WO2021244629A1 - 检测SARS-CoV-2病毒中和抗体的免疫层析装置及其应用 - Google Patents

检测SARS-CoV-2病毒中和抗体的免疫层析装置及其应用 Download PDF

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WO2021244629A1
WO2021244629A1 PCT/CN2021/098272 CN2021098272W WO2021244629A1 WO 2021244629 A1 WO2021244629 A1 WO 2021244629A1 CN 2021098272 W CN2021098272 W CN 2021098272W WO 2021244629 A1 WO2021244629 A1 WO 2021244629A1
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cov
sars
protein
signal
labeled
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PCT/CN2021/098272
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French (fr)
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覃喜建
虎慧珍
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南京金斯瑞生物科技有限公司
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    • 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
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • 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
    • 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
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention relates to the field of antibody detection, in particular to a method for detecting SARS-CoV-2 virus neutralizing antibodies by immunochromatography technology and an immunochromatographic detection device for SARS-CoV-2 virus neutralizing antibodies.
  • the SARS-CoV-2 virus the pathogen of the novel coronavirus pneumonia (COVID-19), also known as the 2019 Novel Coronavirus (2019-nCoV), is an RNA coronavirus.
  • COVID-19 the pathogen of the novel coronavirus pneumonia
  • 2019-nCoV 2019 Novel Coronavirus
  • RNA coronavirus RNA coronavirus
  • the SARS-CoV-2 virus is a type of RNA virus with an envelope and a linear single-stranded positive-stranded genome.
  • the entry of SARS-CoV-2 virus into host cells is mediated by transmembrane spike (S) glycoprotein (S protein).
  • S protein transmembrane spike (S) glycoprotein (S protein).
  • S protein is divided into two subunits, S1 and S2.
  • S1 subunit is responsible for binding to the host cell receptor
  • the S2 subunit is responsible for the fusion of the viral membrane and the cell membrane.
  • S1 promotes viral infection by binding to the host receptor. It contains two domains, the N-terminal domain and the C-terminal RBD domain that directly interact with the host receptor.
  • the C-terminal RBD domain is located on the SARS-CoV-2 virus subunit, which is the interaction site with the human ACE2 (Angiotensin Converting Enzyme 2) receptor, and plays an important role in the process of virus infection.
  • ACE2 is the receptor for SARS-CoV to enter the cell. It is a zinc metalloprotease, which belongs to type 1 transmembrane protein. Its structure includes a signal peptide, a transmembrane domain and a metalloprotease active site containing the HEMGH zinc binding domain.
  • the full-length ACE2 receptor is composed of 805 amino acids and is located on the luminal surface of epithelial cells.
  • the New York Blood Center used mouse models to prove that the RBD domain of the SARS virus S protein expressed in mammalian, insect and E. coli cells can elicit effective neutralizing antibodies and protective immunity. Studies have shown that during the treatment of patients with new coronavirus pneumonia, plasma therapy for patients in convalescence has achieved good results, showing the potential of neutralizing antibodies in the treatment of new coronavirus pneumonia. S protein is an ideal and effective diagnostic and drug development target for vaccine and antibody research.
  • SARS-CoV-2 virus detection methods mainly include 2019-nCoV virus nucleic acid detection method and serum detection method.
  • SARS-CoV-2 serum detection methods such as chemiluminescence, enzyme-linked immunosorbent assay (ELISA), and colloidal gold detection methods all analyze SARS-CoV-2 total antibody (Ab) and SARS-CoV-2IgM antibody, but The SARS-CoV-2 neutralizing antibody cannot be distinguished from the non-neutralizing antibody, and the immune protection effect cannot be accurately evaluated. Therefore, the development of a kit for detecting SARS-CoV-2 virus neutralizing antibodies can effectively evaluate the protective effect and has an auxiliary diagnostic effect on SARS-CoV-2 infection.
  • ELISA enzyme-linked immunosorbent assay
  • Immunochromatographic test paper has the advantages of more sensitivity, specificity, simplicity, and speed. It is an ideal point-of-care test (POCT) and on-site detection technology. It can determine the results within 1-15 minutes and is widely used in various Qualitative and semi-quantitative rapid detection of analytes, so the development of SARS-CoV-2 virus neutralizing antibody immunochromatographic detection kit is of great significance.
  • POCT point-of-care test
  • SARS-CoV-2 virus neutralizing antibody immunochromatographic detection kit is of great significance.
  • the purpose of the present invention is to develop a SARS-CoV-2 virus neutralizing antibody immunochromatographic device suitable for detecting human serum and plasma.
  • the detection test paper is specific, sensitive, fast, simple, and easy to practice in production. Promotion and application.
  • the present invention provides an immunochromatographic device for detecting neutralizing antibodies to coronaviruses, characterized in that the immunochromatographic device includes a spike protein portion of a coronavirus that specifically binds to the spike protein of the coronavirus Part of the ACE2 protein or its functional fragments and a solid support, on the solid support to detect the influence of the sample to be tested on the binding of the coronavirus spike protein part to the ACE2 protein or its functional fragments, and determine whether the sample is There are coronavirus neutralizing antibodies.
  • the coronavirus spike protein portion or the ACE2 protein or functional fragment thereof is connected to a signal substance.
  • the ACE2 protein or functional fragments thereof, or the coronavirus spike protein portion is immobilized on a solid support.
  • the coronavirus spike protein is partially connected to a signal substance, and the ACE2 protein or functional fragment thereof is immobilized on a solid support; or (2) the ACE2 protein or The functional fragment is connected to the signal substance, and the coronavirus spike protein is partially fixed on the solid support.
  • the influence of the sample to be tested on the binding of the coronavirus spike protein portion to the ACE2 protein or functional fragments thereof is judged by the signal generated by the signal.
  • the signal is selected from at least one of a visual signal, a fluorescent signal, and a magnetic signal.
  • the visual signal includes colloidal gold, colloidal silver, colored latex microspheres and colored dyes, preferably colloidal gold.
  • the fluorescent signal includes fluorescent microspheres and quantum dots.
  • the immunochromatographic device in the present invention is selected from a colloidal gold immunochromatographic device, a quantum dot immunochromatographic device or an immunofluorescence detection device, preferably a colloidal gold immunochromatographic device and an immunofluorescence detection device.
  • the colloidal gold immunochromatographic device includes a gold-labeled coronavirus spike protein portion, and the ACE2 protein or functional fragment thereof is immobilized at a specific position on a solid support, and the The coronavirus neutralizing antibody blocks the binding of the viral spike protein part and the ACE2 protein or its functional fragments to generate a detection signal.
  • the colloidal gold immunochromatographic device includes gold-labeled ACE2 protein or functional fragments thereof, and the coronavirus spike protein is partially immobilized on a solid support at a specific position and passed through the sample to be tested. The coronavirus neutralizing antibody blocks the combination of the viral spike protein part and the ACE2 protein or its functional fragments to generate a detection signal.
  • the quantum dot immunochromatographic device includes a coronavirus spike protein portion labeled with quantum dots, and the ACE2 protein or a functional fragment thereof is fixed on a specific position on a solid support and passes through the sample to be tested.
  • the coronavirus neutralizing antibody blocks the combination of the viral spike protein part and the ACE2 protein or its functional fragments to generate a detection signal.
  • the quantum dot immunochromatographic device includes a quantum dot-labeled ACE2 protein or a functional fragment thereof, and the coronavirus spike protein is partially fixed on a specific position on a solid support and passes through the sample to be tested.
  • the virus-neutralizing antibody in the virus blocks the combination of the viral spike protein part and the ACE2 protein or its functional fragments to generate a detection signal.
  • the quantum dots are selected from compounds composed of groups IV and VI or quantum dot particles assembled from quantum dot compounds and other chemical substances.
  • the immunofluorescence detection device is a time-resolved immunofluorescence detection device, the device includes a coronavirus spike protein portion labeled with immunofluorescence microspheres, and the ACE2 protein or a functional fragment thereof is immobilized on a solid support At a specific location on the object, a detection signal is generated by blocking the combination of the viral spike protein part and the ACE2 protein or its functional fragments by the virus-neutralizing antibody in the sample to be tested.
  • the immunofluorescence detection device is a time-resolved immunofluorescence detection device, and the device includes an immunofluorescence microsphere labeled ACE2 protein or a functional fragment thereof, and the coronavirus spike protein is partially immobilized on a solid phase.
  • a detection signal is generated by blocking the combination of the viral spike protein part and the ACE2 protein or its functional fragments by the virus-neutralizing antibody in the sample to be tested.
  • the immunofluorescence microspheres are selected from immunofluorescence microspheres containing samarium (SM), immunofluorescence microspheres containing europium (Eu), immunofluorescence microspheres containing dysprosium (Dy) or immunofluorescence microspheres containing technetium (Te).
  • SM immunofluorescence microspheres containing samarium
  • Eu immunofluorescence microspheres containing europium
  • Dy dysprosium
  • Te technetium
  • the coronavirus in the present invention is selected from SARS-CoV-2 or its variants, SARS-CoV or its variants, or MERS-Cov or its variants, preferably SARS-CoV-2 or its variants.
  • the coronavirus is a SARS-CoV-related virus.
  • the coronavirus is a SARS-CoV-2 related virus.
  • the coronavirus is SARS-CoV-2 or a variant thereof.
  • the coronavirus is SARS-CoV-2.
  • the coronavirus spike protein in the present invention is partly selected from coronavirus S protein, coronavirus S1 protein, coronavirus RBD protein or coronavirus RBD protein with His or Fc tag, preferably coronavirus S1 protein or coronavirus RBD
  • the protein is more preferably the SARS-CoV-2 virus RBD protein.
  • the coronavirus spike protein portion is a coronavirus S1 protein or a coronavirus RBD protein.
  • the coronavirus spike protein part is SARS-CoV-2 virus S1 protein or SARS-CoV-2 virus RBD protein. More preferably, the coronavirus spike protein portion is the SARS-CoV-2 virus RBD protein.
  • the coronavirus spike protein portion comprises a sequence that is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 or 2. In other embodiments, the coronavirus spike protein portion comprises at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87% of the amino acid sequence shown in SEQ ID NO: 1 or 2. %, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical sequences. In some embodiments, the coronavirus spike protein portion includes the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the coronavirus spike protein portion includes the amino acid sequence shown in SEQ ID NO: 2. In a specific embodiment, the partial amino acid sequence of the coronavirus spike protein is shown in SEQ ID NO:1. In a specific embodiment, the partial amino acid sequence of the coronavirus spike protein is shown in SEQ ID NO: 2.
  • the ACE2 protein or its functional fragments are derived from humans.
  • the ACE2 protein or functional fragment thereof is selected from a recombinant ACE2 protein or an Fc-tagged ACE2 protein.
  • the ACE2 protein is selected from human recombinant ACE2 protein.
  • the ACE2 protein or functional fragment thereof comprises a sequence that is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the ACE2 protein or functional fragment thereof comprises at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least the amino acid sequence shown in SEQ ID NO: 3 Sequences that are 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical. In some embodiments, the ACE2 protein or functional fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 3. In a specific embodiment, the amino acid sequence of the ACE2 protein or functional fragment thereof is shown in SEQ ID NO: 3.
  • the solid support in the present invention is selected from nitrocellulose membranes and cellulose acetate membranes, preferably nitrocellulose membranes.
  • the solid support is a nitrocellulose membrane.
  • the nitrocellulose membrane can be any commercial nitrocellulose membrane, including S&SAE99, whatman 8um, Millipore M135, Sartoirus CN140, etc.
  • the present invention provides an immunochromatographic device for detecting neutralizing antibodies to coronavirus, which is characterized in that the device includes a detection test strip, and the test strip includes a sample loading pad, a binding pad, and a reaction pad that are sequentially overlapped And an absorbent pad, the sample loading pad, the binding pad, the reaction pad and the absorbent pad are sequentially adhered to the bottom plate, and the reaction pad includes a test line and a quality control line arranged in sequence along the sample flow direction, and the test line includes Coronavirus spike protein part or ACE2 protein or functional fragments thereof, the device further comprises a signal-labeled ACE2 protein or functional fragments thereof, or a signal-labeled coronavirus spike protein part, and the coronavirus spike protein part is compatible with ACE2 protein or its functional fragments combine to produce a detection signal on the detection line.
  • the device when the detection line contains the ACE2 protein or a functional fragment thereof, the device includes a signal-labeled coronavirus spike protein portion, and the signal-labeled coronavirus spike protein portion is connected to the ACE2 protein or its functional fragments combine to generate a detection signal.
  • the device when the detection line includes the coronavirus spike protein portion, the device includes a signal-labeled ACE2 protein or a functional fragment thereof, and the signal-labeled ACE2 protein or a functional fragment thereof is The coronavirus spike protein binds to produce a detection signal.
  • the ACE2 protein or its functional fragments are selected from recombinant ACE2 protein or ACE2 protein with Fc tag.
  • the ACE2 protein or functional fragment thereof is a recombinant human ACE2 protein.
  • the ACE2 protein or functional fragment thereof contains a sequence that is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 3.
  • the ACE2 protein or functional fragment thereof comprises at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least the amino acid sequence shown in SEQ ID NO: 3 Sequences that are 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical.
  • the ACE2 protein or functional fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 3.
  • the amino acid sequence of the ACE2 protein or functional fragment thereof is shown in SEQ ID NO: 3.
  • the coronavirus spike protein portion includes a coronavirus S protein, a coronavirus S1 protein, a coronavirus RBD protein, or a coronavirus RBD protein with a His or Fc tag.
  • the coronavirus spike protein portion is a SARS-CoV-2 virus spike protein portion. More preferably, the coronavirus spike protein portion is SARS-CoV-2 virus S1 protein or SARS-CoV-2 virus RBD protein. More preferably, the spike protein portion of the SARS-CoV-2 virus contains a sequence that is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • the SARS-CoV-2 viral spike protein portion comprises at least 70%, at least 75%, at least 80%, at least 83%, at least 85% of the amino acid sequence shown in SEQ ID NO: 1 or 2. , At least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical sequence.
  • the SARS-CoV-2 virus spike protein portion includes SARS-CoV-2 virus S1 protein or SARS-CoV-2 virus RBD protein.
  • the SARS-CoV-2 virus RBD protein comprises at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87%, at least the amino acid sequence shown in SEQ ID NO:1 Sequences that are 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical.
  • the SARS-CoV-2 virus S1 protein comprises at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, at least 87% of the amino acid sequence shown in SEQ ID NO: 2. %, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical sequences.
  • the SARS-CoV-2 virus RBD protein comprises the amino acid sequence shown in SEQ ID NO:1. In other specific embodiments, the SARS-CoV-2 virus S1 protein comprises the amino acid sequence shown in SEQ ID NO: 2. In some specific embodiments, the amino acid sequence of the SARS-CoV-2 virus RBD protein is shown in SEQ ID NO:1. In other specific embodiments, the amino acid sequence of the SARS-CoV-2 virus RBD protein is shown in SEQ ID NO: 2.
  • the quality control line includes a first molecule
  • the loading pad includes a second molecule or a second molecule is added to the loading pad during detection, and the second molecule can bind to the first molecule Generate a detection signal.
  • the first molecule and the second molecule are respectively selected from any one of an immunoglobulin and an anti-immunoglobulin antibody, a receptor and a ligand, or a pair of molecules composed of biotin and avidin.
  • the first molecule is selected from anti-immunoglobulin antibodies, ligands, or biotin
  • the second molecule is selected from signal-labeled immunoglobulins, signal-labeled receptors or Avidin.
  • the first molecule is selected from immunoglobulin, receptor or avidin
  • the second molecule is selected from signal-labeled anti-immunoglobulin antibody, signal-labeled ligand Or biotin.
  • the first molecule is an anti-immunoglobulin antibody
  • the second molecule is an immunoglobulin labeled with a signal.
  • the first molecule is an immunoglobulin
  • the second molecule is an anti-immunoglobulin antibody labeled with a signal.
  • the first molecule is a ligand and the second molecule is a receptor labeled with a signal.
  • the first molecule is a receptor and the second molecule is a ligand labeled with a signal.
  • the first molecule is biotin and the second molecule is avidin.
  • the first molecule is avidin and the second molecule is biotin.
  • the ACE2 protein or functional fragments thereof are uniformly streaked or spray coated at 0.2-1.0ug/cm to form a detection line. In some embodiments, the ACE2 protein or a functional fragment thereof is uniformly streaked at 0.2-1.0ug/cm to form a detection line at the position of the detection line.
  • the ACE2 protein or functional fragment thereof is at a rate of about 0.2ug/cm, 0.3ug/cm, 0.4ug/cm, 0.5ug/cm, 0.6ug/cm, 0.7ug/cm, 0.8ug/cm cm, 0.9ug/cm or 1.0ug/cm uniformly scribed and coated at the position of the detection line to form a detection line.
  • the ACE2 protein or functional fragments thereof are uniformly streaked or spray coated at 0.3 ⁇ g/cm to form a detection line.
  • the ACE2 protein or functional fragments thereof are uniformly streaked or spray coated at 0.5 ⁇ g/cm to form a detection line.
  • the amount of the viral spike protein portion labeled by the signal substance is 1.5 ⁇ g-5 mg.
  • the viral spike protein portion of the signal marker is the SARS-CoV-2 virus RBD protein.
  • the amount of the colloidal gold-labeled viral spike protein portion is 1.0-5 mg.
  • the amount of the colloidal gold-labeled SARS-CoV-2 virus RBD protein is about 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, or 5.0 mg.
  • the portion of the coronavirus spike protein labeled with fluorescent microspheres is 1.0-5 ⁇ g. In some specific embodiments, the amount of SARS-CoV-2 virus RBD protein labeled with fluorescent microspheres is about 1.0 ⁇ g, 1.5 ⁇ g, 2.0 ⁇ g, 2.5 ⁇ g, 3.0 ⁇ g, 3.5 ⁇ g, 4.0 ⁇ g, 4.5 ⁇ g or 5.0 ⁇ g.
  • the viral spike protein portion is uniformly streaked or spray coated at 0.5-1.5 ⁇ g/cm to form a detection line.
  • the viral spike protein portion is the SARS-CoV-2 virus RBD protein.
  • the viral spike protein portion is 0.5ug/cm, 0.6ug/cm, 0.7ug/cm, 0.8ug/cm, 0.9ug/cm, 1.0ug/cm, 1.1ug/cm, The amount of 1.2ug/cm, 1.3ug/cm, 1.4ug/cm or 1.5ug/cm is evenly scribed or sprayed to form a detection line at the position of the detection line.
  • the viral spike protein portion is uniformly streaked or spray coated at 0.3 ⁇ g/cm to form a detection line.
  • the signal-labeled ACE2 protein or its functional fragments and the signal-labeled immunoglobulin antibody are mixed and sprayed on the binding pad in a volume ratio of 0.5:1 to 1:1.5, wherein the signal-labeled ACE2 protein or The amount of the functional fragment is 0.1-1 mg/ml, and the immunoglobulin antibody labeled with the signal substance is 0.1-1 mg/ml.
  • the signal-labeled ACE2 protein or its functional fragment and the signal-labeled immunoglobulin antibody are mixed and sprayed on the binding pad in a volume ratio of 1:1, wherein the signal-labeled ACE2 protein or its functional fragment The amount is 1mg/ml, and the immunoglobulin antibody labeled with signal is 1mg/ml.
  • the anti-immunoglobulin antibody contained in the quality control line of the present invention is selected from an anti-mouse IgG antibody or an anti-goat IgG antibody; the signal marker immunoglobulin antibody is correspondingly selected from a mouse IgG antibody or a goat IgG antibody.
  • the anti-immunoglobulin antibody contained in the quality control line is an anti-mouse IgG antibody, and the signal marker immunoglobulin antibody corresponds to a mouse IgG antibody.
  • the anti-immunoglobulin antibody contained in the quality control line is an anti-goat IgG antibody, and the signal marker immunoglobulin antibody corresponds to a goat IgG antibody.
  • the signal substance of the present invention is selected from colloidal gold, quantum dots or immunofluorescence microspheres, preferably colloidal gold and immunofluorescence microspheres.
  • the immunofluorescence microspheres are selected from immunofluorescence microspheres containing samarium (SM), immunofluorescence microspheres containing europium (Eu), immunofluorescence microspheres containing dysprosium (Dy), or immunofluorescence microspheres containing technetium (Te) Fluorescent microspheres.
  • the quantum dots are selected from compounds composed of groups IV and VI or quantum dot particles assembled from quantum dot compounds and other chemical substances.
  • the reaction pad of the present invention is a nitrocellulose membrane (NC membrane) or a cellulose acetate membrane, preferably a nitrocellulose membrane.
  • the bottom plate is made of polyethylene material.
  • the immunochromatographic device of the present invention also includes a sample diluent.
  • the sample diluent includes 20-50 mM PBS, 0.01% Tween-20, 0.09% NaCl, 0.03% ProcLin300, and pH 7.0-7.5.
  • the samples in the present invention include:
  • the present invention provides a method for using the above immunochromatographic device, which includes the following steps:
  • the signal marker is a portion of the coronavirus spike protein labeled with colloidal gold, selected from the group consisting of the S1 subunit of the coronavirus spike protein labeled with colloidal gold and the coronavirus labeled with colloidal gold RBD, Coronavirus spike protein S1 subunit labeled with fluorescent microspheres or Coronavirus RBD labeled with fluorescent microspheres.
  • the signal marker is SARS-CoV-2 spike protein S1 subunit labeled with colloidal gold, SARS-CoV-2 RBD labeled with colloidal gold, and SARS-CoV-2 spike protein S1 with fluorescent microspheres.
  • the SARS-CoV-2 RBD labeled with subunits or fluorescent microspheres is preferably SARS-CoV-2 RBD labeled with colloidal gold or SARS-CoV-2 RBD labeled with fluorescent microspheres.
  • the present invention provides another method of using an immunochromatographic device, which includes the following steps:
  • the signal marker in this method is colloidal gold-labeled ACE2 protein or functional fragments thereof, preferably colloidal gold-labeled human recombinant ACE2 protein or functional fragments thereof.
  • the present invention provides a method for preparing the aforementioned immunochromatographic device, which is characterized in that it comprises the following steps:
  • the device in the step (2), when the ACE2 protein or a fragment thereof is coated on the reaction pad to form a detection line, the device further includes a signal-labeled coronavirus spike protein portion.
  • the ACE2 protein or fragments thereof are uniformly streaked or spray coated at the position of the detection line at 0.2-1.0 ug/cm to form a detection line.
  • the amount of the signal-labeled coronavirus spike protein portion contained in the device is 1.0 ⁇ g-5 mg.
  • the amount of the colloidal gold-labeled coronavirus spike protein portion is 1.0 mg-5 mg.
  • the amount of the coronavirus spike protein portion labeled with the fluorescent microsphere is 1.0 ⁇ g-5 ⁇ g.
  • the device in the step (2), when the coronavirus spike protein is partially coated on the reaction pad to form a detection line, the device further includes a signal-labeled ACE2 protein or a fragment thereof. In some embodiments, in the step (2), the 0.5-1.5 ug/cm virus spike protein portion is uniformly streaked or sprayed to form a detection line at the position of the detection line.
  • the step (2) further includes mixing the signal-labeled ACE2 protein or fragments thereof and the signal-labeled immunoglobulin antibody at a ratio of 1:1 and spraying on the binding pad, wherein the signal-labeled ACE2 The protein or its functional fragment is 0.1-1 mg/ml, and the signal-labeled immunoglobulin antibody is 0.1-1 mg/ml.
  • the immunochromatographic device prepared by the invention is an immunochromatographic test strip.
  • the test strip is put into the box to make an immunoassay kit.
  • the present invention provides the application of the above immunochromatographic device in detecting a subject infected or suspected of being infected with the SARS-CoV-2 virus or its variants.
  • the present invention provides the application of the above immunochromatographic device in detecting the neutralizing antibody of SARS-CoV-2 virus or its variants in a sample.
  • the sample is derived from a subject infected or suspected of being infected with the SARS-CoV-2 virus or a variant thereof, and a person who has recovered from the infection with the SARS-CoV-2 virus or a variant thereof, and is vaccinated against SARS -Subjects of vaccines of CoV-2 or variants thereof, or antibodies against SARS-CoV-2 or variants thereof obtained by biological methods.
  • the aforementioned immunochromatographic device is used to detect whether a subject has an effective immunity after being vaccinated with a vaccine against SARS-CoV-2 or a variant thereof.
  • the above immunochromatographic device is used to detect the application of anti-SARS-CoV-2 antibodies obtained by biological methods.
  • the principle of the present invention can be expressed as: a detection signal to detect SARS in a sample by blocking the binding of SARS-CoV-2 virus RBD protein and ACE2 receptor by immunochromatographic action with SARS-CoV-2 virus neutralizing antibody CoV-2 virus neutralizing antibody. It fixes the recombinant ACE2 protein on the detection membrane.
  • the neutralizing antibody binds to the signal-labeled SARS-CoV-2 virus RBD protein to block the recombinant ACE2 protein on the membrane and the signal-labeled SARS-CoV- 2
  • the virus RBD protein is bound, no detection signal (or band) appears, and the detection result is positive.
  • the recombinant ACE2 receptor on the membrane binds to the SARS-CoV-2 virus RBD protein labeled with the signal. A detection signal (or band) appears, and the test result is negative (see Figure 1).
  • the principle of the present invention can also be expressed as: a detection signal to detect SARS in a sample by blocking the binding of ACE2 receptor and SARS-CoV-2 virus RBD protein by immunochromatographic action with SARS-CoV-2 virus neutralizing antibody -CoV-2 virus neutralizing antibody. It fixes the recombinant SARS-CoV-2 virus RBD protein on the detection membrane.
  • the SARS-CoV-2 virus RBD protein neutralizing antibody in the sample competes with the signal-labeled recombinant ACE2 receptor to bind to the recombinant SARS-CoV-2 virus RBD protein immobilized on the carrier membrane.
  • the neutralizing antibody binds to the signal-labeled SARS-CoV-2 virus RBD protein to block the recombinant ACE2 protein on the membrane and the signal-labeled SARS-CoV- 2
  • the virus RBD protein is bound, and there is no detection signal (or band) on the detection line, and the detection result is positive.
  • the recombinant SARS-CoV-2 virus RBD protein on the membrane binds to the recombinant ACE2 protein labeled with the signal.
  • a detection signal (or band) appears on the detection line, and the detection result is negative (see Figure 2).
  • the immunochromatographic device of the present invention refers to an application form formed by the combination of immunochromogenic labeling technology and antigen-antibody reaction, and includes immunochromatographic test papers.
  • the immunological test papers are packaged into a box, such as a cartridge forming an immune system. Chromatography kit.
  • the specific device of the present invention can be embodied as being composed of a reaction tube, a support plate (bottom plate), a sample addition pad, a binding pad, a detection film (reaction pad) and a water absorption pad.
  • the reaction tube contains signal-labeled SARS-CoV-2 virus RBD protein
  • the binding pad is sprayed with signal-labeled mouse monoclonal antibody
  • the detection membrane contains a detection line (T line) and a quality control line (C line).
  • the detection line (T line) was sprayed with recombinant human ACE2 receptor, and the quality control line (C line) was sprayed with rabbit anti-mouse antibody (see Figure 3).
  • the invention provides an immunochromatographic device for detecting neutralizing antibodies of coronavirus, and specifically relates to a neutralizing antibody immunochromatographic detection kit of SARS-CoV-2 virus or its variants, which can be embodied as SARS-CoV-2 virus Neutralizing antibody colloidal gold detection kit of or its variants, neutralizing antibody quantum dot detection kit of SARS-CoV-2 virus or its variants, immunofluorescence of neutralizing antibody of SARS-CoV-2 virus or its variants Detection kit, time-resolved immunofluorescence detection kit for neutralizing antibody of SARS-CoV-2 virus or its variants, and blocking the interaction between SARS-CoV-2 virus RBD protein and ACE2 protein using the principle of chromatography and neutralizing antibody Kit for signal generation.
  • SARS-CoV-2 virus Neutralizing antibody colloidal gold detection kit of or its variants neutralizing antibody quantum dot detection kit of SARS-CoV-2 virus or its variants
  • the neutralizing antibody immunochromatographic kit for detecting SARS-CoV-2 virus or its variants includes the spike protein part of SARS-CoV-2 virus or its variants, and specifically binds to the coronavirus spike The ACE2 protein or its functional fragments of the protein part and a solid support. Further, the effect of the sample to be tested on the binding of the spike protein portion of the coronavirus with the ACE2 protein or functional fragments thereof is detected on the solid support to determine whether the SARS-CoV-2 coronavirus or its variants are present in the sample Of neutralizing antibodies.
  • the present invention provides a neutralizing antibody immunochromatographic kit for detecting SARS-CoV-2 virus
  • the kit includes the spike protein portion of SARS-CoV-2 virus, and specifically binds to the coronavirus spike protein Part of the ACE2 protein or its functional fragments and a solid support, on the solid support to detect the influence of the sample to be tested on the binding of the coronavirus spike protein part to the ACE2 protein or its functional fragments, and determine whether the sample is There are neutralizing antibodies to SARS-CoV-2 coronavirus or its variants.
  • the spike protein of the SARS-CoV-2 virus or a variant thereof is connected to a signal, and the ACE2 protein or a functional fragment thereof is immobilized on a solid-phase carrier.
  • the ACE2 protein or a functional fragment thereof is connected to a signal, and the spike protein of the SARS-CoV-2 virus or a variant thereof is immobilized on a solid carrier.
  • the neutralizing antibody colloidal gold detection kit for SARS-CoV-2 virus or its variants of the present invention can be embodied as a gold particle-labeled SARS-CoV-2 virus or its variant RBD protein, and the ACE2 protein is fixed on a solid support In a specific location, a detection kit that blocks the binding of RBD protein and ACE2 protein to generate a detection signal by SARS-CoV-2 virus neutralizing antibody.
  • the SARS-CoV-2 virus or its variant neutralizing antibody colloidal gold detection kit includes a detection test strip, the test strip including a sample pad, a binding pad, a reaction pad, and water absorption are sequentially adhered to the bottom plate
  • the reaction pad includes a detection line and a quality control line arranged in sequence, the detection line is coated with ACE2 protein, and the quality control line is coated with an anti-immunoglobulin antibody.
  • the colloidal gold detection kit includes a test strip, the test strip including a sample loading pad, a bonding pad, a reaction pad, and an absorbent pad that are sequentially overlapped, the sample loading pad, The binding pad, the reaction pad, and the absorbent pad are sequentially adhered to the bottom plate.
  • the reaction pad is sequentially provided with a detection line and a quality control line along the flow direction of the sample.
  • the detection line contains recombinant human ACE2 protein, and the quality control line contains anti- Immunoglobulin antibodies.
  • the ACE2 protein 0.2-1.0 ⁇ g/cm is uniformly coated on the detection line (T line) of the reaction pad to form a detection line.
  • the ACE2 protein is uniformly coated on the detection line position of the reaction pad at 0.5 ⁇ g/cm to form a detection line.
  • the concentration of the anti-immunoglobulin antibody is 0.5-3.6 ⁇ g/cm and is uniformly coated on the position of the quality control line (line C) of the reaction pad to form a quality control line. Dry the reaction pad coated with the detection line and the quality control line for later use.
  • the kit includes colloidal gold-labeled SARS-CoV-2 virus RBD protein and colloidal gold-labeled immunoglobulin.
  • the colloidal gold labeled immunoglobulin is sprayed on the binding pad.
  • Spray the colloidal gold-labeled immunoglobulin (concentration 1 mg/ml) according to conventional operations in the art to ensure that a signal reaction occurs in the chromatographic reaction.
  • the colloidal gold-labeled immunoglobulin 3.75 ⁇ l/ml was sprayed onto the treated binding pad.
  • the colloidal gold-labeled SARS-CoV-2 virus RBD protein is dried and used as a gold label tube to be mixed with the sample solution.
  • the amount of the colloidal gold-labeled SARS-CoV-2 virus RBD protein is 1.0-5 mg.
  • the amount of the colloidal gold-labeled SARS-CoV-2 virus RBD protein contained in the gold label tube is 1.5 mg, which is to be mixed with the sample solution.
  • the kit also includes a sample diluent.
  • the sample diluent includes 20-50mM PBS, 0.01% Tween-20, 0.09% NaCl, 0.03% ProcLin300, pH 7.0-7.5
  • the preparation method of the neutralizing antibody colloidal gold detection kit for SARS-CoV-2 virus or its variants of the present invention includes the following steps:
  • Step (1) includes drying the colloidal gold-labeled SARS-CoV-2 virus or its variant RBD into a gold label tube.
  • the amount of the colloidal gold-labeled SARS-CoV-2 virus RBD protein is 1.0-5.0 mg, preferably 1.5 mg.
  • the ACE2 protein is uniformly coated on the detection line (T line) of the reaction pad at 0.2-1.0 ⁇ g/cm to form a detection line.
  • T line detection line
  • 0.5 ⁇ g/cm of the ACE2 protein is uniformly coated on the detection line position of the reaction pad to form a detection line.
  • 0.5-3.6 ⁇ g/cm of the anti-immunoglobulin antibody is uniformly coated on the position of the quality control line (C line) of the reaction pad to form a quality control line. Dry the reaction pad coated with the detection line and the quality control line for later use.
  • the concentration of the colloidal gold-labeled immunoglobulin is 1 mg/ml, and 3.75 ⁇ l/ml is sprayed on the treated binding pad and dried for use.
  • Step (3) includes cutting the assembled sheet into test strips, and assemble the cut test strips into the prepared test strip card housing, the sample loading window corresponds to the sample pad of the test strip, and the result display window corresponds to the test strip
  • the detection area is sealed with a desiccant in an aluminum foil bag.
  • the prepared gold marker tube, the SARS-CoV-2 virus neutralizing antibody colloidal gold test strip and the sample diluent are placed in the box and assembled into a kit.
  • the neutralizing antibody colloidal gold detection kit for SARS-CoV-2 virus or its variants of the present invention can also be embodied as gold particle-labeled ACE2 protein, and SARS-CoV-2 virus or its variant RBD protein is immobilized on a solid support It is a detection kit that uses SARS-CoV-2 virus neutralizing antibody to block the binding of RBD protein and ACE2 protein to produce a detection signal at a specific location.
  • the SARS-CoV-2 virus or its variant neutralizing antibody colloidal gold detection kit includes a test strip including a sample pad, a binding pad, a reaction pad, and an absorbent pad that are sequentially overlapped
  • the sample loading pad, the binding pad, the reaction pad, and the water-absorbing pad are sequentially adhered to the bottom plate, the reaction pad is sequentially provided with a detection line and a quality control line along the sample flow direction, and the detection line is coated with SARS-CoV -2 virus or its variant RBD protein, the quality control line is coated with an anti-immunoglobulin antibody.
  • the colloidal gold detection kit includes a test strip, the test strip including a sample loading pad, a bonding pad, a reaction pad, and an absorbent pad that are sequentially overlapped, the sample loading pad, The binding pad, the reaction pad, and the absorbent pad are sequentially adhered to the bottom plate.
  • the reaction pad is sequentially provided with a detection line and a quality control line along the sample flow direction.
  • the detection line contains the recombinant SARS-CoV-2 virus RBD protein.
  • the quality control line contains anti-mouse IgG antibodies.
  • the RBD protein concentration of the SARS-CoV-2 virus is 0.5-1.5 ⁇ g/cm and is uniformly coated on the detection line (T line) of the reaction pad to form a detection line.
  • the SARS-CoV-2 virus RBD protein is uniformly coated on the detection line position of the reaction pad at 1.0 ⁇ g/cm to form a detection line.
  • the concentration of the anti-immunoglobulin antibody is 0.5-3.6 ⁇ g/cm and is uniformly coated on the position of the quality control line (line C) of the reaction pad to form a quality control line. Dry the reaction pad coated with the detection line and the quality control line for later use.
  • the kit includes colloidal gold-labeled ACE2 protein (concentration between 0.1-1 mg/ml) and colloidal gold-labeled immunoglobulin (concentration between 0.1-1.0 mg/ml).
  • the gold-labeled ACE2 protein and the colloidal gold-labeled immunoglobulin are thoroughly mixed at a volume ratio of 1:1, and sprayed on the binding pad at 3.75 ⁇ l/ml to dry, wherein the colloidal gold-labeled ACE2 protein is 0.1-1 mg/ ml, the immunoglobulin antibody labeled with colloidal gold is 0.1-1 mg/ml.
  • the kit also includes a sample diluent.
  • the sample diluent includes 20-50 mM PBS, 0.01% Tween-20, 0.09% NaCl, 0.03% ProcLin300, and pH 7.0-7.5.
  • the preparation method of the neutralizing antibody colloidal gold detection kit for SARS-CoV-2 virus or its variants of the present invention includes the following steps:
  • the SARS-CoV-2 virus RBD protein 0.5-1.5 ⁇ g/cm is uniformly coated on the detection line (T line) of the reaction pad to form a detection line.
  • the SARS-CoV-2 virus RBD protein 1.0 ⁇ l/cm is uniformly coated on the detection line position of the reaction pad to form a detection line.
  • the concentration of the anti-immunoglobulin antibody is 0.5-3.6 ⁇ g/cm and is uniformly coated on the position of the quality control line (line C) of the reaction pad to form a quality control line.
  • the gold-labeled ACE2 protein (1mg/ml) and colloidal gold-labeled immunoglobulin (1mg/ml) described in step (2) are mixed thoroughly at a volume ratio of 1:1, sprayed on the binding pad at 3.75 ⁇ l/ml and dried.
  • the colloidal gold-labeled ACE2 protein is 1 mg/ml
  • the colloidal gold-labeled immunoglobulin antibody is 1 mg/ml.
  • Step (3) includes cutting the assembled sheet into test strips, and assemble the cut test strips into the prepared test strip card housing, the sample loading window corresponds to the sample pad of the test strip, and the result display window corresponds to the test strip
  • the detection area is sealed with a desiccant in an aluminum foil bag.
  • the prepared SARS-CoV-2 virus neutralizing antibody colloidal gold test strips and the sample diluent are placed in the box in order and assembled into a kit.
  • the neutralizing antibody time-resolved immunofluorescence detection kit of the SARS-CoV-2 virus or its variants of the present invention can be embodied as immunofluorescence microspheres labeling the SARS-CoV-2 virus RBD protein, and the ACE2 protein is fixed on a solid support At a specific location, a detection kit that blocks the binding of RBD protein and ACE2 protein to generate a detection signal by the neutralizing antibody of the SARS-CoV-2 virus or its variants.
  • the SARS-CoV-2 virus or its variant neutralizing antibody colloidal gold detection kit includes a test strip including a sample pad, a binding pad, a reaction pad, and an absorbent pad that are sequentially overlapped
  • the sample loading pad, the binding pad, the reaction pad and the water-absorbing pad are sequentially adhered to the bottom plate, the reaction pad is sequentially provided with a detection line and a quality control line along the sample flow direction, and the detection line is coated with ACE2 protein, The quality control line is coated with an anti-immunoglobulin antibody.
  • the colloidal gold detection kit includes a test strip, the test strip including a sample loading pad, a bonding pad, a reaction pad, and an absorbent pad that are sequentially overlapped, the sample loading pad, The binding pad, the reaction pad, and the absorbent pad are sequentially adhered to the bottom plate.
  • the reaction pad is sequentially provided with a detection line and a quality control line along the sample flow direction.
  • the detection line is coated with recombinant ACE2 protein, and the quality control line includes It has anti-mouse IgG antibody.
  • the ACE2 protein is uniformly coated on the detection line (T line) of the reaction pad at 0.2-1.0 ⁇ g/cm to form a detection line.
  • the ACE2 protein is uniformly coated on the detection line position of the reaction pad at 0.3 ⁇ g/cm to form a detection line.
  • the anti-immunoglobulin antibody is uniformly coated on the position of the quality control line (C line) of the reaction pad at 0.5-3.6 ⁇ g/cm to form a quality control line. Dry the reaction pad coated with the detection line and the quality control line for later use.
  • kits includes SARS-CoV-2 virus RBD protein labeled with fluorescent microspheres and immunoglobulin labeled with fluorescent microspheres.
  • the immunoglobulin labeled with the fluorescent microspheres (at a concentration of 0.1 mg/ml) is sprayed on the binding pad.
  • the immunoglobulin labeled with the fluorescent microspheres was sprayed on the treated binding pad at 3.75 ⁇ l/ml.
  • the SARS-CoV-2 virus RBD protein labeled by the fluorescent microspheres is dried and used as a marker tube to be mixed with the sample solution.
  • the amount of SARS-CoV-2 virus RBD protein labeled with fluorescent microspheres is 1.0 ⁇ g-5 ⁇ g. In a preferred embodiment, the amount of SARS-CoV-2 virus RBD protein labeled with fluorescent microspheres is 1.5 ⁇ g.
  • the kit also includes a sample diluent.
  • the sample diluent includes 20-50 mM PBS, 0.01% Tween-20, 0.09% NaCl, 0.03% ProcLin300, and pH 7.0-7.5.
  • the preparation method of the neutralizing antibody colloidal gold detection kit for SARS-CoV-2 virus or its variants of the present invention includes the following steps:
  • Step (1) includes drying the SARS-CoV-2 virus or its variant RBD labeled with fluorescent microspheres into a fluorescent microsphere marker tube.
  • the amount of SARS-CoV-2 virus RBD protein labeled with fluorescent microspheres is 1.0 ⁇ g-5 ⁇ g. In a preferred embodiment, the amount of SARS-CoV-2 virus RBD protein labeled with fluorescent microspheres is 1.5 ⁇ g.
  • the ACE2 protein is uniformly coated on the detection line (T line) of the reaction pad at 0.2-1.0 ⁇ g/cm to form a detection line.
  • the ACE2 protein is uniformly coated on the detection line position of the reaction pad at 0.3 ⁇ g/cm to form a detection line.
  • the anti-immunoglobulin antibody is uniformly coated on the position of the quality control line (C line) of the reaction pad at 0.5-3.6 ⁇ g/cm to form a quality control line. Dry the reaction pad coated with the detection line and the quality control line for later use.
  • the colloidal gold-labeled immunoglobulin is sprayed on the treated binding pad at a concentration of 0.1 mg/ml and 3.75 ⁇ l/ml for drying.
  • Step (3) includes cutting the assembled sheet into test strips, and assemble the cut test strips into the prepared test strip card housing, the sample loading window corresponds to the sample pad of the test strip, and the result display window corresponds to the test strip
  • the detection area is sealed with a desiccant in an aluminum foil bag.
  • the prepared fluorescent microsphere marker tube, SARS-CoV-2 virus neutralizing antibody immunofluorescence paper strip, and sample diluent are placed in the box body and assembled into a kit.
  • the time-resolved immunofluorescence detection kit for neutralizing antibodies of SARS-CoV-2 virus or its variants can also be embodied as immunofluorescence microspheres labeled ACE2 protein, and the RBD protein of SARS-CoV-2 virus or its variants is fixed on a solid phase.
  • the present invention also provides a neutralizing antibody quantum dot detection kit for SARS-CoV-2 virus or its variants, which can be embodied as quantum dots labeling the RBD protein of SARS-CoV-2 virus or its variants, and the ACE2 protein is fixed on a solid A detection kit that blocks the binding of RBD protein and ACE2 protein to a detection signal at a specific position of the phase support by the neutralizing antibody of the SARS-CoV-2 virus or its variants.
  • the present invention also provides a neutralizing antibody quantum dot detection kit for SARS-CoV-2 virus or its variants, which can be embodied as quantum dots labeled ACE2 protein, and SARS-CoV-2 virus RBD protein is fixed at a specific position on a solid support , A detection kit that blocks the binding of RBD protein and ACE2 protein to generate a detection signal by the neutralizing antibody of SARS-CoV-2 virus or its variants.
  • the present invention provides a method for using the above immunochromatographic device, which includes the following steps:
  • the result of the immunochromatographic kit of the present invention is judged: when the SARS-CoV-2 virus neutralizing antibody is present in the sample, the signal (or band) appears on the quality control line and the signal (or band) does not appear on the detection line; when When there is no SARS-CoV-2 virus neutralizing antibody in the sample, a signal (or band) appears on the detection line and the quality control line at the same time.
  • a SARS-CoV-2 virus neutralizing antibody detection kit (colloidal gold method 1) consisting of SARS-CoV-2 virus neutralizing antibody colloidal gold detection test strips, SARS-CoV-2 RBD gold standard tube and The composition of the sample diluent.
  • the SARS-CoV-2 virus neutralizing antibody test paper is composed of a support plate, a sample pad, a binding pad, a detection membrane, and an absorbent pad.
  • the binding pad absorbs colloidal gold-labeled mouse monoclonal antibodies.
  • the detection membrane contains a detection line (T line).
  • the SARS-CoV-2 virus neutralizing antibody sample tube contains colloidal gold-labeled SARS-CoV-2 RBD recombinant protein.
  • the kit detects the SARS-CoV-2 virus neutralizing antibody by blocking the binding mode of the human ACE2 receptor and the SARS-CoV-2 virus RBD protein, if it appears on the detection line (T line) and quality control line (C line)
  • ” means SARS-CoV-2 virus neutralizing antibody is negative; if only the quality control line (C line) shows a red band "
  • a SARS-CoV-2 virus neutralizing antibody detection kit time-resolved immunofluorescence method time-resolved immunochromatographic test strips of SARS-CoV-2 virus neutralizing antibodies, SARS-CoV-2 RBD protein fluorescence Microsphere marker tube and sample diluent composition.
  • the SARS-CoV-2 virus neutralizing antibody test paper is composed of a support plate, a sample pad, a binding pad, a detection membrane, and an absorbent pad.
  • the binding pad absorbs mouse monoclonal antibodies labeled with fluorescent microspheres.
  • the detection membrane contains a detection line (T Line) "
  • SARS-CoV-2 virus neutralizing antibody sample tube contains SARS-CoV-2 RBD recombinant protein labeled with fluorescent microspheres.
  • the test paper detects the SARS-CoV-2 virus neutralizing antibody by blocking the binding mode of the human ACE2 receptor and the SARS-CoV-2 virus RBD protein. Judging the presence of neutralizing antibodies based on the inhibition rate.
  • a SARS-CoV-2 virus neutralizing antibody detection kit (colloidal gold method 2) is composed of SARS-CoV-2 virus neutralizing antibody test strips and sample diluent.
  • the SARS-CoV-2 virus neutralizing antibody test paper is composed of a support plate, a sample pad, a binding pad, a detection membrane, and an absorbent pad.
  • the binding pad absorbs colloidal gold-labeled mouse monoclonal antibodies and colloidal gold-labeled ACE2 receptors for detection
  • the membrane contains the detection line (T line) "
  • the kit detects the SARS-CoV-2 virus neutralizing antibody by blocking the binding mode of the human ACE2 receptor and the SARS-CoV-2 virus RBD protein, if it appears on the detection line (T line) and quality control line (C line)
  • ” means SARS-CoV-2 virus neutralizing antibody is negative; if only the quality control line (C line) shows a red band "
  • the present invention provides the application of the aforementioned immunochromatographic device in detecting infection or suspected infection of a subject with SARS-CoV-2 virus or its variants.
  • the body After the body is infected with the new type of coronavirus SARS-CoV-2, at first, there will be no time to cause the body's immune response in the future. Therefore, only the new type of coronavirus SARS-CoV-2 antigen can be detected at this time. After that, it causes the body's immune response.
  • the first immunoglobulin to appear is IgM antibody, and then IgG antibody appears.
  • the body by detecting the presence or absence of the new coronavirus COVID-19 antigen in the body and its specific IgM antibodies and IgG antibodies, the body’s immune response to the new coronavirus SARS-CoV-2 antigen can be diagnosed.
  • the presence of SARS-CoV-2 virus neutralizing antibodies in serum can be detected by the kit of the present invention to determine whether the subject is infected with SARS-CoV-2 virus.
  • Infected subjects can detect the presence of SARS-CoV-2 virus neutralizing antibodies in serum through the kit of the present invention to determine whether effective immunity is generated in the body.
  • the present invention provides the application of the above immunochromatographic device in detecting whether a subject has an effective immunity after being vaccinated with a vaccine against SARS-CoV-2 or a variant thereof.
  • vaccines for the SARS-CoV-2 virus are being actively developed in the clinic, and the development of some vaccines has entered the clinical stage.
  • the kit of the present invention can be used to quickly detect whether a subject vaccinated with SARS-CoV-2 vaccine has developed effective immunity, and to determine whether a neutralizing antibody is produced in the serum of the vaccinated subject. This plays an important role in the development of the SARS-CoV-2 vaccine.
  • the present invention provides the application of the above immunochromatographic device for detecting SARS-CoV-2 neutralizing antibodies obtained by biological methods.
  • SARS-CoV-2 neutralizing antibodies obtained by biological methods.
  • the immunochromatographic device of the invention can be used for the detection of SARS-CoV-2 neutralizing antibodies in the serum of immunized animals. It can also be used for the detection of neutralizing antibodies in SARS-CoV-2 antibodies purified by other different methods.
  • coronavirus refers to a type of zoonotic RNA virus that spreads between animals and humans. Coronavirus can infect mammals and birds, causing digestive tract diseases in cattle and pigs or upper respiratory tract diseases in chickens. Common in nature, there are seven types of coronaviruses known to infect humans, which can cause respiratory infections in humans, and can cause common colds, and even Middle East Respiratory Syndrome (MERS), severe acute respiratory syndrome (SARS), and 2019 coronavirus diseases. (COVID-19 or SARS-CoV-2) and other serious diseases.
  • the coronavirus in the present invention is selected from SARS-CoV-2 or its variants, SARS-CoV or its variants, or MERS-Cov or its variants. Preferably, the coronavirus is SARS-CoV-2 or a variant thereof.
  • the "new coronavirus” (SARS-CoV-2), also known as 2019-nCoV, belongs to the ⁇ -coronavirus, has an envelope, and the particles are round or elliptical, often pleomorphic, with a diameter of 60 -140nm. Its genetic characteristics are significantly different from SARSr-Cov and MERSr-CoV. Studies have shown that it has more than 85% homology with bat SARS-like coronavirus (bat-SL-CoVZC45). When isolated and cultured in vitro, 2019-nCov can be found in human respiratory epithelial cells in about 96 hours, while isolation and culture in Vero E6 and Huh-7 cell lines takes about 6 days.
  • the "SARS-CoV-2 virus variant" in the present invention refers to a virus with a high degree of sequence homology to the newly discovered coronavirus.
  • a coronavirus isolated from Malayan pangolin by a research team has shown 100%, 98.2%, 96.7% and 90.4% amino acid identity with 2019-nCoV in the E, M, N, and S genes, respectively.
  • the receptor binding domain of the S protein of pangolin coronavirus is actually the same as the receptor binding domain of the S protein of 2019-nCoV, with only one amino acid difference (https://www.biorxiv.org/content/10.1101/ 2020.02.17.951335v1).
  • the gene sequence of the SARS-CoV-2 virus variant described in the present invention and the gene sequence of the new coronavirus have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the treasure trove of SARS-CoV-2 virus variants of the present invention is the recently discovered British virus mutant B.1.1.7, South African virus mutant B.1.351, and the new coronavirus mutant strain B.1.617 that appeared in India.
  • SARS-CoV-2 virus antigen refers to the antigen of SARS-CoV-2 whole virus lysate or the recombinantly expressed SARS-CoV-2 antigen.
  • SARS-CoV-2 virus includes spike protein (S), envelope protein (E), matrix protein (M) and nucleocapsid protein (N) antigens, among which S protein is the largest structural protein of SARS-CoV-2.
  • S protein can be cleaved into S1 and S2 subunits under the action of host enzymes.
  • the S1 subunit contains the receptor binding region RBD and is the main target antigen.
  • the SARS-CoV-2 virus antigen is selected from spike protein (S), envelope protein (E), matrix protein (M) and/or nucleocapsid protein (N) antigen.
  • the SARS-CoV-2 virus antigen is SARS-CoV-2S1 subunit or SARS-CoV-2S RBD antigen.
  • the SARS-CoV-2S RBD antigen in the present invention can be produced by conventional recombinant expression methods, by constructing a plasmid expressing SARS-CoV-2S RBD, such as pFastBac1, pTT5, and transfecting expression cells with expression vectors containing the target gene, such as CHO cells, SF9 cells, expressed and purified SARS-CoV-2S RBD recombinant protein.
  • neutralizing antibody refers to antibodies that prevent cells from being attacked by a certain antigen or source of infection by binding to virus molecules, and its principle is to inhibit or even neutralize their certain biochemical effects.
  • SARS-CoV-2 virus neutralizing antibody refers to an antibody that blocks the SARS-CoV-2 virus RBD from binding to the human ACE2 receptor by binding to the SARS-CoV-2 virus RBD protein.
  • ACE2 or "ACE2 protein” can be embodied as recombinant ACE2 protein, can be embodied as soluble ACE2 protein, and can be embodied as Fc tag ACE2 protein.
  • a functional fragment of ACE2 protein refers to a fragment of the ACE2 protein that can bind to the spike protein of the coronavirus, especially the SARS-CoV-2 virus RBD protein. It may be the binding function of the ACE2 protein. partial area.
  • the "coronavirus spike protein portion” refers to the spike protein portion that includes the function of the coronavirus, which can be virus RBD protein, virus S1 protein and virus S protein. It is preferably SARS-CoV-2 virus RBD protein, SARS-CoV-2 virus S1 protein and SARS-CoV-2 virus S protein, more preferably SARS-CoV-2 virus RBD protein or SARS-CoV-2 virus S1 protein .
  • SARS-CoV-2 virus RBD protein herein can be embodied as recombinant SARS-CoV-2 virus RBD protein, SARS-CoV-2 virus RBD protein with His tag, and SARS-CoV-2 with Fc tag Viral RBD protein, including molecules containing SARS-CoV-2 virus RBD protein, such as SARS-CoV-2 virus S1 subunit (sequence shown in SEQ ID NO: 2) and SARS-CoV-2 virus S protein.
  • signal herein may refer to at least one of a visual signal, a fluorescent signal, and a magnetic signal.
  • Visual signals include colloidal gold, colloidal silver, colored latex microspheres, and colored dyes.
  • Fluorescent signals include fluorescent microspheres, quantum dots, and so on. Fluorescent microspheres include up-conversion fluorescent microspheres, time-resolved fluorescent microspheres, etc.
  • immunofluorescence microspheres in the present invention can be embodied as immunofluorescence microspheres containing samarium (SM), immunofluorescence microspheres containing europium (Eu), immunofluorescence microspheres containing dysprosium (Dy), immunofluorescence containing technetium (Te) Microspheres.
  • SM samarium
  • Eu europium
  • Dy dysprosium
  • Te technetium
  • the quantum dots in the present invention can choose the following particles: (1) Quantum dots of a single compound, such as any one of InAs, InP, InGaAs, AlGaAs, InAlAs, BaSe, BaTe, ZnS, ZnSe, ZnTe, CSiC, SiGe; Compounds of group IV and group VI. (2) Quantum dot particles assembled from quantum dot compounds and other chemical substances, such as ZnS coated CdSe, silica coated CdSe, which can be assembled from organic polymer and quantum dot compounds, such as polypropylene Any one of amide, polystyrene, polypropylene, cross-linked dextran, dendrimer, etc. wraps one kind of quantum dot or particles made of several kinds of quantum dots.
  • Quantum dots of a single compound such as any one of InAs, InP, InGaAs, AlGaAs, InAlAs, BaSe, BaTe, ZnS, Z
  • the material of the bonding pad of the present invention is not particularly limited, and materials well known to those skilled in the art can be used.
  • the bonding pad is a glass fiber membrane.
  • the sample loading pad of the present invention and the binding pad are glass fiber membranes or non-woven fabrics or filter paper immersed in a treatment liquid, and the treatment liquid contains the following components: Tween 20, Triton x-405, Casein, BSA, PEG- 20000, PVP and NaCl.
  • the reaction pad in the present invention includes but is not limited to nitrocellulose membrane and cellulose acetate membrane, preferably nitrocellulose membrane.
  • the nitrocellulose membrane can be any commercial nitrocellulose membrane, including S&SAE99, whatman 8um, Millipore M135, Sartoirus CN140, etc.
  • the "detection line” is set on the reaction pad, and is a line formed by sprayed or drawn compounds, such as proteins. For example, first determine the position of the detection line on the reaction pad along the direction perpendicular to the sample flow, and then evenly fix the protein and other molecules at the position of the detection line by spraying or scribing to form the detection line.
  • the test strip or test kit will indicate the position of the test line. After adding the sample to be tested, you can determine whether the molecules in the sample react with the molecules contained in the test line by observing the signal displayed on the test line.
  • the "quality control line" is set on the reaction pad, a line formed by sprayed or marked control compounds, such as protein, biotin, biotin, ligand, etc., is usually set on the test line and flows along the sample The direction is downstream and parallel to the detection line. First, select the downstream position of the detection line along the sample flow direction on the reaction pad, and then fix the protein, biotin or avidin and other molecules at the position of the quality control line by spraying or scribing to form the quality control line.
  • a line formed by sprayed or marked control compounds such as protein, biotin, biotin, ligand, etc.
  • the absorbent pad of the present invention can be made of any material that can absorb liquid, but the absorption capacity should be large enough. Materials that can be used include, but are not limited to, absorbent cotton pads, silicone absorbent pads or sponge absorbent pads.
  • the bottom plate of the present invention is used to carry the sample pad, the bonding pad, the reaction pad and the absorbent pad; the bottom plate can be a variety of non-absorbent and supporting sheets.
  • the bottom plate can be polyvinyl chloride ( PVC) board, polypropylene (PP) board, polyethylene (PE) board or polyurethane (PU) board, preferably PVC board.
  • the test strip in the present invention includes a sample loading pad, a bonding pad, a reaction pad and a water absorbing pad that are sequentially overlapped, and the sample loading pad, the bonding pad, the reaction pad, and the water absorbing pad are sequentially adhered to the bottom plate.
  • a reaction pad such as nitrocellulose membrane
  • a bottom plate such as a polyvinyl chloride plate
  • an absorbent pad such as absorbent paper
  • the bonding pad On the right side of the pad (nitrocellulose membrane), the bonding pad is flatly attached between the sample pad and the nitrocellulose membrane, so that one end is pressed under the sample pad and the other end is covered on the reaction pad.
  • “lap joint” refers to the connection where the adjacent two parts of the sample loading pad, the bonding pad, the reaction pad and the absorbent pad overlap each other.
  • Such an overlapping structure can ensure that the sample can flow through sequentially during the sample loading chromatography process. Sampling pads, binding pads, reaction pads and absorbent pads.
  • the absorbent pad may not be included when ensuring that the sample flows through the loading pad, the binding pad, and the reaction pad.
  • the bottom plate may not be included when ensuring that the sample loading pad, binding pad, reaction pad, and absorbent pad complete the chromatography of the sample.
  • subject refers to an animal in need of alleviation, prevention and/or treatment of a disease or condition such as a viral infection, preferably a mammal, more preferably a human.
  • a disease or condition such as a viral infection
  • the term includes human subjects who have a coronavirus such as SARS-CoV-2 infection or are at risk of having a coronavirus such as SARS-CoV-2 infection.
  • a healthy subject refers to a healthy animal that has not been infected with the SARS-CoV-2 virus, preferably a mammal, and more preferably a human.
  • the samples in the present invention include but are not limited to (1) plasma, serum or whole blood infected or suspected of being infected with coronavirus; (2) plasma, serum or whole blood after inoculation with coronavirus vaccine; (3) coronavirus spike protein immunity Plasma, serum or whole blood after animals; or (4) Anti-coronavirus antibody samples obtained by biological methods.
  • the sample can be derived from the plasma, serum, whole blood, pleural effusion, cerebrospinal fluid or tissue specimens of humans or animals infected or suspected of being infected with the SARS-CoV-2 virus, and can also be derived from animal serum prepared by biological methods such as animal immunization , Plasma, whole blood or a solution of antibodies against SARS-CoV-2 coronavirus.
  • the "percent (%) amino acid sequence identity" of a peptide or polypeptide sequence is defined as comparing the sequences and introducing gaps when necessary to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Candidates The percentage of amino acid residues in the sequence that are identical to the amino acid residues in the specific peptide or polypeptide sequence. Sequence comparisons can be performed in a variety of ways within the skill of the art to determine percent amino acid sequence identity, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required to obtain the maximum comparison over the entire length of the sequence being compared.
  • SARS-CoV-2 virus serological detection methods are to detect SARS-CoV-2 total antibody (Ab) and SARS-CoV-2IgM antibody and SARS-CoV-2IgG antibody.
  • the value of rapid diagnosis of SARS-CoV-2 neutralizing antibody in auxiliary diagnosis and vaccine evaluation has not yet been developed.
  • the method of developing diagnostic kits based on the interaction between SARS-CoV-2 RBD protein fragments and human soluble ACE2 receptors is relatively novel and is still being developed and utilized.
  • the inhibition rate of neutralizing antibody can be calculated by reading the detection line signal, etc., which is convenient and fast. Use chromatography test strips to achieve rapid diagnosis, avoid the use of large instruments and site restrictions.
  • the immunochromatographic device provided by the present invention has high accuracy and strong specificity for detecting virus neutralizing antibodies, fast detection speed, simple operation, no professional operation, and can be used in communities, grassroots hospitals, airports, customs and even families Preliminary screening in many other places can judge the results within a few minutes, providing a simpler and faster on-site detection method for the investigation of suspected infections and the screening of asymptomatic infections, so as to prevent the spread of the epidemic early.
  • FIG. 1 is a schematic diagram of the SARS-CoV-2 virus neutralizing antibody immunochromatography kit.
  • the human ACE2 protein is fixed on the detection line (T line), and the signal is labeled SARS CoV-2 RBD protein;
  • FIG. 2 is a schematic diagram of the SARS-CoV-2 virus neutralizing antibody immunochromatography kit.
  • the SARS CoV-2 RBD protein is fixed on the detection line (T line), and the signal is labeled with the human ACE2 protein;
  • FIG. 3 is a schematic diagram of the top view structure of the SARS-CoV-2 virus neutralizing antibody immunochromatographic kit, which in turn includes the bottom plate, the sample pad (sample hole), the binding pad (gold label pad), the detection line (T line), Quality control line (C line) and absorbent pad;
  • Figure 4 shows the detection effect of the SARS-CoV-2 virus neutralizing antibody immunochromatographic kit.
  • Figure 4A uses the animal serum immunized with the recombinant SARS-CoV-2 virus RBD protein as a positive quality control.
  • the positive quality control product has a band on the quality control line (C line), but no band on the test line (T line);
  • Figure 4B uses pre-immune animal blank serum as a negative control product to evaluate SARS-CoV-2 Virus neutralizing antibody immunochromatography kit, and negative control products have bands on the detection line (T line) and quality control line (C line).
  • Dissolve chloroauric acid in ultrapure water to make a 10% solution Then mix with pure water to make 1L 0.01% concentration and put it into the beaker. Place the beaker on the heatable mixer and adjust the speed to 2000r/min to make the solution fully rotate. Quickly add 0.561g of trisodium citrate solution in one go. When the solution turns into a stable purple-red, reduce the speed to 200r/min, turn off the heating, and continue the reaction for 20 minutes. After the gold solution returns to room temperature, it is stored in a clean container with a lid and stored at 4°C.
  • NC membrane Nitrocellulose membrane
  • the human ACE2 recombinant protein (Swiss-Prot: Q9BYF1.2, the sequence is shown in SEQ ID NO: 3) was coated with a coating buffer (15g Na 2 HPO 4 , 0.23g NaH 2 PO 4 , 5-10g sucrose, 0.1-0.5g Methanol dissolved in 1L ultrapure water) was diluted to 0.5mg/ml, 1mg/ml, 2mg/ml with different concentration gradients, preferably 1mg/ml, using a three-dimensional flat spot film spraying machine (gold standard) to uniformly distribute at 0.3 ⁇ g/cm It is coated on the T-line position of the nitrocellulose membrane (Sartorius, 1MN14ER100025NT) (the T-line position of the detection line as shown in Figure 2).
  • a coating buffer 15g Na 2 HPO 4 , 0.23g NaH 2 PO 4 , 5-10g sucrose, 0.1-0.5g Methanol dissolved in 1L ultrapure water
  • the rabbit anti-mouse Ig antibody (GenScript: V90301) was dissolved in 1L ultrapure water with coating buffer (15g Na 2 HPO 4 , 0.23g NaH 2 PO 4 , 5-10g sucrose, and 0.1-0.5g methanol ) Dilute to 1-3mg/ml, and coat it on the line C of the nitrocellulose membrane at 0.5-1.2 ⁇ L/cm with a three-dimensional flat spot film spraying machine (the line C of the quality control line as shown in Figure 2) , And then put it in an oven at 37°C overnight to dry.
  • coating buffer 15g Na 2 HPO 4 , 0.23g NaH 2 PO 4 , 5-10g sucrose, and 0.1-0.5g methanol
  • the prepared colloidal gold-labeled mouse IgG resuspension was sprayed onto the treated binding pad with a three-dimensional plane spot film spray gold sprayer (gold label) with a spray volume of 3.75 ⁇ l/ml. Place it in a 37°C drying oven overnight to dry.
  • the sample pad (Shanghai Jieyi, GL-b02), binding pad (Shanghai Jieyi, 8951), marked with recombinant human ACE2 protein as the detection line and marked with rabbit anti-mouse lgG as
  • the nitrocellulose membrane (reaction pad) of the quality control line and the absorbent pad were adhered to the polyethylene backing (bottom plate) in order. Cut the assembled sheet into test strips with a width of 4mm, and assemble the cut test strips into the prepared test strip cassette.
  • the sample loading window corresponds to the sample pad of the test strip
  • the result display window corresponds to the detection of the test strip. Zone, and then sealed with desiccant in an aluminum foil bag.
  • the temperature should be controlled at 20-30°C, and the humidity should be controlled at 20-30%.
  • the serum of 40 healthy people was tested by the SARS-CoV-2 virus neutralizing antibody immunochromatographic kit, and the test results were all Is negative.
  • the negative coincidence rate of the kit of the present invention is 100%.
  • 4 positive quality control products containing matrix serum and anti-SARS-CoV-2S protein antibody 9A9C9 (recombinant neutralizing antibody, Nanjing GenScript)) (P1, P2, P3, P4), the test results were all positive.
  • the positive coincidence rate of the kit of the present invention is 100%. Where "+" means negative, Means positive.
  • Table 1 SARS-CoV-2 virus neutralizing antibody detection kit detects the specificity of healthy human serum
  • microsphere activation buffer (5-50mM MES PH5-6.5) and 10 ⁇ l of 5% fluorescent microspheres (Microtest, MD001) into a 2.0mL centrifuge tube, shake and mix. Centrifuge at 20,000 g for 20 min, and discard the supernatant. Resuspend the pellet with 100 ⁇ L microsphere activation buffer (5-50mM MES, pH5.0-6.5), and sonicate the pellet for 50S with an sonicator. Add 1-3 ⁇ L of 2mg/mL EDC (Sigmaaldrich, E6383) diluent, place it on the mixer and mix for 25min at a speed of 200r/min.
  • EDC Sigmaaldrich, E6383
  • microsphere activation buffer (5-50mM MES, pH5-6.5) and 10 ⁇ l of 5% fluorescent microspheres (Microtest, MD001) into a 2.0mL centrifuge tube, shake to mix well. Centrifuge at 20,000 g for 20 min, and discard the supernatant. Resuspend the pellet with 100 ⁇ L microsphere activation buffer (5-50mM MES, pH5.0-6.5), and sonicate the pellet for 50S. Add 1-3 ⁇ L of 2mg/mL EDC (Sigmaaldrich, E6383) diluent. Place it on the mixer and mix for 25min, the reaction temperature is 30°C, and the rotation speed is 200r/min.
  • EDC Sigmaaldrich, E6383
  • NC membrane Nitrocellulose membrane
  • Dilute human ACE2 recombinant protein with coating buffer (15g Na 2 HPO 4 , 0.23g NaH 2 PO 4 , 5-10g sucrose, 0.1-0.5g methanol dissolved in 1L ultrapure water) to 0.5mg/ml, 1mg /ml, 2mg/ml with different concentration gradients, preferably 1mg/ml, uniformly coated on the T-line position of the nitrocellulose membrane (Sartorius, 1 ⁇ N14ER100025NT) at 0.3 ⁇ g/cm (the detection line T-line shown in Figure 2 Location).
  • coating buffer 15g Na 2 HPO 4 , 0.23g NaH 2 PO 4 , 5-10g sucrose, 0.1-0.5g methanol dissolved in 1L ultrapure water
  • sample pad (Shanghai Jieyi, GL-b02), the binding pad (Shanghai Jieyi, 8951), the line marked with recombinant human ACE2 protein as the test line, and the line marked with rabbit anti-mouse lgG as the quality control
  • the nitrocellulose membrane of the thread and the absorbent pad are adhered to the polyethylene backing (bottom plate) in order. Cut the assembled sheet into test strips with a width of 4mm, and assemble the cut test strips into the prepared test strip cassette.
  • the sample loading window corresponds to the sample pad of the test strip, and the result display window corresponds to the detection of the test strip. Zone, and then sealed with desiccant in an aluminum foil bag.
  • the temperature should be controlled at 20-30°C, and the humidity should be controlled at 20-30%.
  • the SARS-CoV-2 RBD protein fluorescent microsphere marker tube, the SARS-CoV-2 virus neutralizing antibody immunochromatographic detection test strip, and the sample diluent (20mM PBS, 0.01% Tween- 20, 0.09% NaCl, 0.03% ProcLin300, pH 7.0-7.5) are placed in the box in order, assembled into a kit, and stored at 4-25°C in the dark.
  • the SARS-CoV-2 virus neutralizing antibody immunochromatographic kit was used to detect 50 healthy human sera and 4 positive quality control products (containing matrix serum and anti-SARS-CoV-2S protein antibody 9A9C9, P1 , P2, P3, P4) and 16 positive rabbit sera (serum obtained by immunizing rabbits with SARS-CoV-2S protein fragment, R1-R16), calculate the inhibition rate, the inhibition rate is less than 30%, the result is judged as negative, the inhibition rate ⁇ 30%, the result is judged as positive.
  • the inhibition rate of 50 healthy people's serum detection was less than 30%, and the negative coincidence rate was 100%.
  • the detection inhibition rate of 4 positive quality control products and 16 positive rabbit sera was ⁇ 30%, and the positive coincidence was 100%.
  • the test results are shown in Table 2.
  • Negative sample number Test results 1 -5% 26 18% 2 18% 27 4% 3 7% 28 15% 4 25% 29 8% 5 twenty three% 30 9% 6 20% 31 twenty two% 7 0% 32 19% 8 -6% 33 -1% 9 1% 34 twenty four% 10 twenty one% 35 14% 11 3% 36 10% 12 9% 37 15% 13 6% 38 20% 14 -3% 39 -2% 15 -1% 40 12% 16 15% 41 14% 17 16% 42 11% 18 11% 73 12% 19 20% 44 2% 20 6% 45 twenty one% twenty one -14% 46 1% twenty two 6% 47 28% twenty three 2% 48 4% twenty four 9% 49 3% 25 twenty four% 50 11% P1 43% R7 33% P2 59% R8 93% P3 84% R9 88% P4 87% R10 75% R1 62% R11 56% R2 33% R12 72% R3 61% R13 52% R4 45% R14 58% R5 76% R15 32% R6 53% R16 83%
  • Dissolve chloroauric acid in ultrapure water to make a 10% solution Then mix with pure water to make 1L 0.01% concentration and put it into the beaker. Place the beaker on the heatable mixer and adjust the speed to 2000r/min to make the solution fully rotate. Quickly add 0.561g of trisodium citrate solution in one go. When the solution turns into a stable purple-red, reduce the speed to 200r/min, turn off the heating, and continue the reaction for 20 minutes. After the gold solution returns to room temperature, it should be stored in a clean container with a lid and stored at 4°C. The validity period is 1 year.
  • NC membrane Nitrocellulose membrane
  • Dissolve human SARS-CoV-2 RBD protein (GenScript, T80302) with coating buffer (15g Na 2 HPO 4 , 0.23g NaH 2 PO 4 , 5-10g sucrose, and 0.1-0.5g methanol in 1L ultrapure water) Medium) diluted to 0.1mg/ml, 0.3mg/ml, 0.6mg/ml with different concentration gradients, preferably 0.3mg/ml, uniformly coated on nitrocellulose membrane (Sartorius, 1 ⁇ N14ER100025NT) at 0.5 ⁇ l/cm T Line position (detection line T line position as shown in Figure 2).
  • the rabbit anti-mouse Ig antibody (GenScript: V90301) was dissolved in IL ultrapure water with coating buffer (15g Na 2 HPO 4 , 0.23 g NaH 2 PO 4 , 5-10 g sucrose and 0.1-0.5 g methanol) ) Dilute to 1-3mg/ml, coat it evenly on the line C of the nitrocellulose membrane at 0.5-1.2 ⁇ L/cm (the line C of the quality control line as shown in Figure 2), and then put it in a 37°C oven overnight drying.
  • coating buffer 15g Na 2 HPO 4 , 0.23 g NaH 2 PO 4 , 5-10 g sucrose and 0.1-0.5 g methanol
  • the prepared human ACE2 recombinant protein gold standard substance resuspension and colloidal gold-labeled mouse IgG weight-to-volume ratio suspension were mixed thoroughly at 1:1, and the three-dimensional plane spot membrane spray gold sprayer sprayed 3.75 ⁇ l/ml to the binding Put it on the mat, and then put it in a 37°C drying oven overnight to dry.
  • the sample pad (Shanghai Jieyi, GL-b02), the binding pad (Shanghai Jieyi, 8951), the human SARS-CoV-2 RBD protein as the detection line and the rabbit antibody
  • the nitrocellulose membrane with mouse lgG as the quality control line and the absorbent pad were adhered to the polyethylene backing (bottom plate) in order. Cut the assembled sheet into test strips with a width of 4mm, and assemble the cut test strips into the prepared test strip cassette.
  • the sample loading window corresponds to the sample pad of the test strip, and the result display window corresponds to the detection of the test strip. Zone, and then sealed with desiccant in an aluminum foil bag.
  • the temperature should be controlled at 20-30°C, and the humidity should be controlled at 20-30%.
  • Aspirate 20 ⁇ L of serum sample add it to a tube of 80 ⁇ L diluent sample, and mix well. Take 100 ⁇ L of all the mixed solution and drop it vertically to the sample point of the detection card, start timing, and observe the color development of the band for 10-15min.
  • the serum of healthy humans was tested by the SARS-CoV-2 virus neutralizing antibody immunochromatographic kit, and the test results were all negative.
  • the specificity of the kit of the present invention is 100%. Where "+" means negative, Means positive.
  • SARS-CoV-2 spike protein RBD protein amino acid sequence SARS-CoV-2 spike protein RBD protein amino acid sequence (SEQ ID NO:1)
  • SARS-CoV-2 spike protein S1 subunit amino acid sequence SEQ ID NO: 2

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Abstract

涉及抗体检测领域，具体涉及检测SARS-CoV-2病毒中和抗体的免疫层析装置及其应用，进一步包括SARS-CoV-2病毒中和抗体的免疫层析装置的制备和使用方法。所述冠状病毒中和抗体的免疫检测装置，包括冠状病毒的刺突蛋白部分、特异性结合所述冠状病毒刺突蛋白部分的ACE2蛋白或其功能片段以及固相支持物，在所述固相支持物上检测待测样品对所述冠状病毒刺突蛋白部分与ACE2蛋白或其功能片段结合的影响，判断样品中是否存在冠状病毒中和抗体。免疫层析检测装置，如检测试纸特异、敏感、快捷、简便，易在生产实践中推广应用。

Description

检测SARS-CoV-2病毒中和抗体的免疫层析装置及其应用 技术领域
本发明涉及抗体检测领域，具体涉及免疫层析技术对SARS-CoV-2病毒中和抗体进行检测的方法以及针对SARS-CoV-2病毒中和抗体的免疫层析检测装置。
背景技术
新型冠状病毒肺炎(COVID-19)病原体SARS-CoV-2病毒，又称2019新型冠状病毒(2019 Novel coronavirus,2019-nCoV)，是一种RNA冠状病毒。目前全球已有220多个国家和地区累计报告逾470万名确诊病例，逾30万名患者死亡。2019冠状病毒病疫情逐渐变成一场全球性大瘟疫。全球至今尚未出现用于SARS-CoV-2病毒疾病的疫苗和特效药。
SARS-CoV-2病毒是一类具有囊膜、基因组为线性单股正链的RNA病毒。SARS-CoV-2病毒进入宿主细胞是由跨膜刺突(S)糖蛋白(S蛋白)介导的。S蛋白分为S1和S2两个亚基，其中S1亚基负责与宿主细胞受体结合，S2亚基负责病毒膜和细胞膜融合。S1通过结合宿主受体促进病毒感染。它包含两个结构域，即与宿主受体直接相互作用的N端结构域和C端RBD结构域。C端RBD结构域位于SARS-CoV-2病毒亚基上，是与人ACE2(血管紧张素转化酶2)受体的相互作用位点，在病毒的传染过程中发挥着重要作用。ACE2是SARS-CoV进入细胞的受体。它是一种锌金属蛋白酶，属于1型跨膜蛋白。它的结构包括一个信号肽、一个跨膜结构域和一个含有HEMGH锌结合结构域的金属蛋白酶活性位点。全长ACE2受体由805个氨基酸组成，位于上皮细胞的腔面。
2009年纽约血液中心使用小鼠模型研究证明在哺乳动物、昆虫和大肠杆菌细胞中表达的SARS病毒S蛋白RBD结构域能引发有效的中和抗体和保护性免疫。研究表明，在新型冠状病毒肺炎患者治疗过程中，康复期病人血浆治疗取得了较好的疗效，显示出中和抗体在新冠病毒肺炎治疗方面的潜力。S蛋白是进行疫苗和抗体研究的一个比较理想和有效的诊断和药物开发靶点。
SARS-CoV-2病毒的检测方法主要有2019-nCoV病毒核酸检测方法和血清检测方法。SARS-CoV-2血清检测法如化学发光法、酶联免疫吸附试验法(ELISA)、胶体金法检测法均为分析SARS-CoV-2总抗体(Ab)和SARS-CoV-2IgM抗体，但不能区分SARS-CoV-2中和抗体与非中和抗体，无法准确评价免疫保护效果。因此，开发用于检测SARS-CoV-2病毒中和抗体的试剂盒可有效评估保护效果，对SARS-CoV-2感染状况具有辅助诊断作用。
免疫层析试纸具有更加灵敏、特异、简便、快速等优点，是理想的即时检测(point-of-care test,POCT)和现场检测技术,能在1-15min内判定结果，广泛应用于各种分析物的定性和半定量快速检测，因此研制SARS-CoV-2病毒中和抗体免疫层析检测试剂盒具有重要意义。
发明内容
针对现有技术的不足，本发明的目的是研制适用于检测人血清和血浆的SARS-CoV-2病毒中和抗体免疫层析装置，如检测试纸特异、敏感、快捷、简便，易在生产实践中推广应用。
一方面，本发明提供了一种检测冠状病毒中和抗体的免疫层析装置，其特征在于，所述免疫层析装置包括冠状病毒的刺突蛋白部分、特异性结合所述冠状病毒刺突蛋白部分的ACE2蛋白或其功能片段以及固相支持物，在所述固相支持物上检测待测样品对所述冠状病毒刺突蛋白部分与ACE2蛋白或其功能片段结合的影响，判断样品中是否存在冠状病毒中和抗体。
在一些实施方案中，所述冠状病毒刺突蛋白部分或者所述ACE2蛋白或其功能片段连接信号物。在另一些实施方案中，所述ACE2蛋白或其功能片段或者所述冠状病毒刺突蛋白部分固定在固相支持物上。优选地，所述免疫层析装置中(1)所述冠状病毒刺突蛋白部分连接信号物，所述ACE2蛋白或其功能片段固定在固相支持物上；或(2)所述ACE2蛋白或其功能片段连接信号物，所述冠状病毒刺突蛋白部分固定在固相支持物上。
在一些实施方案中，所述待测样品对冠状病毒刺突蛋白部分与ACE2蛋白或其功能片段结合的影响情况通过信号物产生信号判断。
在一些实施方案中，所述信号物选自可视化信号物、荧光信号物、磁信号物中的至少一种。其中，所述可视化信号物包括胶体金、胶体银、彩色乳胶微球和彩色染料，优选为胶体金。在另一些实施方案中，所述荧光信号物包括荧光微球和量子点。
本发明中所述免疫层析装置选自胶体金免疫层析装置、量子点免疫层析装置或免疫荧光检测装置，优选为胶体金免疫层析装置和免疫荧光检测装置。
在一些实施方案中，所述胶体金免疫层析装置包括金标记的冠状病毒刺突蛋白部分，所述ACE2蛋白或其功能片段固定在固相支持物上的特定位置，通过待测样品中的冠状病毒中和抗体阻断病毒刺突蛋白部分和ACE2蛋白或其功能片段的结合产生检测信号。在另一些实施方案中，所述胶体金免疫层析装置包括金标记的ACE2蛋白或其功能片段，所述冠状病毒刺突蛋白部分固定在固相支持物上的特定位置，通过待测样品中的冠状病毒中和抗体阻断病毒刺突蛋白部分和ACE2蛋白或其功能片段的结合产生检测信号。
在一些实施方案中，所述量子点免疫层析装置包括量子点标记的冠状病毒刺突蛋白部分，所述ACE2蛋白或其功能片段固定在固相支持物上的特定位置，通过待测样品中的冠状病毒中和抗体阻断病毒刺突蛋白部分和ACE2蛋白或其功能片段的结合产生检测信号。在另一些实施方案中，所述量子点免疫层析装置包括量子点标记的ACE2蛋白或其功能片段，所述冠状病毒刺突蛋白部分固定在固相支持物上的特定位置，通过待测样品中的病毒中和抗体阻断病毒刺突蛋白部分和ACE2蛋白或其功能片段的结合产生检测信号。优选地，所述量子点选自第IV族和第VI族组成的化合物或由量子点化合物与其它化学物质组装而成的量子点颗粒。在一些实施方案中，所述免疫荧光检测装置为时间分辨免疫荧光检测装置，所述装置包括免疫荧光微球标记的冠状病毒刺突蛋白部分，所述ACE2蛋白或其功能片段固定在固相支持物上的特定位置，通过待测样品中的病毒中和抗体阻断病毒刺突蛋白部分和ACE2蛋白或其功能片段的结合产生检测信号。在另一些实施方案中，所述免疫荧光检测装置为时间分辨免疫荧光检测装置，所述装置包括免疫荧光微球标记的ACE2蛋白或其功能片段，所述冠状病毒刺突蛋白部分固定在固相支持物上的特定位置，通过待测样品中的病毒中和抗体阻断病毒刺突蛋白部分和ACE2蛋白或其功能片段的结合产生检测信号。优选地，所述免疫微球选自含 钐(SM)免疫荧光微球，含铕(Eu)免疫荧光微球，含镝(Dy)免疫荧光微球或含锝(Te)免疫荧光微球。
本发明中所述冠状病毒选自SARS-CoV-2或其变体、SARS-CoV或其变体或者MERS-Cov或其变体，优选为SARS-CoV-2或其变体。在一些实施方案中，所述冠状病毒为SARS-CoV相关的病毒。在另一些实施方案中，所述冠状病毒为SARS-CoV-2相关的病毒。在一些实施方案中，所述冠状病毒为SARS-CoV-2或其变体。在一个具体实施方案中，所述冠状病毒为SARS-CoV-2。
本发明中所述冠状病毒刺突蛋白部分选自冠状病毒S蛋白、冠状病毒S1蛋白、冠状病毒RBD蛋白或者带有His或者Fc标签的冠状病毒RBD蛋白，优选为冠状病毒S1蛋白或冠状病毒RBD蛋白，更优选为SARS-CoV-2病毒RBD蛋白。在一些实施方案中，所述冠状病毒刺突蛋白部分为冠状病毒S1蛋白或冠状病毒RBD蛋白。优选地，所述冠状病毒刺突蛋白部分为SARS-CoV-2病毒S1蛋白或SARS-CoV-2病毒RBD蛋白。更优选地，所述冠状病毒刺突蛋白部分为SARS-CoV-2病毒RBD蛋白。
在一些实施方案中，冠状病毒刺突蛋白部分包含与SEQ ID NO:1或2所示氨基酸序列至少70％一致性的序列。在另一些实施方案中，所述冠状病毒刺突蛋白部分包含与SEQ ID NO:1或2所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一些实施方案中，所述冠状病毒刺突蛋白部分包含与SEQ ID NO:1所示氨基酸序列。在一些实施方案中，所述冠状病毒刺突蛋白部分包含与SEQ ID NO:2所示氨基酸序列。在一个具体实施方案中，所述冠状病毒刺突蛋白部分氨基酸序列如SEQ ID NO:1所示。在一个具体实施方案中，所述冠状病毒刺突蛋白部分氨基酸序列如SEQ ID NO:2所示。
本发明中所述ACE2蛋白或其功能片段来源于人。在一些实施方案中，所述ACE2蛋白或其功能片段选自重组ACE2蛋白或带Fc标签ACE2蛋白。在另一些实施方案中，所述ACE2蛋白选自人重组ACE2蛋白。
在一些实施方案中，所述ACE2蛋白或其功能片段包含与SEQ ID NO:3所示氨基酸序列至少70％一致性的序列。在一些实施方案中，所述ACE2蛋白或其功能片段包含与SEQ ID NO:3所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一些实施方案中，所述ACE2蛋白或其功能片段包含与SEQ ID NO:3所示氨基酸序列。在一个具体实施方案中，所述ACE2蛋白或其功能片段的氨基酸序列如SEQ ID NO:3所示。
本发明中所述固相支持物选自硝酸纤维素膜和醋酸纤维素膜，优选为硝酸纤维素膜。在一些实施方案中，所述固相支持物为硝酸纤维素膜。其中的硝酸纤维素膜可以为任何商品化的硝酸纤维素膜，包括S&SAE99、whatman 8um、Millipore M135、Sartoirus CN140等。
本发明提供一种检测冠状病毒中和抗体的免疫层析装置，其特征在于，所述装置包括检测试纸条，所述试纸条包括顺次搭接的加样垫、结合垫、反应垫和吸水垫，所述加样垫、结合垫、反应垫和吸水垫依次粘附在底板上，所述反应垫上沿样品流动方向包含依次设置的检测线和质控线，所述检测线上包含冠状病毒刺突蛋白部分或者ACE2蛋白或其功能片段，所述装置还包括信号物标记的ACE2蛋白或其功能片段或者信号物标记的冠状病毒刺突蛋白部分，所述冠状病毒刺突蛋白部分与ACE2蛋白或其功能片段结合后在检测线上产生检测信号。
在一些实施方案中，所述检测线上包含ACE2蛋白或其功能片段时，所述装置包括信号物标记的冠状病毒刺突蛋白部分，所述信号物标记的冠状病毒刺突蛋白部分与所述ACE2蛋白或其功能片段结合产生检测信号。
在另一些实施方案中，所述检测线上包含冠状病毒刺突蛋白部分时，所述装置包括信号物标记的ACE2蛋白或其功能片段，所述信号物标记的ACE2蛋白或其功能片段与所述冠状病毒刺突蛋白结合产生检测信号。
本发明中所述ACE2蛋白或其功能片段选自重组ACE2蛋白或带Fc标签ACE2蛋白。在一些实施方案中，所述ACE2蛋白或其功能片段为重组人ACE2蛋白。在另一些方案中，所述ACE2蛋白或其功能片段包含与SEQ ID NO:3所示氨基酸序列至少70％一致性的序列。在一些实施方案中，所述ACE2蛋白或其功能片段包含与SEQ ID NO:3所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一些实施方案中，所述ACE2蛋白或其功能片段包含SEQ ID NO:3所示氨基酸序列。在一个具体实施方案中，所述ACE2蛋白或其功能片段的氨基酸序列如SEQ ID NO:3所示。
在一些实施方案中，所述冠状病毒刺突蛋白部分包括冠状病毒S蛋白、冠状病毒S1蛋白、冠状病毒RBD蛋白或者带有His或者Fc标签的冠状病毒RBD蛋白。优选地，所述冠状病毒刺突蛋白部分为SARS-CoV-2病毒刺突蛋白部分。更优选地，所述冠状病毒刺突蛋白部分为SARS-CoV-2病毒S1蛋白或SARS-CoV-2病毒RBD蛋白。更优选地，所述SARS-CoV-2病毒刺突蛋白部分包含与SEQ ID NO:1或2所示氨基酸序列至少70％一致性的序列。在一些实施方案中，所述SARS-CoV-2病毒刺突蛋白部分包含与SEQ ID NO:1或2所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在另一些实施方案中，所述SARS-CoV-2病毒刺突蛋白部分包括SARS-CoV-2病毒S1蛋白或SARS-CoV-2病毒RBD蛋白。在一些实施方案中，SARS-CoV-2病毒RBD蛋白包含与SEQ ID NO:1所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在另一些实施方案中，所述SARS-CoV-2病毒S1蛋白包含与SEQ ID NO:2所示氨基酸序列至少70％、至少75％、至少80％、至少83％、至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％或至少99％一致性的序列。在一些具体实施方案中，所述SARS-CoV-2病毒RBD蛋白包含SEQ ID NO:1所示氨基酸序列。在另一些具体实施方案中，所述SARS-CoV-2病毒S1蛋白包含SEQ ID NO:2所示氨基酸序列。在一些具体实施方案中，所述SARS-CoV-2病毒RBD蛋白的氨基酸序列如SEQ ID NO:1所示。在另一些具体实施方案中，所述SARS-CoV-2病毒RBD蛋白的氨基酸序列如SEQ ID NO:2所示。
在一些实施方案中，所述质控线包含第一分子，所述加样垫上包含第二分子或检测时所述加样垫上另添加第二分子，所述第二分子能与第一分子结合产生检测信号。在另一些实施方案中，所述第一分子和第二分子分别选自免疫球蛋白和抗免疫球蛋白抗体、受体和配体或生物素和亲和素组成分子对中的任一个。在一些具体实施方案中，所述第一分子选自抗免疫球蛋白抗体、配体或者生物素，对应地所述第二分子选自信号物标记的免疫球蛋白、信号物标记的受体或者亲和素。在另一些实施方案中，所述第一分子选自免疫球蛋白、受体或者亲 和素，对应地所述第二分子选自信号物标记的抗免疫球蛋白抗体、信号物标记的配体或者生物素。在一个具体实施方案中，所述第一分子为抗免疫球蛋白抗体，所述第二分子为信号物标记的免疫球蛋白。在另一个具体实施方案中，所述第一分子为免疫球蛋白，所述第二分子为信号物标记的抗免疫球蛋白抗体。在一个具体实施方案中，所述第一分子为配体，所述第二分子为信号物标记的受体。在另一个具体实施方案中，所述第一分子为受体，所述第二分子为信号物标记的配体。在一个具体实施方案中，所述第一分子为生物素，所述第二分子为亲和素。在另一个具体实施方案中，所述第一分子为亲和素，所述第二分子为生物素。
在一些实施方案中，所述ACE2蛋白或其功能片段以0.2-1.0ug/cm均匀划线或喷洒包被在检测线位置形成检测线。在一些实施方案中，所述ACE2蛋白或其功能片段以0.2-1.0ug/cm均匀划线包被在检测线位置形成检测线。在一些具体实施方案中，所述ACE2蛋白或其功能片段以约0.2ug/cm、0.3ug/cm、0.4ug/cm、0.5ug/cm、0.6ug/cm、0.7ug/cm、0.8ug/cm、0.9ug/cm或1.0ug/cm均匀划线包被在检测线位置形成检测线。在一些优选实施方案中，所述ACE2蛋白或其功能片段，以0.3μg/cm均匀划线或喷洒包被在检测线位置形成检测线。在另一些优选实施方案中，所述ACE2蛋白或其功能片段，以0.5μg/cm均匀划线或喷洒包被在检测线位置形成检测线。其中，所述信号物标记的病毒刺突蛋白部分的量为1.5μg-5mg。在一些实施方案中，所述信号物标记的病毒刺突蛋白部分为SARS-CoV-2病毒RBD蛋白。在一些实施方案中，所述胶体金标记的病毒刺突蛋白部分的量为1.0-5mg。在一些具体实施方案中，所述胶体金标记的SARS-CoV-2病毒RBD蛋白的量约为1.0mg、1.5mg、2.0mg、2.5mg、3.0mg、3.5mg、4.0mg、4.5mg或5.0mg。在另一些实施方案中，所述荧光微球标记的冠状病毒刺突蛋白部分为1.0-5μg。在一些具体实施方案中，所述荧光微球标记的SARS-CoV-2病毒RBD蛋白的量约为1.0μg、1.5μg、2.0μg、2.5μg、3.0μg、3.5μg、4.0μg、4.5μg或5.0μg。
在另一些实施方案中，所述病毒刺突蛋白部分以0.5-1.5μg/cm均匀划线或喷洒包被在检测线位置形成检测线。在一些实施方案中，所述病毒刺突蛋白部分为SARS-CoV-2病毒RBD蛋白。在一些具体实施方案中，所述病毒刺突蛋白部分以0.5ug/cm、0.6ug/cm、0.7ug/cm、0.8ug/cm、0.9ug/cm、1.0ug/cm、1.1ug/cm、1.2ug/cm、1.3ug/cm、1.4ug/cm或1.5ug/cm的量均匀划线或喷洒包被在检测线位置形成检测线。在一些优选实施方案中，所述病毒刺突蛋白部分以0.3μg/cm均匀划线或喷洒包被在检测线位置形成检测线。其中，所述信号物标记的ACE2蛋白或其功能片段与信号物标记的免疫球蛋白抗体按体积比0.5:1～1:1.5混合喷洒在结合垫上，其中，所述信号物标记的ACE2蛋白或其功能片段的量为0.1-1mg/ml，信号物标记的免疫球蛋白抗体为0.1-1mg/ml。优选地，所述信号物标记的ACE2蛋白或其功能片段与信号物标记的免疫球蛋白抗体按体积比1:1混合喷洒在结合垫上，其中，所述信号物标记的ACE2蛋白或其功能片段的量为1mg/ml，信号物标记的免疫球蛋白抗体为1mg/ml。
本发明中所述质控线包含的抗免疫球蛋白抗体选自抗小鼠IgG抗体或抗羊IgG抗体；所述信号物标记免疫球蛋白抗体对应的选自小鼠IgG抗体或羊IgG抗体。在一些实施方案中，所述质控线包含的抗免疫球蛋白抗体为抗小鼠IgG抗体，所述信号物标记免疫球蛋白抗体对应为小鼠IgG抗体。在另一些实施方案中，所述质控线包含的抗免疫球蛋白抗体为抗羊IgG抗体，所述信号物标记免疫球蛋白抗体对应的为羊IgG抗体。
本发明所述信号物选自胶体金、量子点或免疫荧光微球，优选为胶体金和免疫荧光微球。在一些实施方案中，所述免疫荧光微球选自含钐(SM)免疫荧光微球，含铕(Eu)免疫荧光微球，含镝(Dy)免疫荧光微球或含锝(Te)免疫荧光微球。在另一些实施方案中，所述量子点选自第IV族和第VI族组成的化合物或由量子点化合物与其它化学物质组装而成的量子点颗粒。
本发明所述反应垫为硝酸纤维素膜(NC膜)或醋酸纤维素膜，优选为硝酸纤维素膜。所述底板为聚乙烯材料。
本发明所述免疫层析装置中还包括样品稀释液。所述样品稀释液包括20-50mM PBS，0.01％Tween-20，0.09％NaCl，0.03％ProcLin300，pH7.0-7.5。
本发明中所述样品包括：
(1)感染或疑似感染冠状病毒的血浆、血清或全血；
(2)接种冠状病毒疫苗后的血浆、血清或全血；
(3)冠状病毒刺突蛋白免疫的动物血浆、血清或全血；或
(4)生物学方法获得的抗冠状病毒的抗体样品。
另一方面，本发明提供了上述免疫层析装置的使用方法，包括如下步骤：
(1)待测样品中加入样品稀释液稀释后，加入信号标记物混匀；
(2)将混合物滴加至所述免疫层析装置的加样垫上；
(3)待一段时间后，根据检测线和质控线的显色情况，判断样品中是否有冠状病毒中和抗体，判断方法如下：
(a)阳性：所述质控线呈现信号，检测线不呈现信号，说明样品中含有冠状病毒中和抗体；
(b)阴性：所述质控线和检测线均呈现信号，说明样品中不含有冠状病毒中和抗体；
(c)失效：所述质控线和检测线均不呈现信号，说明免疫层析装置失效。
本发明所述免疫层析装置使用方法中，所述信号标记物为胶体金标记的冠状病毒刺突蛋白部分，选自胶体金标记的冠状病毒刺突蛋白S1亚基、胶体金标记的冠状病毒RBD、荧光微球标记的冠状病毒刺突蛋白S1亚基或荧光微球标记的冠状病毒RBD。优选地，所述信号标记物为胶体金标记的SARS-CoV-2刺突蛋白S1亚基、胶体金标记的SARS-CoV-2 RBD、荧光微球标记的SARS-CoV-2刺突蛋白S1亚基或荧光微球标记的SARS-CoV-2 RBD，优选为胶体金标记的SARS-CoV-2 RBD或荧光微球标记的SARS-CoV-2 RBD。
本发明提供了另一种免疫层析装置的使用方法，包括如下步骤：
(1)待测样品中加入样品稀释液稀释；
(2)将稀释的样品液滴加至所述免疫层析装置的加样垫上；
(3)待一段时间后，根据检测线和质控线的显示信号情况，判断样品中是否有冠状病毒中和抗体，判断方法如下：
(a)阳性：所述质控线呈现信号，检测线不呈现信号，说明样品中含有冠状病毒中和抗体；
(b)阴性：所述质控线和检测线均呈现信号，说明样品中不含有冠状病毒中和抗体；
(c)失效：所述质控线和检测线均不呈现信号，说明免疫层析装置失效。
本方法中所述信号标记物为胶体金标记的ACE2蛋白或其功能片段，优选为胶体金标记的人重组的ACE2蛋白或其功能片段。
又一方面，本发明提供了上述的免疫层析装置的制备方法，其特征在于，包括以下步骤：
(1)制备信号物标记的冠状病毒刺突蛋白或信号物标记的ACE2蛋白或其片段和信号物标记的免疫球蛋白抗体；
(2)将ACE2蛋白或其片段包被在反应垫上形成检测线或将冠状病毒刺突蛋白包被在反应垫上形成检测线，将抗免疫球蛋白抗体包被在反应垫上形成质控线，将信号物标记的免疫球蛋白抗体喷洒在结合垫上；
(3)将样品垫、结合垫、带有检测线和质控线的反应垫和吸水垫按照顺序依次粘附在底板上，组装后得到免疫层析装置。
在一些实施方案中，所述步骤(2)中将ACE2蛋白或其片段包被在反应垫上形成检测线时，所述装置中还包括信号物标记的冠状病毒刺突蛋白部分。在另一些实施方案中，所述步骤(2)中所述ACE2蛋白或其片段以0.2-1.0ug/cm均匀划线或喷洒包被在检测线位置形成检测线。其中，所述装置中包含的信号物标记的冠状病毒刺突蛋白部分的量为1.0μg-5mg。在一些实施方案中，所述胶体金标记的冠状病毒刺突蛋白部分的量为1.0mg-5mg。在另一些实施方案中，所述荧光微球标记的冠状病毒刺突蛋白部分的量为1.0μg-5μg。
在另一些实施方案中，所述步骤(2)中将冠状病毒刺突蛋白部分包被在反应垫上形成检测线时，所述装置中还包括信号物标记的ACE2蛋白或其片段。在一些实施方案中，所述步骤(2)病毒刺突蛋白部分0.5-1.5ug/cm均匀划线或喷洒包被在检测线位置形成检测线。进一步，所述步骤(2)还包括将信号物标记的ACE2蛋白或其片段与信号物标记的免疫球蛋白抗体以1：1的比例混合喷洒在结合垫上，其中，所述信号物标记的ACE2蛋白或其功能片段为0.1-1mg/ml，信号物标记的免疫球蛋白抗体为0.1-1mg/ml。
本发明制备的免疫层析装置为免疫层析试纸条。优选地，所述试纸条放入盒体内制成免疫检测试剂盒。
又一方面，本发明提供了上述免疫层析装置在检测受试者感染或疑似感染SARS-CoV-2病毒或其变体中的应用。
另一方面，本发明提供了上述免疫层析装置在检测样品中SARS-CoV-2病毒或其变体的中和抗体中的应用。在一些实施方案中，所述所述样品来源于感染或疑似感染SARS-CoV-2病毒或其变体的受试者，感染SARS-CoV-2病毒或其变体后康复者，接种针对SARS-CoV-2或其变体的疫苗的受试者，或者通过生物学方法获得的抗SARS-CoV-2或其变体的抗体。进一步，上述免疫层析装置在检测受试者接种针对SARS-CoV-2或其变体的疫苗后是否产生有效免疫中的应用。上述免疫层析装置在检测受试者感染SARS-CoV-2或其变体后是否产生有效免疫中的应用。上述免疫层析装置用于检测通过生物学方法获得的抗SARS-CoV-2抗体的应用。
发明详述
本发明的原理可以表述为：一种通过免疫层析作用与SARS-CoV-2病毒中和抗体阻断SARS-CoV-2病毒RBD蛋白与ACE2受体结合产生检测信号来检测样本中的SARS-CoV-2病毒中和抗体。它是将重组ACE2蛋白固定在检测膜上。当检测样本中存在SARS-CoV-2病毒中和抗体时，中和抗体与信号物标记的SARS-CoV-2病毒RBD蛋白结合阻断膜上的重组ACE2蛋白与信号物标记的SARS-CoV-2病毒RBD蛋白结合，不出现检测信号(或条带)，检测结果呈阳性。当检测样本中不存在SARS-CoV-2病毒中和抗体时，膜上的重组ACE2受 体与信号物标记的SARS-CoV-2病毒RBD蛋白结合。出现检测信号(或条带)，检测结果呈阴性(见图1)。
本发明的原理又可以表述为：一种通过免疫层析作用与SARS-CoV-2病毒中和抗体阻断ACE2受体与SARS-CoV-2病毒RBD蛋白结合产生检测信号来检测样本中的SARS-CoV-2病毒中和抗体。它是将重组SARS-CoV-2病毒RBD蛋白固定在检测膜上。样本中的SARS-CoV-2病毒RBD蛋白中和抗体与信号物标记的重组ACE2受体竞争结合固定在载体膜上的重组SARS-CoV-2病毒RBD蛋白。当检测样本中存在SARS-CoV-2病毒中和抗体时，中和抗体与信号物标记的SARS-CoV-2病毒RBD蛋白结合阻断膜上的重组ACE2蛋白与信号物标记的SARS-CoV-2病毒RBD蛋白结合，检测线不出现检测信号(或条带)，检测结果呈阳性。当检测样本中不存在SARS-CoV-2病毒中和抗体时，膜上的重组SARS-CoV-2病毒RBD蛋白与信号物标记的重组ACE2蛋白结合。检测线出现检测信号(或条带)，检测结果呈阴性(见图2)。
免疫层析装置
本发明的免疫层析装置是指利用免疫显色标记技术和抗原抗体反应相结合而形成的一种应用形式，包括免疫层析的检测试纸，免疫检测试纸包装入盒体内，如卡盒形成免疫层析试剂盒。
本发明的具体装置可以体现为由反应管、支撑板(底板)、加样垫、结合垫、检测膜(反应垫)和吸水垫构成。反应管中含有信号物标记的SARS-CoV-2病毒RBD蛋白，结合垫喷洒有信号物标记的小鼠单克隆抗体，检测膜上含有检测线(T线)和质控线(C线)。检测线(T线)喷洒重组人ACE2受体，质控线(C线)喷洒兔抗小鼠抗体(见图3)。
本发明提供了一种检测冠状病毒中和抗体的免疫层析装置，具体涉及SARS-CoV-2病毒或其变体的中和抗体免疫层析检测试剂盒，可以体现为SARS-CoV-2病毒或其变体的中和抗体胶体金检测试剂盒，SARS-CoV-2病毒或其变体的中和抗体量子点检测试剂盒，SARS-CoV-2病毒或其变体的中和抗体免疫荧光检测试剂盒，SARS-CoV-2病毒或其变体的中和抗体时间分辨免疫荧光检测试剂盒，以及利用层析原理和中和抗体阻断SARS-CoV-2病毒RBD蛋白和ACE2蛋白相互作用产生信号的试剂盒。
本发明提供的检测SARS-CoV-2病毒或其变体的中和抗体免疫层析试剂盒包括SARS-CoV-2病毒或其变体的刺突蛋白部分、特异性结合所述冠状病毒刺突蛋白部分的ACE2蛋白或其功能片段以及固相支持物。进一步，在所述固相支持物上检测待测样品对所述冠状病毒刺突蛋白部分与ACE2蛋白或其功能片段结合的影响，判断样品中是否存在SARS-CoV-2冠状病毒或其变体的中和抗体。
优选地，本发明提供检测SARS-CoV-2病毒的中和抗体免疫层析试剂盒，所述试剂盒包括SARS-CoV-2病毒的刺突蛋白部分、特异性结合所述冠状病毒刺突蛋白部分的ACE2蛋白或其功能片段以及固相支持物，在所述固相支持物上检测待测样品对所述冠状病毒刺突蛋白部分与ACE2蛋白或其功能片段结合的影响，判断样品中是否存在SARS-CoV-2冠状病毒或其变体的中和抗体。
在一些实施方案中，所述SARS-CoV-2病毒或其变体的刺突蛋白连接信号物，所述ACE2蛋白或其功能片段固定在固相载体上。
在另一些实施方案中，所述ACE2蛋白或其功能片段连接信号物，所述SARS-CoV-2病毒或其变体的刺突蛋白固定在固相载体上。
(一)SARS-CoV-2病毒或其变体的中和抗体胶体金检测试剂盒及其制备
本发明中SARS-CoV-2病毒或其变体的中和抗体胶体金检测试剂盒可以体现为金颗粒标记的SARS-CoV-2病毒或其变体RBD蛋白，ACE2蛋白固定在固相支持物特定位置，通过SARS-CoV-2病毒中和抗体阻断RBD蛋白和ACE2蛋白结合产生检测信号的检测试剂盒。
具体地，SARS-CoV-2病毒或其变体中和抗体胶体金检测试剂盒包括检测试纸条，所述试纸条包括加样垫、结合垫、反应垫和吸水依次粘附在底板上，所述反应垫上包含依次设置的检测线和质控线，所述检测线包被有ACE2蛋白，所述质控线包被有抗免疫球蛋白抗体。
在一些具体实施方案中，所述胶体金检测试剂盒包括检测试纸条，所述试纸条包括顺次搭接的加样垫、结合垫、反应垫和吸水垫，所述加样垫、结合垫、反应垫和吸水垫依次粘附在底板上，所述反应垫上沿样品流动方向依次设有检测线和质控线，所述检测线包含重组人ACE2蛋白，所述质控线包含抗免疫球蛋白抗体。
其中，所述ACE2蛋白0.2-1.0μg/cm均匀包被在反应垫的检测线(T线)位置形成检测线。优选地，所述ACE2蛋白以0.5μg/cm均匀包被在反应垫的检测线位置形成检测线。其中，所述抗免疫球蛋白抗体浓度为0.5-3.6μg/cm均匀包被在反应垫的质控线(C线)位置形成质控线。将包被好检测线和质控线的反应垫烘干待用。
进一步，所述试剂盒包括胶体金标记的SARS-CoV-2病毒RBD蛋白和胶体金标记的免疫球蛋白。
其中，所述胶体金标记的免疫球蛋白喷洒在所述结合垫上。根据本领域的常规操作对所述胶体金标记的免疫球蛋白(浓度1mg/ml)进行喷洒，保证在层析反应中发生信号反应即可。所述胶体金标记的免疫球蛋白3.75μl/ml喷洒到处理过的结合垫上。
所述胶体金标记的SARS-CoV-2病毒RBD蛋白烘干后作为金标物管，待与样品溶液混合。所述胶体金标记的SARS-CoV-2病毒RBD蛋白的量为1.0-5mg。优选地，所述金标管中包含胶体金标记的SARS-CoV-2病毒RBD蛋白的量为1.5mg，待与样品溶液混合。
进一步所述试剂盒中还包括样品稀释液。所述样品稀释液包括20-50mM PBS，0.01％Tween-20，0.09％NaCl，0.03％ProcLin300，pH7.0-7.5
本发明的SARS-CoV-2病毒或其变体的中和抗体胶体金检测试剂盒的制备方法包括以下步骤：
(1)制备胶体金标记的SARS-CoV-2病毒或其变体RBD和胶体金标记的免疫球蛋白；
(2)将ACE2蛋白包被在反应垫上形成检测线，将抗免疫球蛋白抗体包被在反应垫上形成质控线，将胶体金标记的免疫球蛋白喷洒在结合垫上；
(3)将样品垫、结合垫、带有检测线和质控线的反应垫和吸水垫按照顺序依次粘附在底板上，组装后得到免疫层析装置。
步骤(1)包括将胶体金标记的SARS-CoV-2病毒或其变体RBD烘干制成金标记物管。所述胶体金标记的SARS-CoV-2病毒RBD蛋白的量为1.0-5.0mg，优选为1.5mg。
步骤(2)中所述ACE2蛋白以0.2-1.0μg/cm均匀包被在反应垫的检测线(T线)位置形成检测线。优选地，所述ACE2蛋白0.5μg/cm均匀包被在反应垫的检测线位置形成检测线。其中，所述抗免疫球蛋白抗体0.5-3.6μg/cm均匀包被在反应垫的质控线(C线)位置形 成质控线。将包被好检测线和质控线的反应垫烘干待用。其中，所述胶体金标记的免疫球蛋白浓度为1mg/ml，3.75μl/ml喷洒到处理过的结合垫上，烘干待用。
步骤(3)包括将组装完成的片材切割成试纸条，切好的试纸条组装到备好的试纸卡壳中，加样窗口对应试纸条的样品垫，结果显示窗对应试纸条的检测区，再与干燥剂封存在铝箔袋中。
进一步，将制备好的金标记物管、SARS-CoV-2病毒中和抗体胶体金试纸条和样本稀释液放置在盒体内，组装成试剂盒。
(二)SARS-CoV-2病毒或其变体的中和抗体胶体金检测试剂盒及其制备
本发明中SARS-CoV-2病毒或其变体的中和抗体胶体金检测试剂盒还可以体现为金颗粒标记的ACE2蛋白，SARS-CoV-2病毒或其变体RBD蛋白固定在固相支持物特定位置，通过SARS-CoV-2病毒中和抗体阻断RBD蛋白和ACE2蛋白结合产生检测信号的检测试剂盒。
具体地，SARS-CoV-2病毒或其变体中和抗体胶体金检测试剂盒包括检测试纸条，所述试纸条包括顺次搭接的加样垫、结合垫、反应垫和吸水垫，所述加样垫、结合垫、反应垫和吸水垫依次粘附在底板上，所述反应垫上沿样品流动方向依次设有检测线和质控线，所述检测线包被有SARS-CoV-2病毒或其变体RBD蛋白，所述质控线包被有抗免疫球蛋白抗体。
在一些具体实施方案中，所述胶体金检测试剂盒包括检测试纸条，所述试纸条包括顺次搭接的加样垫、结合垫、反应垫和吸水垫，所述加样垫、结合垫、反应垫和吸水垫依次粘附在底板上，所述反应垫上沿样品流动方向依次设有检测线和质控线，所述检测线包含重组SARS-CoV-2病毒RBD蛋白，所述质控线包含抗小鼠IgG抗体。
其中，所述SARS-CoV-2病毒RBD蛋白浓度为0.5-1.5μg/cm均匀包被在反应垫的检测线(T线)位置形成检测线。优选地，所述SARS-CoV-2病毒RBD蛋白以1.0μg/cm均匀包被在反应垫的检测线位置形成检测线。其中，所述抗免疫球蛋白抗体浓度为0.5-3.6μg/cm均匀包被在反应垫的质控线(C线)位置形成质控线。将包被好检测线和质控线的反应垫烘干待用。
进一步，所述试剂盒包括胶体金标记的ACE2蛋白(浓度在0.1-1mg/ml之间)和胶体金标记的免疫球蛋白(浓度在0.1-1.0mg/ml之间)。所述金标记的ACE2蛋白和胶体金标记免疫球蛋白按体积比1：1充分混合，以3.75μl/ml喷洒到结合垫上烘干，其中，所述胶体金标记的ACE2蛋白为0.1-1mg/ml，胶体金标记的免疫球蛋白抗体为0.1-1mg/ml。
所述试剂盒还包括样品稀释液。所述样品稀释液包括20-50mM PBS，0.01％Tween-20，0.09％NaCl，0.03％ProcLin300，pH7.0-7.5。
本发明的SARS-CoV-2病毒或其变体的中和抗体胶体金检测试剂盒的制备方法包括以下步骤：
(1)制备胶体金标记ACE2蛋白的和胶体金标记的免疫球蛋白；
(2)将SARS-CoV-2病毒或其变体RBD包被在反应垫上形成检测线，将抗免疫球蛋白抗体包被在反应垫上形成质控线，将胶体金标记ACE2蛋白和胶体金标记的免疫球蛋白喷洒在结合垫上；
(3)将样品垫、结合垫、带有检测线和质控线的反应垫和吸水垫按照顺序依次粘附在底板上，组装后得到免疫层析装置。
步骤(2)中所述SARS-CoV-2病毒RBD蛋白0.5-1.5μg/cm均匀包被在反应垫的检测线(T线)位置形成检测线。优选地，所述SARS-CoV-2病毒RBD蛋白1.0μl/cm均匀包被在反应垫的检测线位置形成检测线。其中，所述抗免疫球蛋白抗体浓度为0.5-3.6μg/cm均匀包被在反应垫的质控线(C线)位置形成质控线。
步骤(2)中所述金标记的ACE2蛋白(1mg/ml)和胶体金标记免疫球蛋白(1mg/ml)按体积比1：1充分混合，以3.75μl/ml喷洒到结合垫上烘干，其中，所述胶体金标记的ACE2蛋白为1mg/ml，胶体金标记的免疫球蛋白抗体为1mg/ml。
步骤(3)包括将组装完成的片材切割成试纸条，切好的试纸条组装到备好的试纸卡壳中，加样窗口对应试纸条的样品垫，结果显示窗对应试纸条的检测区，再与干燥剂封存在铝箔袋中。
进一步，将制备好的SARS-CoV-2病毒中和抗体胶体金试纸条和样本稀释液按次序放置在盒体内，组装成试剂盒。
(三)SARS-CoV-2病毒或其变体的中和抗体时间分辨免疫荧光检测试剂盒及其制备方法
本发明SARS-CoV-2病毒或其变体的中和抗体时间分辨免疫荧光检测试剂盒，可以体现为免疫荧光微球标记SARS-CoV-2病毒RBD蛋白，ACE2蛋白固定在固相支持物的特定位置上，通过SARS-CoV-2病毒或其变体的中和抗体阻断RBD蛋白和ACE2蛋白结合产生检测信号的检测试剂盒。
具体地，SARS-CoV-2病毒或其变体中和抗体胶体金检测试剂盒包括检测试纸条，所述试纸条包括顺次搭接的加样垫、结合垫、反应垫和吸水垫，所述加样垫、结合垫、反应垫和吸水垫依次粘附在底板上，所述反应垫上沿样品流动方向依次设有检测线和质控线，所述检测线包被有ACE2蛋白，所述质控线包被有抗免疫球蛋白抗体。
在一些具体实施方案中，所述胶体金检测试剂盒包括检测试纸条，所述试纸条包括顺次搭接的加样垫、结合垫、反应垫和吸水垫，所述加样垫、结合垫、反应垫、吸水垫依次粘附在底板上，所述反应垫上沿样品流动方向依次设有检测线和质控线，所述检测线包被有重组ACE2蛋白，所述质控线包被有抗小鼠IgG抗体。
其中，所述ACE2蛋白以0.2-1.0μg/cm均匀包被在反应垫的检测线(T线)位置形成检测线。优选地，所述ACE2蛋白以0.3μg/cm均匀包被在反应垫的检测线位置形成检测线。其中，所述抗免疫球蛋白抗体以0.5-3.6μg/cm均匀包被在反应垫的质控线(C线)位置形成质控线。将包被好检测线和质控线的反应垫烘干待用。
进一步，所述试剂盒包括荧光微球标记的SARS-CoV-2病毒RBD蛋白和荧光微球标记的免疫球蛋白。
其中，所述荧光微球标记的免疫球蛋白(浓度为0.1mg/ml)喷洒在所述结合垫上。根据本领域的常规操作对所述荧光微球标记的免疫球蛋白进行喷洒，保证在层析反应中发生信号反应即可。所述荧光微球标记的免疫球蛋白3.75μl/ml喷洒到处理过结合垫上。
所述荧光微球标记的SARS-CoV-2病毒RBD蛋白烘干后作为标记物管，待与样品溶液混合。在一些实施方案中，所述荧光微球标记的SARS-CoV-2病毒RBD蛋白的量为1.0μg-5μg。在一个优选实施方案中，所述荧光微球标记的SARS-CoV-2病毒RBD蛋白的量为1.5μg。
进一步所述试剂盒中还包括样品稀释液。所述样品稀释液包括20-50mM PBS，0.01％Tween-20，0.09％NaCl，0.03％ProcLin300，pH7.0-7.5。
本发明的SARS-CoV-2病毒或其变体的中和抗体胶体金检测试剂盒的制备方法包括以下步骤：
(1)制备荧光微球标记的SARS-CoV-2病毒或其变体RBD和荧光微球标记的免疫球蛋白；
(2)将ACE2蛋白包被在反应垫上形成检测线，将抗免疫球蛋白抗体包被在反应垫上形成质控线，将荧光微球标记的免疫球蛋白喷洒在结合垫上；
(3)将样品垫、结合垫、带有检测线和质控线的反应垫和吸水垫按照顺序依次粘附在底板上，组装后得到免疫层析装置。
步骤(1)包括将荧光微球标记的SARS-CoV-2病毒或其变体RBD烘干制成荧光微球标记物管。在一些实施方案中，所述荧光微球标记的SARS-CoV-2病毒RBD蛋白的量为1.0μg-5μg。在一个优选实施方案中，所述荧光微球标记的SARS-CoV-2病毒RBD蛋白的量为1.5μg。
步骤(2)中所述ACE2蛋白以0.2-1.0μg/cm均匀包被在反应垫的检测线(T线)位置形成检测线。优选地，所述ACE2蛋白，以0.3μg/cm均匀包被在反应垫的检测线位置形成检测线。其中，所述抗免疫球蛋白抗体以0.5-3.6μg/cm均匀包被在反应垫的质控线(C线)位置形成质控线。将包被好检测线和质控线的反应垫烘干待用。其中，所述胶体金标记的免疫球蛋白以0.1mg/ml浓度，3.75μl/ml喷洒到处理过的结合垫上烘干。
步骤(3)包括将组装完成的片材切割成试纸条，切好的试纸条组装到备好的试纸卡壳中，加样窗口对应试纸条的样品垫，结果显示窗对应试纸条的检测区，再与干燥剂封存在铝箔袋中。
进一步，将制备好的荧光微球标记物管、SARS-CoV-2病毒中和抗体免疫荧光纸条、和样本稀释液放置在盒体内，组装成试剂盒。
SARS-CoV-2病毒或其变体的中和抗体时间分辨免疫荧光检测试剂盒还可以体现为免疫荧光微球标记ACE2蛋白，SARS-CoV-2病毒或其变体的RBD蛋白固定在固相支持物特定位置上，通过SARS-CoV-2病毒中和抗体阻断RBD蛋白和ACE2蛋白结合产生检测信号的检测试剂盒。
本发明还提供了SARS-CoV-2病毒或其变体的中和抗体量子点检测试剂盒，可以体现为量子点标记SARS-CoV-2病毒或其变体的RBD蛋白，ACE2蛋白固定在固相支持物特定位置上，通过SARS-CoV-2病毒或其变体的中和抗体阻断RBD蛋白和ACE2蛋白结合产生检测信号的检测试剂盒。
本发明也提供了SARS-CoV-2病毒或其变体的中和抗体量子点检测试剂盒，可以体现为量子点标记ACE2蛋白，SARS-CoV-2病毒RBD蛋白固定在固相支持物特定位置，通过SARS-CoV-2病毒或其变体的中和抗体阻断RBD蛋白和ACE2蛋白结合产生检测信号的检测试剂盒。
免疫层析装置的使用方法
本发明提供了上述免疫层析装置的使用方法，包括如下步骤：
(1)待测样品中加入样品稀释液稀释后，加入信号标记物混匀；
(2)将混合物滴加至所述免疫层析装置的加样垫上；
(3)待一段时间后，根据检测线和质控线的显色情况，判断样品中是否有冠状病毒中和抗体，判断方法如下：
(a)阳性：所述质控线呈现信号，检测线不呈现信号，说明样品中含有冠状病毒中和抗体；
(b)阴性：所述质控线和检测线均呈现信号，说明样品中不含有冠状病毒中和抗体；
(c)失效：所述质控线和检测线均不呈现信号，说明免疫层析装置失效。
本发明的免疫层析试剂盒的结果判断：当样本中存在SARS-CoV-2病毒中和抗体时，质控线出现信号(或条带)和检测线不出现信号(或条带)；当样本中不存在SARS-CoV-2病毒中和抗体时，检测线和质控线同时出现信号(或条带)。
为了实现上述目的，本发明所采用以下三种技术方案是：
1)一种SARS-CoV-2病毒中和抗体检测试剂盒(胶体金法1)由SARS-CoV-2病毒中和抗体胶体金检测试纸条、SARS-CoV-2 RBD金标物管和样本稀释液组成。SARS-CoV-2病毒中和抗体试纸由支撑板、加样垫、结合垫、检测膜和吸水垫构成，结合垫吸附胶体金标记的小鼠单克隆抗体，检测膜上含有检测线(T线)“|”和质控线(C线)“|”印迹，检测线T为重组人ACE2受体印迹，质控线(C线)为兔抗小鼠IgG印迹。SARS-CoV-2病毒中和抗体加样管中含有胶体金标记的SARS-CoV-2 RBD重组蛋白。所述试剂盒通过阻断人ACE2受体与SARS-CoV-2病毒RBD蛋白结合模式检测SARS-CoV-2病毒中和抗体，若在检测线(T线)和质控线(C线)显现红色条带“|”，则为SARS-CoV-2病毒中和抗体阴性；若仅质控线(C线)显现一条红色条带“|”，则为SARS-CoV-2病毒中和抗体阳性。
2)一种SARS-CoV-2病毒中和抗体检测试剂盒(时间分辨免疫荧光法)由SARS-CoV-2病毒中和抗体时间分辨免疫层析试纸条、SARS-CoV-2 RBD蛋白荧光微球标记物管和样本稀释液组成。SARS-CoV-2病毒中和抗体试纸由支撑板、加样垫、结合垫、检测膜和吸水垫构成，结合垫吸附荧光微球标记的小鼠单克隆抗体，检测膜上含有检测线(T线)“|”和质控线(C线)“|”印迹，检测线(T线)为重组人ACE2受体印迹，质控线(C线)为兔抗小鼠IgG印迹。SARS-CoV-2病毒中和抗体加样管中含有荧光微球标记的SARS-CoV-2 RBD重组蛋白。所述试纸通过阻断人ACE2受体与SARS-CoV-2病毒RBD蛋白结合模式检测SARS-CoV-2病毒中和抗体。根据抑制率判断中和抗体的存在情况。
3)一种SARS-CoV-2病毒中和抗体检测试剂盒(胶体金法2)由SARS-CoV-2病毒中和抗体试纸条和样本稀释液组成。SARS-CoV-2病毒中和抗体试纸由支撑板、加样垫、结合垫、检测膜和吸水垫构成，结合垫吸附胶体金标记的小鼠单克隆抗体和胶体金标记的ACE2受体，检测膜上含有检测线(T线)“|”和质控线C“|”印迹，检测线(T线)为重组SARS-CoV-2 RBD印迹，质控线(C线)为兔抗小鼠IgG印迹。所述试剂盒通过阻断人ACE2受体与SARS-CoV-2病毒RBD蛋白结合模式检测SARS-CoV-2病毒中和抗体，若在检测线(T线)和质控线(C线)显现红色条带“|”，则为SARS-CoV-2病毒中和抗体阴性；若仅质控线(C线)显现一条红色条带“|”，则为SARS-CoV-2病毒中和抗体阳性。
免疫层析装置的应用
本发明提供了上述免疫层析装置在检测受试者感染或疑似感染SARS-CoV-2病毒或其变体中的应用。机体感染新型冠状病毒SARS-CoV-2之后，起初，未来得及引起机体的免疫反应，因此，此时只能检测到新型冠状病毒SARS-CoV-2抗原。之后，引起机体的免疫反应，最早出现的免疫球蛋白是IgM抗体，之后出现IgG抗体。因此，通过检测机体内新型冠状病毒COVID-19抗原，及其特异性的IgM抗体和IgG抗体的存在与否，可以诊断机体对新型冠状病毒SARS-CoV-2抗原的免疫反应状态。对于疑似感染的受试者可以通过本发明的试剂盒检测血清中SARS-CoV-2病毒中和抗体的存在情况，以确定受试者是否感染SARS-CoV-2病毒。已感染的受试者可以通过本发明的试剂盒检测血清中SARS-CoV-2病毒中和抗体的存在情况，以确定是否在机体内产生有效的免疫。
本发明提供了上述免疫层析装置在检测受试者接种针对SARS-CoV-2或其变体的疫苗后是否产生有效免疫中的应用。目前临床正在积极开发SARS-CoV-2病毒的疫苗，部分疫苗的研发已进入临床阶段。本发明的试剂盒可用于快速检测接种SARS-CoV-2疫苗的受试者是否已产生有效的免疫，确定接种疫苗的受试者血清中是否产生中和抗体。这对于SARS-CoV-2疫苗的研发有重要作用。
又一方面，本发明提供了上述免疫层析装置用于检测通过生物学方法获得的SARS-CoV-2中和抗体的应用。目前大量针对SARS-CoV-2或其变体的抗体正在开发中，通过本领域常见的方法，包括动物免疫、噬菌体展示库等方法获得的抗SARS-CoV-2抗体，需要快速有效的方法筛选出有中和能力的抗体。本发明的免疫层析装置可用于测定免疫的动物血清中SARS-CoV-2中和抗体的检测。也可用于其他不同方式纯化得到的SARS-CoV-2抗体中中和抗体的检测。
本发明中“冠状病毒”是指是一类在动物与人类之间传播的人畜共患的RNA病毒。冠状病毒可感染哺乳动物、鸟类，引起牛和猪的消化道疾病或鸡的上呼吸道疾病。自然界常见，已知可感染人类的冠状病毒共有七种，会引起人类的呼吸道感染，可引发普通感冒，乃至中东呼吸综合征(MERS)和严重急性呼吸综合征(SARS)、2019年冠状病毒疾病(COVID-19或SARS-CoV-2)等较严重疾病。本发明中所述冠状病毒选自SARS-CoV-2或其变体、SARS-CoV或其变体或者MERS-Cov或其变体。优选地，所述冠状病毒为SARS-CoV-2或其变体。
本发明中“新型冠状病毒”(SARS-CoV-2)，亦称为2019-nCoV，其属于β属冠状病毒，有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60-140nm。其基因特征与SARSr-Cov和MERSr-CoV有明显区别。研究显示，其与蝙蝠SARS样冠状病毒(bat-SL-CoVZC45)同源性达85％以上。体外分离培养时，2019-nCov 96个小时左右即可在人呼吸道上皮细胞内发现，而在Vero E6和Huh-7细胞系中分离培养需约6天。
本发明中“SARS-CoV-2病毒变体”是指目前发现的新型冠状病毒具有序列高度同源性的病毒。目前已有研究团队从马来亚穿山甲中分离出的一种冠状病毒在E，M，N和S基因中分别与2019-nCoV表现出100％，98.2％，96.7％和90.4％的氨基酸一致性。特别地，穿山甲冠状病毒的S蛋白的受体结合域实际上与2019-nCoV的S蛋白的受体结合域相同，仅具有一个氨基酸差异(https://www.biorxiv.org/content/10.1101/2020.02.17.951335v1)。本发明中所述SARS-CoV-2病毒变体的基因序列与新型冠状病毒的基因序列具有至少85％、86％、 87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列一致性。本发明所述SARS-CoV-2病毒变体宝库偶近期发现的英国病毒突变体B.1.1.7、南非病毒突变体B.1.351以及印度出现的新冠病毒突变株B.1.617等。
“SARS-CoV-2病毒抗原”是指SARS-CoV-2全病毒裂解液的抗原或者重组表达的SARS-CoV-2抗原。SARS-CoV-2病毒包括刺突蛋白(S)、包膜蛋白(E)、基质蛋白(M)和核衣壳蛋白(N)抗原，其中S蛋白是SARS-CoV-2最大的结构蛋白。S蛋白在宿主酶的作用下能裂解为S1和S2亚单位，其中S1亚单位含有受体结合区RBD，是主要的靶抗原。本发明中，所述SARS-CoV-2病毒抗原选自刺突蛋白(S)、包膜蛋白(E)、基质蛋白(M)和/或核衣壳蛋白(N)抗原。优选地，所述SARS-CoV-2病毒抗原为SARS-CoV-2S1亚基或SARS-CoV-2S RBD抗原。本发明中所述SARS-CoV-2S RBD抗原可采用常规重组表达的方法生产，通过构建表达SARS-CoV-2S RBD的质粒，如pFastBac1、pTT5，包含目的基因的表达载体转染表达细胞，如CHO细胞、SF9细胞，表达纯化得到SARS-CoV-2S RBD重组蛋白。
本文所用的术语“中和抗体”指通过与病毒分子结合防止细胞被某种抗原或感染源侵害的抗体，其原理是通过抑制乃至中和它们的某种生化作用。本文所用的术语“SARS-CoV-2病毒中和抗体”是指通过与SARS-CoV-2病毒RBD蛋白结合来阻断SARS-CoV-2病毒RBD与人ACE2受体结合的抗体。
本发明中“ACE2”或“ACE2蛋白”可以体现为重组ACE2蛋白，可以体现为可溶性ACE2蛋白，可以体现为Fc标签ACE2蛋白。本发明中“ACE2蛋白的功能片段”是指能发挥与冠状病毒的刺突蛋白结合，特别是SARS-CoV-2病毒RBD蛋白结合作用的ACE2蛋白的片段，可以是ACE2蛋白中发挥结合功能的部分区域。
本发明中“冠状病毒刺突蛋白部分”是指包括冠状病毒能发挥功能的刺突蛋白部分，可以是病毒RBD蛋白，病毒S1蛋白和病毒S蛋白。优选地为SARS-CoV-2病毒RBD蛋白，SARS-CoV-2病毒S1蛋白和SARS-CoV-2病毒S蛋白，更优选为SARS-CoV-2病毒RBD蛋白或SARS-CoV-2病毒S1蛋白。
本文术语“SARS-CoV-2病毒RBD蛋白”，可以体现为重组SARS-CoV-2病毒RBD蛋白，带有His标签的SARS-CoV-2病毒RBD蛋白，带有Fc标签的SARS-CoV-2病毒RBD蛋白，包含有SARS-CoV-2病毒RBD蛋白的分子，比如SARS-CoV-2病毒S1亚基(如SEQ ID NO:2所示序列)和SARS-CoV-2病毒S蛋白。
本文术语“信号物”可以指可视化信号物、荧光信号物、磁信号物中的至少一种。可视化信号物包括胶体金、胶体银、彩色乳胶微球和彩色染料等。荧光信号物包括荧光微球、量子点等。荧光微球包括上转换荧光微球、时间分辨荧光微球等。
本发明中“免疫荧光微球”,可以体现为含钐(SM)免疫荧光微球，含铕(Eu)免疫荧光微球，含镝(Dy)免疫荧光微球,含锝(Te)免疫荧光微球。
本发明中量子点可选用以下颗粒：(1)单一化合物的量子点，如InAs，InP，InGaAs，AlGaAs，InAlAs，BaSe，BaTe，ZnS，ZnSe，ZnTe，CSiC，SiGe中的任意一种；第IV族和第VI族组成的化合物。(2)由量子点化合物与其它化学物质组装而成的量子点颗粒，如ZnS包覆CdSe，二氧化硅包覆CdSe，可以是有机高分子聚合物与量子点化合物组装而成，如聚丙烯酰胺、聚苯乙烯、聚丙烯、交联葡聚糖、树形分子等任意一种包裹一种量子点或几种量子点制成的颗粒。
本发明所述结合垫的材料不作特别限定，可采用本领域技术人员熟知的材料即可，作为优选，所述结合垫为玻璃纤维膜。
本发明所述加样垫，结合垫是经过处理液浸渍处理的玻璃纤维膜或无纺布或滤纸，所述处理液含有如下组分：Tween 20、Triton x-405、Casein、BSA、PEG-20000，PVP和NaCl。
本发明中反应垫包括但不限于硝酸纤维素膜和醋酸纤维素膜，优选为硝酸纤维素膜。其中的硝酸纤维素膜可以为任何商品化的硝酸纤维素膜，包括S&SAE99、whatman 8um、Millipore M135、Sartoirus CN140等。
本发明中“检测线”是设在反应垫上的，由喷洒或划上的化合物，如蛋白等形成的一条线。例如，首先在反应垫上沿垂直于样品流动的方向确定检测线位置，再将蛋白等分子通过喷洒或划线等方法均匀固定在检测线位置形成检测线。在试纸条或检测试剂盒中会指示出检测线位置，加入待检测样本后，可通过观察检测线显示信号情况判断样品中的分子是否与检测线上包含的分子发生反应。
本发明中“质控线”是设在反应垫上的，由喷洒或划上的对照化合物，如蛋白、生物素、生物素、配体等，形成的一条线，通常设在检测线沿样品流动方向的下游且与检测线平行。首先在反应垫上选择检测线沿样品流动方向的下游位置，再将蛋白、生物素或亲和素等分子通过喷洒或划线等方法固定在质控线位置形成质控线。
本发明中吸水垫可以用任何能吸收液体的材料制成，但吸收能力应足够大。可使用的材料包括但不局限于脱脂棉吸水垫、硅胶吸水垫或海绵吸水垫。
本发明所述底板用于承载所述样品垫、结合垫、反应垫和吸水垫；所述底板可以是各种不吸水的具有支撑作用的薄片，作为举例，所述底板可以为聚氯乙烯(PVC)板、聚丙烯(PP)板、聚乙烯(PE)板或聚氨酯(PU)板，优选为PVC板。
本发明中试纸条包括顺次搭接的加样垫、结合垫、反应垫和吸水垫，所述加样垫、结合垫、反应垫、吸水垫依次粘附在底板上。具体地，试纸条结构为反应垫(如硝酸纤维素膜)置于底板(如聚氯乙烯板)上面，样品垫平贴于反应垫左侧，吸收垫(如吸水纸)平贴于反应垫(硝酸纤维素膜)右侧，结合垫平贴于样品垫与硝酸纤维素膜之间，使其一端压于样品垫之下，另一端覆于反应垫之上。本发明中“搭接”是指加样垫、结合垫、反应垫和吸水垫 相邻两个部分相互重合的连接，这样的搭接结构能保证在加样层析过程中样品能依次流经加样垫、结合垫、反应垫和吸水垫。在一些情况下，在保证样品流经加样垫、结合垫和反应垫时，可不包括吸水垫。在另一些情况下，在保证加样垫、结合垫、反应垫和吸水垫完成样品的层析时，可不包括底板。
如本文使用的术语“受试者”指需要缓解、预防和/或治疗疾病或病症如病毒感染的动物，优选哺乳动物，更优选人。术语包括具有冠状病毒如SARS-CoV-2感染或处于具有冠状病毒如SARS-CoV-2感染风险的人受试者。健康受试者是指未感染SARS-CoV-2病毒的健康动物，优选哺乳动物，更优选人。
本发明中样品包括但不限于(1)感染或疑似感染冠状病毒的血浆、血清或全血；(2)接种冠状病毒疫苗后的血浆、血清或全血；(3)冠状病毒刺突蛋白免疫动物后的血浆、血清或全血；或(4)生物学方法获得的抗冠状病毒的抗体样品。样品可来源于感染或疑似感染SARS-CoV-2病毒的人或动物的血浆、血清、全血、胸腹腔积液、脑脊液或组织标本，还可来源于生物学方法如动物免疫制备的动物血清、血浆、全血或抗SARS-CoV-2冠状病毒的抗体的溶液。
关于肽或多肽序列的“百分比(％)氨基酸序列一致性”定义为对比序列并在必要时引入缺口以获取最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分，候选序列中与特定肽或多肽序列中的氨基酸残基相同的氨基酸残基的百分率。可以本领域技术范围内的多种方式进行序列对比以测定百分比氨基酸序列同一性，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可决定测量对比的适宜参数，包括对所比较的序列全长获得最大对比所需的任何算法。
有益技术效果
SARS-CoV-2病毒血清学检测方法均为检测SARS-CoV-2总抗体(Ab)和SARS-CoV-2IgM抗体和SARS-CoV-2IgG抗体。快速诊断SARS-CoV-2中和抗体在辅助诊断中、疫苗评价的价值尚未被开发。通过SARS-CoV-2 RBD蛋白片段与人可溶性ACE2受体的相互作用开发诊断试剂盒的方法比较新颖，尚为被开发利用。通过读取检测线信号等方式可以计算出中和抗体的抑制率水平，方便、快捷。用层析试纸条实现快速诊断，避免使用大型仪器和场地限制。
本发明提供的免疫层析装置检测病毒中和抗体的准确性高、特异性强，而且检测速度快、操作简便，不需要专业人员的操作，可应用于社区、基层医院、机场、海关甚至家庭等多种场所的初步筛查，能够在数分钟内判断结果，为疑似感染者排查和无症状感染者筛查提供更简便、更快速的现场检测手段，从而及早预防疫情扩散。
附图说明
图1为SARS-CoV-2病毒中和抗体免疫层析试剂盒的原理图，人ACE2蛋白固定在检测线(T线)，信号物标记SARS CoV-2 RBD蛋白；
图2为SARS-CoV-2病毒中和抗体免疫层析试剂盒的原理图，SARS CoV-2 RBD蛋白固定在检测线(T线)，信号物标记人ACE2蛋白；
图3为SARS-CoV-2病毒中和抗体免疫层析试剂盒的俯视结构示意图，依次包含底板、加样垫(加样孔)、结合垫(金标垫)、检测线(T线)、质控线(C线)以及吸水垫；
图4为SARS-CoV-2病毒中和抗体免疫层析试剂盒检测效果图，图4A使用重组SARS-CoV-2病毒RBD蛋白免疫的动物血清作为阳性质控品。阳性质控品在质控线(C线)上出现条带，而在检测线(T线)上没有出现条带；图4B使用免疫前动物空白血清作为阴性质控品评估SARS-CoV-2病毒中和抗体免疫层析试剂盒，而阴性质控品在检测线(T线)和质控线(C线)上均出现条带。
具体实施方式
除非另有说明，本发明所用的技术和科学术语具有与本发明所属领域的普通技术员通常所理解的含义。本发明通过以下实施例作进一步举例说明，这些实施例不应解释为对本发明的限制。凡在本发明的精神和原则之内所做的任何修改、等同替换和改进，均应包含在本发明的保护范围之内。
实施例1 SARS-CoV-2病毒中和抗体免疫层析检测试剂盒(胶体金法1)制备及性能评估
1.1胶体金溶液制备
用超纯水溶解氯金酸，配成10％溶液。再用纯水配制成1L 0.01％的浓度放入烧杯中。将烧杯放置于可加热的混匀仪上，转速调到2000r/min，使溶液充分旋转。一次性迅速加入0.561g柠檬酸三钠溶液。待溶液变成稳定的紫红色时，调小转速到200r/min，关掉加热，继续反应20min。待金溶液恢复成室温后，用清洁带盖的器皿盛装，4℃保存。
1.2 SARS-CoV-2 RBD金标物管的制备
用干净的烧杯盛取10ml胶体金溶液，向其中分别加入40μL，50μL，60μL和70μL优选40μL 0.1M碳酸钾溶液(1.38g碳酸钾，100mL超纯水，0.22μM膜滤过)，并迅速充分混匀3分钟。迅速加入50μg，100μg，150μg和200μg，优选100μg SARS-CoV-2 RBD蛋白(金斯瑞，T80302，序列见SEQ ID NO:1)，充分震荡后静置，室温反应30min。加入250μL封闭液(5％BSA，0.1mM PBS)充分搅拌封闭，封闭时间30min。用尖底的离心管装胶体金混合液，10000g离心力下离心30min，充分吸取上清并丢弃，沉淀用20ml的保存液(2％BSA，0.03％ProcLin300，0.01M PBS，pH 7.2)重悬。重悬后的金标物按1.5μl量分装于500μL尖底离心管底部，37℃过夜烘干。放于4℃保存。
1.3小鼠IgG金标物的制备
用干净的烧杯盛取10ml胶体金溶液，向其中加入40μL 0.1M碳酸钾溶液(1.38g碳酸钾，100mL超纯水，0.22μM过滤)，并迅速充分混匀3分钟。迅速加入100-200μg的小鼠 IgG抗体(金斯瑞，A01011)，充分震荡后静置，室温反应30min。加入250μL封闭液(5％BSA，0.1mM PBS)充分震荡后静置封闭，封闭时间30min。用尖底的离心管装胶体金混合液，10000g离心30min，充分吸取上清并丢弃，将沉淀用2ml保存液(2％BSA，0.03％ProcLin300，0.01M PBS，pH 7.2)重悬，放于4℃保存。
1.4硝酸纤维素膜(NC膜)检测线和质控线包被及胶体金结合垫制备
将人ACE2重组蛋白(Swiss-Prot:Q9BYF1.2，序列见SEQ ID NO:3)用包被缓冲液(15g Na 2HPO 4，0.23g NaH 2PO 4，5-10g蔗糖，0.1-0.5g甲醇溶于1L超纯水中)稀释成0.5mg/ml，1mg/ml，2mg/ml不同浓度梯度，优选1mg/ml，用三维平面点膜喷金仪(金标)以0.3μg/cm均匀包被在硝酸纤维素膜(赛多利斯，1ΜN14ER100025NT)的T线位置(如图2所示的检测线T线位置)。
将兔抗小鼠Ig抗体(金斯瑞：V90301)用包被缓冲液(15g Na 2HPO 4，0.23g NaH 2PO 4，5-10g蔗糖，0.1-0.5g甲醇溶于1L超纯水中)稀释成1-3mg/ml，用三维平面点膜喷金仪以0.5-1.2μL/cm均匀包被在硝酸纤维素膜的C线位置(如图2所示的质控线C线位置)，然后放于37℃烘箱过夜烘干。
将上述制备好的胶体金标记小鼠IgG重悬液，用三维平面点膜喷金仪(金标)喷量3.75μl/ml喷洒到处理过结合垫上。放于37℃干燥箱中过夜烘干。
1.5 SARS-CoV-2病毒中和抗体胶体金试纸条的组装、包装
如图1和图3所示，将样品垫(上海杰一，GL-b02)、结合垫(上海杰一，8951)、划有重组人ACE2蛋白作为检测线和划有兔抗小鼠lgG作为质控线的硝酸纤维素膜(反应垫)以及吸收垫按顺序依次粘附在聚乙烯背衬(底板)上。将组装完成的片材切割成宽度4mm的试纸条，切好的试纸条组装到备好的试纸卡壳中，加样窗口对应试纸条的样品垫，结果显示窗对应试纸条的检测区，再与干燥剂封存在铝箔袋中。温度应该控制在20-30℃，湿度控制在20-30％。
1.6 SARS-CoV-2病毒中和抗体胶体金检测试剂盒的组装
按照上述步骤制备好的金标记物管、SARS-CoV-2病毒中和抗体胶体金试纸条、和样本稀释液(20mM PBS，0.01％Tween-20，0.09％NaCl，0.03％ProcLin300，pH7.0-7.5)按次序放置在盒体内，组装成试剂盒，于4-25℃避光保存。
1.7 SARS-CoV-2病毒中和抗体胶体金检测试剂盒的质控
吸取20μL(2滴)阳性对照(金斯瑞制备动物抗SARS-CoV-2 RBD重组蛋白抗血清)和阴性对照(金斯瑞制备的正常动物抗血清，即免疫前动物空白血清)，分别加入到一管80μL检测缓冲液(20-50mM PBS，0.01％tween-20，0.09％NaCl，0.03％ProcLin300，pH  7.0-7.5)中，充分混匀后，全部加入到重组SARS-CoV-2 RBD金标物管中，充分混匀10min(混匀5-6次)。取所有混合液100μL垂直滴加至检测卡加样处(试纸条加样处)，开始计时，10-15min观察条带显色。结果判定若检测线(T线)和质控线(C线)均显条带，则为SARS-CoV-2抗体检测为阴性，表明检测样品中不包含SARS-CoV-2中和抗体；若检测线(T线)不显条带色而质控线(C线)显条带，则为SARS-CoV-2抗体检测阳性，表明检测样品中包含SARS-CoV-2中和抗体；若质控线不显色，则试纸无效。检测效果如图4所示，结果说明本发明的试剂盒可用于SARS-CoV-2病毒中和抗体的检测。
1.8 SARS-CoV-2病毒中和抗体免疫层析试剂盒性能评估
将实施例1.2制备的金标记物管、SARS-CoV-2病毒中和抗体胶体金检测试纸条、人血清样本、质控品和样本稀释液(20-50mM PBS，0.01％Tween-20，0.09％NaCl，0.03％ProcLin300，pH7.0-7.5)恢复至18-30℃。吸取20μL血清样本，加入到一管80μL稀释液样本中，充分混匀后，全部加入到金标记物管中，充分混匀10min(混匀5-6次)；取所有混合液100μL垂直滴加至检测卡加样处，开始计时，10-15min观察条带显色；检测结果见表1，通过SARS-CoV-2病毒中和抗体免疫层析试剂盒检测40例健康人血清，检测结果均为阴性。本发明的试剂盒的阴性符合率为100％。通过检测4例阳性质控品(含有基质血清及抗SARS-CoV-2S蛋白抗体9A9C9(重组中和抗体，南京金斯瑞))(P1、P2、P3、P4)，检测结果均为阳性。本发明的试剂盒阳性符合率100％。其中“+”表示阴性，
Figure PCTCN2021098272-appb-000001
表示阳性。
表1 SARS-CoV-2病毒中和抗体检测试剂盒检测健康人血清特异性结果
Figure PCTCN2021098272-appb-000002
Figure PCTCN2021098272-appb-000003
实施例2 SARS-CoV-2病毒中和抗体免疫层析检测试剂盒(时间分辨免疫荧光法)制备及性能评估
2.1 SARS-CoV-2 RBD蛋白荧光微球标记物管的制备
移取90μl微球活化缓冲液(5-50mM MES PH5-6.5)和10μl的5％荧光微球(微测，MD001)加入到到2.0mL离心管中，震荡混匀。20000g离心20min，弃上清。用100μL微球活化缓冲液(5-50mM MES，pH5.0-6.5)重悬沉淀，用超声仪超声50S。加入1-3μL的2mg/mL EDC(Sigmaaldrich，E6383)稀释液，放置混匀仪上混匀25min，转速为200r/min。20000g离心20min，弃上清。用100μL微球标记缓冲液(20-100mM Hepes，pH7.0-8.0)重悬沉淀。向重悬后的荧光微球中加入5-20μg SARS-CoV-2 RBD蛋白(金斯瑞，T80302)，混匀仪上混匀3h，温度30℃，转速200r/min。在荧光微球中加入10-30μL 10％BSA，2-10μL的CE510(JSR CE510)放置混匀仪上混匀1h，温度30℃，转速200r/min。20000g离心10min，弃上清。用2000μL荧光微球重悬液(2％BSA，0.03％ProcLin300，0.01M pH 7.2PBS，0.22μm过滤)重悬后的SARS-CoV-2 RBD蛋白荧光微球标记物。按1.5-5μl量分装于500μL尖底离心管底部，37℃过夜烘干，形成SARS-CoV-2 RBD蛋白荧光微球标记物管。放于4℃保存。
2.2小鼠IgG荧光微球标记物的制备
移取90μl微球活化缓冲液(5-50mM MES，pH5-6.5)和10μl 5％荧光微球(微测，MD001)加入到2.0mL离心管中，震荡使其充分混匀。20000g离心20min，弃上清。用100μL微球活化缓冲液(5-50mM MES，pH5.0-6.5)重悬沉淀，超声仪超声50S。加入1-3μL2mg/mL EDC(Sigmaaldrich，E6383)稀释液。放置混匀仪上混匀25min，反应温度为30℃，转速为200r/min。置于离心机中20000g离心20min，弃上清。取100μL微球标记缓冲液(20-100mM Hepes，pH7.0-8.0)重悬沉淀，用超声仪超声50S。在荧光微球中加入10-30μg小鼠IgG抗体(金斯瑞，A01011)，放置混匀仪上混匀3h，温度30℃，转速200r/min。在荧光微球中加入10-30μL 10％BSA，CE510(JSR CE510)2-10μL在涡旋混匀仪上混匀10秒，放置混匀仪上混匀1h，温度30℃，转速200r/min。20000g离心10min，弃上清。加入2000μL荧光微球重悬液(2％BSA，0.03％ProcLin300，0.01MPBS，pH 7.2，0.22μm过滤)，置4℃备用。
2.3硝酸纤维素膜(NC膜)检测线和质控线包被及结合垫制备
将人ACE2重组蛋白用包被缓冲液(15g Na 2HPO 4，0.23g NaH 2PO 4，5-10g蔗糖，0.1-0.5g甲醇溶于1L超纯水中)稀释成0.5mg/ml，1mg/ml，2mg/ml不同浓度梯度，优选1mg/ml，以0.3μg/cm均匀包被在硝酸纤维素膜(赛多利斯，1ΜN14ER100025NT)的T线位置(如图2所示的检测线T线位置)。将兔抗小鼠Ig抗体(金斯瑞：V90301)用包被缓冲液稀释成1-3mg/ml，以0.5-1.2μL/cm均匀包被在硝酸纤维素膜的C线位置(如图2所示的质控线C线位置)，然后放于37℃烘箱过夜烘干。将上述制备好的小鼠IgG荧光微球标记物重悬液，三维平面点膜喷金仪(金标)喷量3.75μl/ml喷洒到结合垫上，然后放于37℃干燥箱中过夜烘干。
2.4 SARS-CoV-2病毒中和抗体免疫层析检测试纸条(时间分辨免疫荧光法)的组装
如图2所示，将样品垫(上海杰一，GL-b02)、结合垫(上海杰一，8951)、划有重组人ACE2蛋白的为检测线和划有兔抗小鼠lgG作为质控线的硝酸纤维素膜以及吸收垫按顺序依次粘附在聚乙烯背衬(底板)上。将组装完成的片材切割成宽度4mm的试纸条，切好的试纸条组装到备好的试纸卡壳中，加样窗口对应试纸条的样品垫，结果显示窗对应试纸条的检测区，再与干燥剂封存在铝箔袋中。温度应该控制在20-30℃，湿度控制在20-30％。
2.5 SARS-CoV-2病毒中和抗体免疫层析检测试剂盒(时间分辨免疫荧光法)组装
按照上述2.1步骤制备好的SARS-CoV-2 RBD蛋白荧光微球标记物管、SARS-CoV-2病毒中和抗体免疫层析检测试纸条、和样本稀释液(20mM PBS，0.01％Tween-20，0.09％NaCl，0.03％ProcLin300，pH7.0-7.5)按次序放置在盒体内，组装成试剂盒，于4-25℃避光保存。
2.6 SARS-CoV-2病毒中和抗体时间分辨免疫荧光试剂盒性能评估
将上述的SARS-CoV-2病毒中和抗体免疫层析检测试剂盒(时间分辨免疫荧光法)和人血清、质控品样本恢复至18-30℃。吸取20μL血清样本，加入到一管80μL稀释液样本中，充分混匀后，全部加入到SARS-CoV-2 RBD蛋白荧光微球标记物管中，充分混匀10min(混匀5-6次)。取所有混合液100μL垂直滴加至检测卡加样处，开始计时，10-15min观察荧光检测仪读数。检测结果见表2，通过SARS-CoV-2病毒中和抗体免疫层析试剂盒检测50例健康人血清、4例阳性质控品(含有基质血清及抗SARS-CoV-2S蛋白抗体9A9C9，P1、P2、P3、P4)和16例阳性兔血清(通过SARS-CoV-2S蛋白片段免疫兔获取的血清，R1-R16)，计算抑制率，抑制率＜30％，结果判定为阴性，抑制率≥30％，结果判定为阳性。50例健康人血清检测抑制率均＜30％，阴性符合率100％，4例阳性质控品和16例阳性兔血清检测抑制率≥30％，阳性符合100％。检测结果见表2。
表2 SARS-CoV-2病毒中和抗体时间分辨免疫荧光试剂盒健康人血清特异性和质控品检测灵敏度结果
阴性样本编号 检测结果 阴性样本编号 检测结果
1 -5％ 26 18％
2 18％ 27 4％
3 7％ 28 15％
4 25％ 29 8％
5 23％ 30 9％
6 20％ 31 22％
7 0％ 32 19％
8 -6％ 33 -1％
9 1％ 34 24％
10 21％ 35 14％
11 3％ 36 10％
12 9％ 37 15％
13 6％ 38 20％
14 -3％ 39 -2％
15 -1％ 40 12％
16 15％ 41 14％
17 16％ 42 11％
18 11％ 73 12％
19 20％ 44 2％
20 6％ 45 21％
21 -14％ 46 1％
22 6％ 47 28％
23 2％ 48 4％
24 9％ 49 3％
25 24％ 50 11％
P1 43％ R7 33％
P2 59％ R8 93％
P3 84％ R9 88％
P4 87％ R10 75％
R1 62％ R11 56％
R2 33％ R12 72％
R3 61％ R13 52％
R4 45％ R14 58％
R5 76％ R15 32％
R6 53％ R16 83％
实施例3 SARS-CoV-2病毒中和抗体免疫层析检测试剂盒(胶体金法2)制备及性能评估
3.1胶体金溶液制备
用超纯水溶解氯金酸，配成10％溶液。再用纯水配制成1L 0.01％的浓度放入烧杯中。将烧杯放置于可加热的混匀仪上，转速调到2000r/min，使溶液充分旋转。一次性迅速加入 0.561g柠檬酸三钠溶液。待溶液变成稳定的紫红色时，调小转速到200r/min，关掉加热，继续反应20min。待金溶液恢复成室温后，用清洁带盖的器皿盛装，4℃保存，有效期1年。
3.2人ACE2重组蛋白金标物的制备
用干净的烧杯盛取10ml胶体金溶液，向其中分别加入40μL，50μL，60μL和70μL，优选60μL 0.1M碳酸钾溶液(1.38g碳酸钾，100mL超纯水，0.22μM过滤)，并迅速充分混匀3分钟。迅速加入50μg，100μg，150μg和200μg，优选100μg的人的ACE2重组蛋白，充分震荡后静置，室温反应30min。加入250μL封闭液(5％BSA，0.1mM PBS)充分搅拌封闭，封闭时间30min。用尖底的离心管装胶体金混合液，10000g离心力下离心30min，充分吸取上清并丢弃，沉淀5ml的保存液(2％BSA，0.03％ProcLin300，0.01M PBS，pH 7.2)重悬。
3.3小鼠IgG金标物的制备：
用干净的烧杯盛取10ml胶体金溶液，向其中加入40μL 0.1M碳酸钾溶液(1.38g碳酸钾，100mL超纯水，0.22μM过滤)，并迅速充分混匀3分钟。迅速加入100-200μg的小鼠IgG抗体(金斯瑞，A01011)，充分震荡后静置，室温反应30min。加入250μL封闭液(5％BSA，0.1mM PBS)充分震荡后静置封闭，封闭时间30min。用尖底的离心管装胶体金混合液，10000g离心力下离心30min，充分吸取上清并丢弃，将沉淀用2ml保存液(2％BSA，0.03％ProcLin300，0.01M PBS，pH 7.2)重悬，放于4℃保存。
3.4硝酸纤维素膜(NC膜)检测线和质控线包被及结合垫制备
将人SARS-CoV-2 RBD蛋白(金斯瑞，T80302)用包被缓冲液(15g Na 2HPO 4，0.23gNaH 2PO 4，5-10g蔗糖，0.1-0.5g甲醇溶于1L超纯水中)稀释成0.1mg/ml，0.3mg/ml，0.6mg/ml不同浓度梯度，优选0.3mg/ml,以0.5μl/cm均匀包被在硝酸纤维素膜(赛多利斯，1ΜN14ER100025NT)的T线位置(如图2所示的检测线T线位置)。将兔抗小鼠Ig抗体(金斯瑞：V90301)用包被缓冲液(15g Na 2HPO 4、0.23g NaH 2PO 4，5-10g蔗糖、0.1-0.5g甲醇溶于IL超纯水中)稀释成1-3mg/ml，以0.5-1.2μL/cm均匀包被在硝酸纤维素膜的C线位置(如图2所示的质控线C线位置)，然后放于37℃烘箱过夜烘干。将上述制备好的人ACE2重组蛋白金标物重悬液和胶体金标记小鼠IgG重体积比悬液按1：1充分混合，三维平面点膜喷金仪喷量3.75μl/ml喷洒到结合垫上，然后放于37℃干燥箱中过夜烘干。
3.5 SARS-CoV-2病毒中和抗体免疫层析试纸条(胶体金法2)的组装，包装
如图2和图3所示，将样品垫(上海杰一，GL-b02)、结合垫(上海杰一，8951)、划有人SARS-CoV-2 RBD蛋白的作为检测线和划有兔抗小鼠lgG作为质控线的硝酸纤维素膜以及吸收垫按顺序依次粘附在聚乙烯背衬(底板)上。将组装完成的片材切割成宽度4mm的 试纸条，切好的试纸条组装到备好的试纸卡壳中，加样窗口对应试纸条的样品垫，结果显示窗对应试纸条的检测区，再与干燥剂封存在铝箔袋中。温度应该控制在20-30℃，湿度控制在20-30％。
3.6 SARS-CoV-2病毒中和抗体免疫层析试剂盒(胶体金法2)的组装
按照上述步骤制备SARS-CoV-2病毒中和抗体试纸条、和样本稀释液(20mM PBS，0.01％Tween-20，0.09％NaCl，0.03％ProcLin300，pH7.0-7.5)按次序放置在盒体内，组装成试剂盒，于4-25℃避光保存。
3.7 SARS-CoV-2病毒中和抗体免疫层析试剂盒(胶体金法2)的质控
吸取20μL(2滴)阳性对照(金斯瑞制备动物抗SARS-CoV-2 RBD重组蛋白抗血清)和阴性对照(金斯瑞制备的正常动物抗血清，即免疫前动物空白血清)，分别加入到一管80μL检测缓冲液(20-50mM PBS，0.01％tween-20，0.09％NaCl，0.03％ProcLin300，pH7.0-7.5)中，充分混匀后，全部加入到重组SARS-CoV-2 RBD金标物管中，充分混匀10min(混匀5-6次)。取所有混合液100μL垂直滴加至检测卡加样处(试纸条加样处)，开始计时，10-15min观察条带显色。结果判定若检测线(T)和质控线(C)均显条带，则为SARS-CoV-2抗体检测为阴性，表明检测样品中不包含SARS-CoV-2中和抗体；若检测(T)线不显条带色而质控线(C)显条带，则为SARS-CoV-2抗体检测阳性，表明检测样品中包含SARS-CoV-2中和抗体；若质控线不显色，则试纸无效。检测效果如图4所示，结果说明本发明的试剂盒可用于SARS-CoV-2病毒中和抗体的检测。
3.8 SARS-CoV-2病毒中和抗体免疫层析试剂盒(胶体金法2)特异性分析
将实施例3.5中制备的SARS-CoV-2病毒中和抗体免疫层析试纸条、人血清样本和样本稀释液(20-50mM PBS，0.01％Tween-20，0.09％NaCl，0.03％ProcLin300，pH7.0-7.5)恢复至18-30℃。吸取20μL血清样本，加入到一管80μL稀释液样本中，充分混匀。取所有混合液100μL垂直滴加至检测卡加样处，开始计时，10-15min观察条带显色。通过SARS-CoV-2病毒中和抗体免疫层析试剂盒检测健康人血清，检测结果均为阴性。本发明的试剂盒的特异性为100％。其中“+“表示阴性，
Figure PCTCN2021098272-appb-000004
表示阳性。
序列信息：
SARS-CoV-2刺突蛋白RBD蛋白氨基酸序列(SEQ ID NO:1)
Figure PCTCN2021098272-appb-000005
SARS-CoV-2刺突蛋白S1亚基氨基酸序列(SEQ ID NO:2)
Figure PCTCN2021098272-appb-000006
人ACE2蛋白氨基酸序列(SEQ ID NO:3)
Figure PCTCN2021098272-appb-000007

Claims (34)

  1. 一种检测冠状病毒中和抗体的免疫层析装置，其特征在于，所述装置包括检测试纸条，所述试纸条包括顺次搭接的加样垫、结合垫、反应垫和吸水垫，所述加样垫、结合垫、反应垫、吸水垫依次粘附在底板上，所述反应垫上沿样品流动方向包含依次设置的检测线和质控线，其中，所述检测线上包含冠状病毒刺突蛋白部分或者ACE2蛋白或其功能片段，所述装置还包括信号物标记的ACE2蛋白或其功能片段或者信号物标记的冠状病毒刺突蛋白部分，所述冠状病毒刺突蛋白部分与ACE2蛋白或其功能片段结合后在检测线上产生检测信号。
  2. 根据权利要求1所述的免疫层析装置，所述检测线上包含ACE2蛋白或其功能片段时，所述装置包括信号物标记的冠状病毒刺突蛋白部分，所述信号物标记的冠状病毒刺突蛋白部分与所述ACE2蛋白或其功能片段结合产生检测信号。
  3. 根据权利要求1所述的免疫层析装置，所述检测线上包含冠状病毒刺突蛋白部分时，所述装置包括信号物标记的ACE2蛋白或其功能片段，所述信号物标记的ACE2蛋白或其功能片段与所述冠状病毒刺突蛋白结合产生检测信号。
  4. 根据权利要求1-3中任一项所述的免疫层析装置，所述ACE2蛋白或其功能片段选自重组ACE2蛋白或带Fc标签ACE2蛋白。
  5. 根据权利要求4所述的免疫层析装置，所述ACE2蛋白或其功能片段包含与SEQ ID NO:3所示氨基酸序列至少70％一致性的序列。
  6. 根据权利要求1-5中任一项所述的免疫层析装置，所述冠状病毒刺突蛋白部分包括冠状病毒S蛋白、冠状病毒S1蛋白、冠状病毒RBD蛋白或者带有His或者Fc标签的冠状病毒RBD蛋白，优选为SARS-CoV-2病毒刺突蛋白部分。
  7. 根据权利要求6中所述的免疫层析装置，所述SARS-CoV-2病毒刺突蛋白部分包含SARS-CoV-2病毒S1蛋白或SARS-CoV-2病毒RBD蛋白。
  8. 根据权利要求6或7所述的免疫层析装置，所述SARS-CoV-2病毒刺突蛋白部分包含与SEQ ID NO:1或2所示氨基酸序列至少70％一致性的序列。
  9. 根据权利要求1-8中任一项所述的免疫层析装置，所述质控线上包含第一分子，所述加样垫上包含第二分子或检测时所述加样垫上另添加第二分子，所述第二分子能与第一分子结合产生检测信号。
  10. 根据权利要求9所述的免疫层析装置，所述第一分子和第二分子分别选自免疫球蛋白和抗免疫球蛋白抗体，受体和配体，或者生物素和亲和素组成的分子对中的任一个。
  11. 根据权利要求10所述的免疫层析装置，其中，
    所述第一分子选自抗免疫球蛋白抗体、配体或者生物素，对应地所述第二分子选自信号物标记的免疫球蛋白、信号物标记的受体或者亲和素；或所述第一分子选自免疫球蛋白、受体或者亲和素，对应地所述第二分子选自信号物标记的抗免疫球蛋白抗体、信号物标记的配体或者生物素。
  12. 根据权利要求1、2和4-11中任一项所述的免疫层析装置，所述信号物标记冠状病毒刺突蛋白部分的量为1.0μg-5mg。
  13. 根据权利要求12所述的免疫层析装置，所述ACE2蛋白或其功能以0.2-1.0ug/cm的量均匀划线或喷洒包被在检测线位置形成检测线。
  14. 根据权利要求1、3-11中任一项所述的免疫层析装置，所述病毒刺突蛋白部分浓度为以0.5-1.5ug/cm均匀划线或喷洒包被在检测线位置形成检测线。
  15. 根据权利要求14所述的免疫层析装置，所述信号物标记的ACE2蛋白或其功能片段与信号物标记的免疫球蛋白抗体按体积比0.5:1～1:1.5混合喷洒在结合垫上，其中，所述信号物标记的ACE2蛋白或其功能片段的浓度为0.1-1mg/ml，信号物标记的免疫球蛋白抗体的浓度为0.1-1mg/ml。
  16. 根据权利要求1-15中任一项所述的免疫层析装置，所述信号物选自胶体金、量子点或免疫荧光微球，优选为胶体金和免疫荧光微球。
  17. 根据权利要求16所述的免疫层析装置，所述免疫荧光微球选自含钐免疫荧光微球，含铕免疫荧光微球，含镝免疫荧光微球或含锝免疫荧光微球。
  18. 根据权利要求16所述的免疫层析装置，所述量子点选自第IV族和第VI族组成的化合物或由量子点化合物与其它化学物质组装而成的量子点颗粒。
  19. 根据权利要求1-18中任一项所述的免疫层析装置，所述装置还包括样品稀释液。
  20. 根据权利要求1-19中任一项所述的免疫层析装置，其中，所述样品包括：
    (1)感染或疑似感染冠状病毒的血浆、血清或全血；
    (2)接种冠状病毒疫苗后的血浆、血清或全血；
    (3)冠状病毒刺突蛋白免疫动物后的血浆、血清或全血；或
    (4)生物学方法获得的抗冠状病毒的抗体样品。
  21. 根据权利要求1-20中任一项所述的免疫层析装置，所述冠状病毒选自SARS-CoV-2或其变体、SARS-CoV或其变体或者MERS-CoV或其变体，优选为SARS-CoV-2或其变体。
  22. 权利要求1-21中任一项免疫层析装置的使用方法，包括如下步骤：
    (1)待测样品中加入样品稀释液稀释后，加入信号标记物混匀；
    (2)将混合物滴加至所述免疫层析装置的加样垫上；
    (3)待一段时间后，根据检测线和质控线的显示信号情况，判断样品中是否有冠状病毒中和抗体，判断方法如下：
    (a)阳性：所述质控线呈现信号，检测线不呈现信号，说明样品中含有冠状病毒中和抗体；
    (b)阴性：所述质控线和检测线均呈现信号，说明样品中不含有冠状病毒中和抗体；
    (c)失效：所述质控线和检测线均不呈现信号，说明免疫层析装置失效。
  23. 权利要求1-21中任一项免疫层析装置的使用方法，包括如下步骤：
    (1)待测样品中加入样品稀释液稀释；
    (2)将稀释的样品液滴加至所述免疫层析装置的加样垫上；
    (3)待一段时间后，根据检测线和质控线的显示信号情况，判断样品中是否有冠状病毒中和抗体，判断方法如下：
    (a)阳性：所述质控线呈现信号，检测线不呈现信号，说明样品中含有冠状病毒中和抗体；
    (b)阴性：所述质控线和检测线均呈现信号，说明样品中不含有冠状病毒中和抗体；
    (c)失效：所述质控线和检测线均不呈现信号，说明免疫层析装置失效。
  24. 根据权利要求22所述的方法，所述信号标记物为胶体金标记的SARS-CoV-2刺突蛋白S1亚基、胶体金标记的SARS-CoV-2 RBD、荧光微球标记的SARS-CoV-2刺突蛋白S1亚基或荧光微球标记的SARS-CoV-2 RBD，优选为胶体金标记的SARS-CoV-2 RBD或荧光微球标记的SARS-CoV-2 RBD。
  25. 根据权利要求23所述的方法，所述信号标记物为胶体金标记的ACE2蛋白或其功能片段，优选为胶体金标记的人重组的ACE2蛋白或其功能片段。
  26. 权利要求1-21中任一项所述的免疫层析装置的制备方法，其特征在于，包括以下步骤：
    (1)制备信号物标记的冠状病毒刺突蛋白或信号物标记的ACE2蛋白或其功能片段和信号物标记的免疫球蛋白抗体；
    (2)将ACE2蛋白或其功能片段包被在反应垫上形成检测线或将冠状病毒刺突蛋白包被在反应垫上形成检测线，将抗免疫球蛋白抗体包被在反应垫上形成质控线，将信号物标记的免疫球蛋白抗体喷洒在结合垫上；
    (3)将样品垫、结合垫、带有检测线和质控线的反应垫和吸水垫按照顺序依次粘附在底板上，组装后得到免疫层析装置。
  27. 根据权利要求26所述的制备方法，所述步骤(2)中将ACE2蛋白或其功能片段包被在反应垫上形成检测线时，所述装置中还包括信号物标记的冠状病毒刺突蛋白部分。
  28. 根据权利要求27所述的制备方法，所述信号物标记的冠状病毒刺突蛋白部分的量为1.0μg-5mg L。
  29. 根据权利要求27或28所述的制备方法，所述步骤(2)中所述ACE2蛋白或其功能片段以0.2-1.0ug/cm的量均匀划线或喷洒包被在检测线位置形成检测线。
  30. 根据权利要求26所述的制备方法，所述步骤(2)中将冠状病毒刺突蛋白部分包被在反应垫上形成检测线时，所述装置中还包括信号物标记的ACE2蛋白或其功能片段。
  31. 根据权利要求30所述的制备方法，所述步骤(2)还包括将信号物标记的ACE2蛋白或其功能片段与信号物标记的免疫球蛋白抗体以体积比1：1的比例混合喷洒在结合垫上，其中，所述信号物标记的ACE2蛋白或其功能片段的量为0.1-1mg/ml，信号物标记的免疫球蛋白抗体为0.1-1mg/ml。
  32. 权利要求1-21中任一项所述的免疫层析装置在检测受试者是否感染SARS-CoV-2病毒或其变体中的应用。
  33. 根据权利要求1-21中任一项所述的免疫层析装置在检测样品中SARS-CoV-2病毒或其变体的中和抗体中的应用。
  34. 根据权利要求33所述的应用，其中，所述样品来源于感染或疑似感染SARS-CoV-2病毒或其变体的受试者，感染SARS-CoV-2病毒或其变体后康复者，接种针对SARS-CoV-2或其变体的疫苗的受试者，或者通过生物学方法获得的抗SARS-CoV-2或其变体的抗体。
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