆抗体可以通过本领域已知的方法进行。在一些实施方式中，本公开鉴定的表位(可以通过例如化学法人工合成)可以与适当的载体蛋白连接，用于免疫动物制备抗体如单克隆抗体。在一些实施方式中，适当的载体蛋白是本领域技术人员已知的，其可以是例如KLH和BSA等。在一些实施方式中，本公开的试剂盒可以包含上述第一组抗体和第二组抗体。
在一些实施方式中，本公开提供包含本文描述的氨基酸片段表位的免疫原性多肽在制备检测SARS-Cov-2的试剂或试剂盒中的用途。
在一些实施方式中，所述方法可以包括将样品与包被于固相上的至少一种N蛋白抗体或其片段接触形成免疫复合物；检测复合物的存在，以确定所述样品中N蛋白的存在。在一些实施方式中，所述方法可以包括将样品与包被于固相上的至少一种N蛋白抗体或其片段接触形成免疫复合物；向产生的复合物中添加与可检测标记连接的第二N蛋白抗体；和检测产生的信号，以确定所述样品中N蛋白的存在和/或含量。在一些实施方式中，本公开提供用于所述方法的试剂盒，其包含1)含有包被于固相上的至少一种N蛋白抗体的容器。在一些实施方式中，所述试剂盒可选地包含与可检测标记连接的第二N蛋白抗体。在一些实施方式中，所述至少一种N蛋白抗体为N蛋白的单克隆抗体在一些实施方式中，本公开提供了一种包含磁珠或试纸条的试剂盒。在一些实施方式中，本公开的试剂盒可以包括适合机械能化学发光检测的试剂。在一些实施方式中，本公开的试剂盒可以利用化学发光全自动仪器高通量、快速准确地检测N蛋白，缩短检测时间，快速的出检验结果。
在一些实施方式中，本公开包括标记在磁珠或试纸条上抗体。在一些实施方式中，本公开的试剂盒可以利用磁珠或试纸条为固相，将抗体直接包被在磁珠或试纸条上，采用双抗体夹心法原理检测N蛋白，提高检出率。
本公开的一些实施方式还提供上述抗体组合，或上述的试剂盒，或所述疫层析试纸条，用于检测SARS-Cov-2的用途。
本公开的一些实施方式还提供一种检测SARS-Cov-2的方法，包括：
A)在足以发生结合反应的条件下，使上述抗体组合，或上述试剂盒，或所述疫层析试纸条与样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
本公开的一些实施方式还提供一种诊断受试者在感染SARS-Cov-2或与SARS-Cov-2感染相关疾病中的方法，包括：
A)在足以发生结合反应的条件下，使上述抗体组合，或上述试剂盒，或所述疫层析试纸条与来自所述受试者的样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
在一些实施方式中，与SARS-Cov-2感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、呼吸困 难、肺炎、严重急性呼吸综合征、肾衰竭。
按本公开提供的抗体组合或制备的检测试剂盒或免疫层析试纸条在检测SARS-Cov-2时具有更高的灵敏度和检出率，可以有效改善突变株漏检问题。
实施例
下面主要结合一些实施例对本公开作进一步详细的说明。提供以下实施例以说明本公开的实施方式，并非意在限制本公开。本公开可以任选包括未通过实施例示出的实施方式。
一、N蛋白单克隆抗体的制备
1.免疫动物
取8～12周龄与骨髓瘤细胞同种系的BALB/c小鼠，以含蛋白质100μg/只的N蛋白抗原与等量福氏完全佐剂充分混匀，注入小鼠腹腔内，每隔2周100μg/只的N蛋白抗原与等量福氏不完全佐剂充分混匀，多次注入小鼠腹腔内加强免疫。经检测小鼠血清(间接ELISA法)，滴度在1∶2000以上者可用于融合，融合前3天经小鼠腹腔内再次加强免疫，剂量为50μg/只。
N蛋白抗原序列：SEQ ID NO:1。
Figure PCTCN2022117381-appb-000007
2.饲养细胞的制备
以BALB/c鼠腹腔巨噬细胞作饲养细胞。在融合前1天，BALB/c鼠拉颈处死，75％酒精全身浸泡，超净台内，无菌操作下用剪刀剪开腹部皮肤，暴露腹膜，用注射器腹腔注入RPMI 1640基础培养液5mL，反复冲洗，回收冲洗液，1000rpm，离心5分钟，留沉淀，用RPMI 1640筛选培养液(含HAT的RPMI 1640完全培养液中)重悬，调整细胞浓度1×10 5个/mL，加入96孔板，150μL/孔，37℃，5％CO 2培养过夜。
3.免疫脾细胞的制备
小鼠末次免疫后三天，在无菌条件下取出脾脏，置于平皿中，RPMI 1640基础培养液冲洗一次，放于小烧杯的尼龙网上磨碎过滤，制成细胞悬液。离心，弃上清，RPMI 1640基础培养液重悬，如此重复三次，计数。
4.细胞融合
(1)取40mL HAT培养液，15mL DMEM无血清培养液和1mL 50％PEG(M12000)分别置于37℃水浴中预温；
(2)分别取小鼠骨髓瘤细胞Sp2/0(菲鹏生物股份有限公司保存)(2-5×10 7)、上述免疫脾细胞(10 8)悬液加入50mL离心管中混匀，并加DMEM无血清培养液至40mL。离心10分钟，倒尽上清液，混匀；
(3)将离心管置于37℃预温的水中，取0.7mL预温的50％PEG溶液，静置90秒钟。立即滴加37℃15mL预温的无血清培养液。
(4)补加DMEM无血清培养液至40mL，离心10分钟，倒尽上清液。加40mL含15％～20％胎牛血清的HAT培养液。用吸管混匀，滴加到已含有饲养细胞的4块96孔细胞培养板的小孔中，每孔2滴，置37℃、7％CO 2的培养箱中培养；
5.杂交瘤细胞的选择培养
免疫小鼠脾细胞与小鼠骨髓瘤细胞，经PEG处理后，形成多种细胞成分的混合体，其中包括未融合的骨髓瘤细胞和免疫脾细胞；骨髓瘤细胞的共核体和免疫脾细胞的共核体，以及骨髓瘤细胞和免疫脾细胞的异核体。仅后者才能形成杂交瘤细胞。为此，在这多种细胞混合体中必须除去未融合的细胞和同种融合的共核体，并选择出真正的杂交细胞。因此，在细胞融合后第1，3，5，7天用上述的HAT培养液换液培养；
6.特异性抗体的检测及杂交瘤细胞克隆化
吸取每个培养孔的上清液，用间接ELISA法检测出培养液中含N蛋白的特异性抗体的培养孔。采用有限稀释法使杂交瘤细胞克隆化。经过培养后单个细胞可增殖为同源性细胞克隆；经通过反应性筛选共得到40株稳定分泌抗N蛋白单克隆抗体的细胞株(详见以下表1)，其效价均在10 5～10 7之间。
表1筛选得到的稳定分泌抗N蛋白单克隆抗体的细胞株
克隆号 1B6 1D9 2A5 2C9 2F7 3A7 3B4 3D6 3G10
效价 5.6×10 5 4.3×10 6 7.2×10 7 4.6×10 6 5.1×10 6 3.3×10 6 5.9×10 7 8.7×10 5 1.8×10 7
克隆号 4B5 4C10 4F1 4F8 4G3 5A1 6B8 6D11 7A3
效价 5.6×10 7 6.8×10 6 8.4×10 5 7.2×10 5 4.8×10 7 6.1×10 6 7.5×10 6 8.4×10 6 6.4×10 6
克隆号 7F1 8B6 8E2 9B7 9F5 10A3 10C6 10F3 11A5
效价 9.1×10 7 6.7×10 5 3.5×10 7 7.4×10 6 3.9×10 6 5.8×10 6 3.6×10 7 9.4×10 5 4.7×10 6
克隆号 11C10 11G2 12H3 13B5 13F1 14C2 14D9 14E3 14H2
效价 5.6×10 6 7.2×10 6 3.2×10 6 7.4×10 6 7.6×10 6 5.3×10 6 6.6×10 6 6.5×10 6 4.1×10 6
克隆号 15A3 15C8 16C4 16D8          
效价 4.9×10 6 5.2×10 6 7.1×10 6 6.7×10 7          
7.反应性筛选
在室温下，在搅拌时微量滴定板(Nunc，Maxisorb)用100μl/孔含2.5μg/ml N蛋白(作为抗原)的包被缓冲液(包被缓冲液，产品目录号0726 559，Scil Diagnostics，GmbH)包被1小时。包被后处理在PBS缓冲液和1％单克隆抗体中进行孵育30分钟。随后，用洗涤缓冲液进行洗涤。在室温下，在搅拌时将100μl/孔抗体样品孵育1小时。然后用洗涤溶液再洗涤2次。然后在室温下，在搅拌时再与100μl/孔按1:40000用PBS缓冲液稀释的检测抗体过氧化物酶缀合的羊抗鼠IgG孵育1小时。用洗涤缓冲液再次洗涤后，用常规方法测定过氧化物酶活性(例如用，室温30分钟，用ELISA读板器读取405nm的消光值的变化，单位为mU)，上述40株单克隆细胞株分泌的抗体均有较好的反应性。
需要说明的是，上表中的细胞株来源于菲鹏生物股份有限公司经筛选后保存细胞株，是通过反应性筛选获得的，本领技术人员通过该方法或者可以通过类似的ELISA方法或者免疫荧光法、免疫印迹法等技术同样可以筛选获得功能类似的上述细胞株，选择的上述细胞株即为反应性较好且稳定分泌抗N蛋 白单克隆抗体的细胞株。上述细胞株的制备方法和选择不以任何途经限制本公开的范围，仅为示例性的举例说明。
二、抗体结合表位鉴定
采用不同种N蛋白短肽抗原分别包被微孔，以PBS+20％NBS为稀释液，稀释单抗为一抗浓度到1μg/ml，以羊抗鼠IgG-HRP为二抗，依据各单抗对不同抗原的反应情况来确定单抗表位。经统计，上述41株抗体分别是针对N蛋白抗原的如下区段(详见下表2)：
表2抗体以及抗体针对N蛋白抗原的区段
单抗号 1-43 44-180 248-361 50-107 74-105
1B6 + - - - -
1D9 - - + - -
2A5 - - - - +
2C9 + - - - -
2F7 + - - - -
3A7 - - - + -
3B4 - + - - -
3D6 - - - + -
3G10 - - + - -
4B5 - + - - -
4C10 - - - + -
4F1 - - - - +
4F8 + - - - -
4G3 - - + - -
5A1 - + - - -
6B8 - - - - +
6D11 + - - - -
7A3 - - - + -
7F1 - - + - -
8B6 - - - + -
8E2 - - - - +
9B7 + - - - -
9F5 - + - - -
10A3 - - + - -
10C6 - - - + -
10F3 + - - - -
11A5 - - + - -
11C10 - - - + -
11G2 - + - - -
12H3 - - - - +
13B5 - + - - -
13F1 - - - - +
14C2 + - - - -
14D9 - - + - -
14E3 - - - - +
14H2 - - - + -
15A3 - + - - -
15C8 + - - - -
16C4 - - - + -
16D8 - + - - -
“+”表示有反应，“-”表示无反应
N蛋白抗原的区段序列如下：
1-43片段序列(即SARS-Cov-2的第1-43位N蛋白氨基酸片段)如SEQ ID NO:2所示：
Figure PCTCN2022117381-appb-000008
44-180片段序列(即SARS-Cov-2的第44-180位N蛋白氨基酸片段)如SEQ ID NO:3所示：
Figure PCTCN2022117381-appb-000009
248-361片段序列(即SARS-Cov-2的第248-361位N蛋白氨基酸片段)如SEQ ID NO:4所示：
Figure PCTCN2022117381-appb-000010
50-107片段序列(即SARS-Cov-2的第50-107位N蛋白氨基酸片段)如SEQ ID NO:5所示：
ASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPR
74-105片段序列(即SARS-Cov-2的第74-105位N蛋白氨基酸片段)如SEQ ID NO:6所示：
Figure PCTCN2022117381-appb-000011
抗N蛋白抗体表位配对：以针对不同表位抗体效价最高的抗体为配对组合，采用ELISA双抗夹心检测方法，以100份核酸检测阳性样本为测试样本，检出率≥95％则为较好的配对效果，详见表3。
表3抗N蛋白抗体表位配对
  包被 标记 检出率
配对1 16D8 6D11 86％
配对2 16D8 7F1 82％
配对3 16D8 16C4 88％
配对4 16D8 8E2 81％
配对5 7F1 8E2 75％
配对6 16D8 16D8+16C4 96％
配对7 16D8+6D11 8E2+16D8 98％
配对8 16D8+7F1 16D8+8E2 96％
配对9 7F1+6D11 8E2+16C4 90％
配对11 7F1+16C4 8E2+6D11 89％
配对12 16D8+16C4 16D8 97％
配对13 8E2+16D8 16D8+6D11 96％
配对14 16D8+8E2 16D8+7F1 96％
上述检测结果中配对6-8和配对12-14的检出率都高于95％，分析检出率提高的因素是：在包被和标记都至少使用了结合44-180aa(16D8)区段的抗体。
用以上配对组合，采用ELISA双抗夹心检测方法学检测突变株以判断是否会有漏检问题，详见表4
表4利用ELISA双抗夹心检测方法检测突变株
Figure PCTCN2022117381-appb-000012
“+”代表检出，“-”代表未检出
上述检测结果中配对6-8和配对12-14针对D103Y和P13L+R203K+G204R+G214C突变株在0.2ng/ml浓度范围都能够有效的检出，部分配对组合在低至50pg/ml时也可以检出结果。
另外，相同表位配对(配对6-8和配对12-14)，不同株抗体的随机替换，采用ELISA双抗夹心检测方法，以100份核酸检测阳性样本为测试样本检测检出率。结果如下表5：
表5测试样本检测检出率
  包被 标记 检出率
配对15 4B5+3A7 9F5 97％
配对16 12H3+13B5 15A3+15C8 96％
配对17 13B5+13F1 5A1+1D9 96％
配对18 3B4+10A3 15A3+2A5 96％
配对19 9F5+4F1 11G2+3G10 96％
配对20 11G2 9F5+14H2 96％
配对21 4B5+2C9 6B8+9F5 98％
配对22 11G2+14D9 4B5+14E4 96％
配对23 3B4+15C8 4F1+13B5 98％
配对24 11G2+3A7 4B5 97％
配对25 4B5+13F1 13B5+11A5 96％
配对26 15A3+2A5 9F5+14D9 96％
配对27 9F5+1D9 15A3+4F1 96％
配对28 3B4+14H2 11G2 97％
配对29 13B5 13B5+4C10 96％
配对30 3B4+14C2 6B8+4B5 98％
配对31 9F5+3G10 13B5+12H3 96％
配对32 5A1+1B6 12H3+3B4 98％
配对33 15A3 13B5+10C6 96％
配对34 11G2+13F1 9F5+4G3 96％
上述配对在替换其他株抗体后依然能够有较高的检出率。
上述检测结果中，配对15至配对34检出率均高于95％，分析检出率高的原因为上述配对方案中包被抗体和标记抗体均包括结合44-180aa区段的抗体。以上数据同时证明，上述方案时只需要选择3种抗体，即可以实现4种抗体联用的技术效果。
上述配对方案15中的抗体9F5、4B5、3A7为例；其中，9F5为抗体1，4B5为抗体2，3A7为抗体3。
经过测序，获得抗体序列的重链、轻链及划分的CDR如下表6所示：
表6抗体序列的重链、轻链及划分的CDR
Figure PCTCN2022117381-appb-000013
对以上抗体CDR区进行个别突变，得到抗体序列如下表7所示：
表7抗体序列
Figure PCTCN2022117381-appb-000014
备注：重链，轻链，CDR中的编号对应为SEQ ID NO:上的序列号，例如7对应SEQ ID NO:7。
用以上抗体替换原配对15中相应的抗体，检出率均在96％以上。
三、单克隆抗体表位鉴定
用0.06M pH9.6碳酸缓冲溶液稀释纯化好的待鉴定单克隆抗体使其终浓度为1μg/mL。每孔0.1mL加入96孔聚苯乙烯板，37℃孵育2小时或4℃过夜。次日，用含10％小牛血清或1％脱脂奶粉的0.02M pH7.2PBS，0.15mL/孔，37℃封闭2小时，加入2000倍稀释的辣根过氧化酶标记的SARS-Cov-2的N蛋白各表位区段的抗原，37℃30分钟，PBST洗5次，拍干，每孔加入100μL含0.1％(M/V)邻苯二胺，0.1％(V/V)双氧水，pH5.0柠檬酸磷酸缓冲液，37℃15分钟，加入稀硫酸溶液，每孔50μL，测450nm吸收值，根据反应区分表位。
抗体是否与其他抗体识别相同的表位，这可以通过两者对表位的竞争来确认。抗体间的竞争可以通过竞争结合测定来评价，其方法有：ELISA、荧光能量转移测定法或荧光微量测定技术等。经过鉴定，9F5、4B5、3A7对应突变抗体与未突变抗体结合表位相同。
四、SARS-Cov-2胶体金免疫层析试纸条及其制备
本实施例中，免疫层析试纸条包括底板、样品垫、结合垫、硝酸纤维素膜和吸水垫，硝酸纤维素膜上设置有T线和C线，结合垫上设置有上文所述的第二组抗体，其中T线上包被有上文所述的第一组抗体。
免疫层析试纸条的制备包括如下：首先将硝酸纤维素膜固定在底板中部，处理过的胶体金结合垫(将胶体金标记的第二组抗体喷涂在结合垫上)的一端压在硝酸纤维素膜的检测T线端(T线喷涂有第一组抗体)，重叠1mm。处理过的样品垫一端压于胶体金结合垫的另一端，重叠2mm。将吸水垫粘在底板上，其中一端压于硝酸纤维素膜的质控C线端，重叠2mm。将装配好的试纸条检测片用切条机切成4mm宽，放入塑料卡扣底座的凹槽里，塑料盖盖上压紧。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本公开的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对公开专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本公开构思的前提下，还可以做出若干变形和改进，这些都属于本公开的保护范围。因此，本公开专利的保护范围应以所附权利要求为准。
工业实用性
本公开提供了一种SARS-Cov-2检测方法和试剂盒，本公开的方法和试剂盒检测可以提高检测灵敏度和检出率，尤其可以检出新冠变异株，可以有效改善突变株漏检问题，具有优异的实用性能，可广泛地应用于新冠病毒检测领域。

Claims (19)

  1. 一种抗体组合，其特征在于，包含第一组抗体和第二组抗体，所述第一组抗体包括选自结合SARS-Cov-2的N蛋白氨基酸片段44-180的抗体1，所述第二组抗体包括选自结合SARS-Cov-2的N蛋白氨基酸片段44-180的抗体2，所述抗体1和所述抗体2可以是相同的抗体或是不同的抗体；
    优选地，所述第一组抗体还可以包括抗体3-抗体6中的至少一种；
    优选地，所述第二组抗体还可以包括抗体3-抗体6中的至少一种；
    优选地，所述第一组抗体或第二组抗体还包括抗体3；
    优选地，所述第一组抗体还包括抗体4或抗体5，所述第二组抗体还包括抗体6；
    抗体3结合N蛋白氨基酸片段50-107；
    抗体4结合N蛋白氨基酸片段1-43；
    抗体5结合N蛋白氨基酸片段248-361；
    抗体6结合N蛋白氨基酸片段74-105。
  2. 根据权利要求1所述的抗体组合，其特征在于，其中所述抗体的反应效价高于10 5。
  3. 根据权利要求1或2所述的抗体组合，其特征在于，所述第一组抗体用于标记，第二组抗体用于包被；或第二组抗体用于标记，第一组抗体用于包被。
  4. 根据权利要求3所述的抗体组合，其特征在于，其中所述用于标记的抗体用可检测标记物标记，例如金属粒子，如胶体金，荧光标记，发色团标记，电子致密标记，化学发光标记，放射性标记，酶标记，例如放射性同位素，荧光团，罗丹明，荧光素酶，荧光素，辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶菌酶，糖类氧化酶，葡萄糖氧化酶，半乳糖氧化酶，葡萄糖-6-磷酸脱氢酶，生物素/抗生物素蛋白，自旋标记，噬菌体标记，例如吖啶酯标记，例如通过接头如生物素-亲和素添加荧光标记如吖啶酯标记。
  5. 根据权利要求3或4所述的抗体组合，其特征在于，其中所述用于包被的抗体连接至固相，例如磁性颗粒，胶乳粒子、ELISA板、微量滴定板、硝酸纤维素膜或微流控芯片。
  6. 根据权利要求1-5中任一项所述的抗体组合，其特征在于，所述抗体1选自以下(1)至(4)所述抗体的任一种：
    (1)所述抗体1的重链互补决定区VH-CDR1包括SEQ ID NO:7或SEQ ID NO:13所示的氨基酸序列，重链互补决定区VH-CDR2包括SEQ ID NO:8所示的氨基酸序列，重链互补决定区VH-CDR3包括SEQ ID NO:9或SEQ ID NO:14所示的氨基酸序列；所述抗体1的轻链互补决定区VL-CDR1包括SEQ ID NO:10所示的氨基酸序列，轻链互补决定区VL-CDR2包括SEQ ID NO:11所示的氨基酸序列，轻链互补决定区VL-CDR3包括SEQ ID NO:12所示的氨基酸序列；
    (2)所述抗体1重链可变区包括SEQ ID NO:15或SEQ ID NO:16所示的氨基酸序列，所述抗体1轻链可变区包括SEQ ID NO:17所示的氨基酸序列；
    (3)所述抗体1重链包括SEQ ID NO:18或SEQ ID NO:19所示的氨基酸序列，所述抗体1轻链包括SEQ ID NO:20所示的氨基酸序列；
    (4)所述抗体1与表位1结合，所述表位1与(2)或(3)所述的抗体所结合的表位相同。
  7. 根据权利要求1-6中任一项所述的抗体组合，其特征在于，所述抗体2选自以下(a)至(d)所 述抗体的任一种：
    (a)所述抗体2的重链互补决定区VH-CDR1包括SEQ ID NO:21或SEQ ID NO:27所示的氨基酸序列，重链互补决定区VH-CDR2包括SEQ ID NO:22所示的氨基酸序列，重链互补决定区VH-CDR3包括SEQ ID NO:23所示的氨基酸序列；所述抗体2的轻链互补决定区VL-CDR1包括SEQ ID NO:24或SEQ ID NO:28所示的氨基酸序列，轻链互补决定区VL-CDR2包括SEQ ID NO:25所示的氨基酸序列，轻链互补决定区VL-CDR3包括SEQ ID NO:26所示的氨基酸序列；
    (b)所述抗体2重链可变区包括SEQ ID NO:29或SEQ ID NO:30所示的氨基酸序列，所述抗体2轻链可变区包括SEQ ID NO:31或SEQ ID NO:32所示的氨基酸序列；
    (c)所述抗体2重链包括SEQ ID NO:33或SEQ ID NO:34所示的氨基酸序列，所述抗体2轻链包括SEQ ID NO:35或SEQ ID NO:36所示的氨基酸序列；
    (d)所述抗体2与表位2结合，所述表位2与(b)或(c)所述的抗体所结合的表位相同。
  8. 根据权利要求1-7中任一项所述的抗体组合，其特征在于，所述抗体3为选自以下(I)至(Ⅳ)所述抗体的任一种：
    (I)所述抗体3的重链互补决定区VH-CDR1包括SEQ ID NO:37所示的氨基酸序列，重链互补决定区VH-CDR2包括SEQ ID NO:38或SEQ ID NO:43所示的氨基酸序列，重链互补决定区VH-CDR3包括SEQ ID NO:39所示的氨基酸序列；所述抗体3的轻链互补决定区VL-CDR1包括SEQ ID NO:40所示的氨基酸序列，轻链互补决定区VL-CDR2包括SEQ ID NO:41或SEQ ID NO:44所示的氨基酸序列，轻链互补决定区VL-CDR3包括SEQ ID NO:42所示的氨基酸序列；
    (II)所述抗体3重链可变区包括SEQ ID NO:45或SEQ ID NO:46所示的氨基酸序列，所述抗体3轻链可变区包括SEQ ID NO:47或SEQ ID NO:48所示的氨基酸序列；
    (III)所述抗体3重链包括SEQ ID NO:49或SEQ ID NO:50所示的氨基酸序列，所述抗体3轻链包括SEQ ID NO:51或SEQ ID NO:52所示的氨基酸序列；
    (Ⅳ)所述抗体3与表位3结合，所述表位3与(II)或(III)所述的抗体所结合的表位相同。
  9. 根据权利要求1-8中任一项所述的抗体组合，其特征在于，所述抗体1或所述抗体2具有以下特征(A)至(B)中的至少一种：
    (A)所述抗体1或所述抗体2结合SARS-Cov-2的N蛋白的构象依赖性表位；
    (B)所述抗体1或所述抗体2不结合由SARS-Cov-2的N蛋白44-180内的6个连续氨基酸组成的任何多肽。
  10. 一种抗原检测试剂盒，其特征在于，包括如权利要求1-9所述的抗体组合。
  11. 一种SARS-Cov-2抗原检测试剂盒，其用于检测来自受试者样品中的SARS-Cov-2 N蛋白，其特征在于，包括如权利要求1-9所述的抗体组合。
  12. 根据权利要求10或11所述的试剂盒，其特征在于，所述试剂盒是免疫层析试纸条、酶联免疫试剂或化学发光试剂。
  13. 根据权利要求10-12所述的试剂盒，其特征在于，其中所述样品包括健康或病理状态的生物组织、细胞或体液，例如唾液、鼻咽拭子。
  14. 一种SARS-Cov-2免疫层析试纸条，其特征在于，所述免疫层析试纸条包括底板、样品垫、结 合垫、硝酸纤维素膜和吸水垫，所述硝酸纤维素膜上设置有T线和C线，所述结合垫上设置有第二组抗体，其中T线上包被有第一组抗体；或所述结合垫上设置有第一组抗体，T线上包被有第二组抗体。
  15. 权利要求1-9所述的抗体组合在制备检测SARS-Cov-2的试剂盒中的应用。
  16. 根据权利要求1-9任一项中所述的抗体组合，或根据权利10-13任一项中所述的试剂盒，或权利要求14所述疫层析试纸条，用于检测SARS-Cov-2的用途。
  17. 一种检测SARS-Cov-2的方法，其特征在于，包括：
    A)在足以发生结合反应的条件下，使权利要求1-9任一项中所述的抗体组合，或根据权利10-13任一项中所述的试剂盒，或权利要求14所述疫层析试纸条与样品接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物。
  18. 一种诊断受试者在感染SARS-Cov-2或与SARS-Cov-2感染相关疾病中的方法，其特征在于，包括：
    A)在足以发生结合反应的条件下，使权利要求1-9任一项中所述的抗体组合，或根据权利10-13任一项中所述的试剂盒，或权利要求14所述疫层析试纸条与来自所述受试者的样品接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物。
  19. 根据权利要求18所述的方法，其中，所述与SARS-Cov-2感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、呼吸困难、肺炎、严重急性呼吸综合征、肾衰竭。
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