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WO2022206401A1 - 一种高灵敏SARS-CoV-2中和抗体的检测方法、检测试剂盒 - Google Patents

一种高灵敏SARS-CoV-2中和抗体的检测方法、检测试剂盒 Download PDF

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Publication number
WO2022206401A1
WO2022206401A1 PCT/CN2022/081424 CN2022081424W WO2022206401A1 WO 2022206401 A1 WO2022206401 A1 WO 2022206401A1 CN 2022081424 W CN2022081424 W CN 2022081424W WO 2022206401 A1 WO2022206401 A1 WO 2022206401A1
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detection
line
neutralizing antibody
protein
cov
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PCT/CN2022/081424
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English (en)
French (fr)
Inventor
张玉基
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南京立顶医疗科技有限公司
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Publication of WO2022206401A1 publication Critical patent/WO2022206401A1/zh

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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the invention belongs to the technical field of biomedical detection, and in particular relates to a detection method and detection kit for a highly sensitive SARS-CoV-2 neutralizing antibody.
  • the pathogen of new coronavirus pneumonia (new coronary pneumonia, COVID-19) is a new type of coronavirus, which belongs to the ⁇ genus. It has 5 essential genes, respectively targeting 4 structural proteins, nucleoprotein (N), viral envelope (E), matrix protein (M) and spike protein (S), and RNA-dependent RNA polymerase (RdRp).
  • the nucleoprotein (N) wraps the RNA genome to form the nucleocapsid, which is surrounded by the viral envelope (E), which is embedded with proteins such as matrix protein (M) and spike protein (S).
  • the spike protein (S) enters the cell by binding to the host's angiotensin-converting enzyme 2 (ACE-2) through the receptor binding site (RBD).
  • ACE-2 angiotensin-converting enzyme 2
  • the S protein is the key for the virus to enter the human body and is the main target of vaccines and therapeutic neutralizing antibodies.
  • the receptor-binding (RBD) domain of the S protein can bind to the host ACE2 receptor, mediate viral invasion into host cells, and is also the binding site for most potent neutralizing antibodies.
  • the neutralizing antibody After the neutralizing antibody binds to the RBD domain, it can make the virus lose its ability to adsorb and invade host cells, thereby inhibiting the replication and spread of the virus in the host.
  • the virus neutralizing antibody plays an important role in eliminating the virus in the host.
  • the "gold standard" of traditional neutralizing antibody detection is infection inhibition test, which is mainly to mix the virus with the antibody to be detected first, and then inoculate the mixture into sensitive animals, embryos or cells, and detect the disease by the lesions of the sensitive animals, embryos or cells.
  • the residual virus infectivity is determined to indirectly determine the neutralizing effect of the antibody to be detected on the virus.
  • PRNT plaque reduction neutralization test
  • CPE cytopathic effct
  • colloidal gold-labeled RBD binds to ACE2 to form a detection line.
  • the neutralizing antibodies block the binding of RBD and ACE2, thereby reducing the color intensity of the detection line.
  • the presence of neutralizing antibodies can be judged by machine interpretation or by the change in the color intensity of the detection line. When interpreting with the naked eye, a large change in the depth of the detection line is required to make a judgment. The sensitivity of this form of detection card needs to be improved.
  • the purpose of the present invention is to solve the technical problems of cumbersome, time-consuming and low-sensitivity detection of SARS-CoV-2 neutralizing antibodies in the prior art, and to provide a simple, fast and highly sensitive method for SARS-CoV-2 neutralizing antibodies. Detection methods and detection kits.
  • the technical scheme of the present invention is as follows:
  • a highly sensitive SARS-CoV-2 neutralizing antibody detection method based on a sandwich method combined with a blocking method to detect SARS-CoV-2 neutralizing antibodies.
  • the highly sensitive SARS-CoV-2 neutralizing antibody detection method enhances the specificity of the subsequent detection of neutralizing antibodies by sandwich method by capturing non-neutralizing antibodies in advance.
  • the highly sensitive SARS-CoV-2 neutralizing antibody detection method uses ACE2 protein as a capture material, blocks non-neutralizing antibodies, and forms a coated RBD protein-neutralizing antibody-RBD protein-labeled carrier complex to achieve Detection of SARS-CoV-2 neutralizing antibodies.
  • a high-sensitivity SARS-CoV-2 neutralizing antibody detection kit based on the above-mentioned detection method includes a capture line, and the capture line is coated with ACE2 protein.
  • a detection line, a quality control line, and a water absorbing pad are sequentially arranged on one side of the capture line, and a binding pad and a sample pad are arranged on the other side of the capture line in sequence;
  • the capture line, detection line and quality control line are all set on the NC membrane;
  • the detection line is coated with RBD protein, and the binding pad is provided with an RBD protein marker, and the RBD protein marker includes a marker carrier and the RBD protein bound to the marker carrier.
  • the labeling carrier comprises one or more of colloidal gold, latex microspheres, nanocarbon, magnetic microspheres or fluorescent microspheres.
  • the sample pad is further provided with a blood filter pad or an anti-RBC antibody.
  • the detection result determination method determines the SARS-CoV-2 neutralizing antibody content by directly observing the reaction results of the capture line, the detection line and the quality control line.
  • the detection result determination method uses an instrument to read the reaction results of the capture line, the detection line and the quality control line, and judges the neutralization of SARS-CoV-2 by the ratio of the detection line and the quality control line antibody content.
  • the detection principle of the present invention is as follows: the present invention detects the neutralizing antibody by adopting the blocking method and the sandwich method, and by adding a capture line, the non-neutralizing antibody targeting RBD is captured in advance, so as to ensure the subsequent detection of neutralization by the sandwich method.
  • Specificity of the antibody Through the design of capture line, detection line and RBD protein label, the RBD protein label is used to capture the non-neutralizing antibody targeting RBD in the sample, and the antibody-RBD protein-labeled carrier complex is formed.
  • the ACE2 protein in the wire captures the RBD protein, thereby immobilizing a non-neutralizing antibody targeting RBD on the capture wire.
  • the neutralizing antibody-RBD protein-labeled carrier complex formed after the neutralizing antibody is captured by the RBD protein marker cannot be captured by the capture line. As the chromatography reaches the detection line, it is captured by the RBD at the detection line to form a packet.
  • the RBD protein-neutralizing antibody-RBD protein-labeled carrier complex causes a corresponding signal to appear on the detection line, and the neutralizing antibody concentration in the sample is judged by this signal.
  • the present invention captures non-neutralizing antibodies targeting RBD in advance by adding a capture line to ensure subsequent detection of the specificity of neutralizing antibodies by the sandwich method, and determines whether there is neutralizing antibody in the sample by the presence or absence of the detection line. and antibody, the sensitivity is better than that by blocking competition method.
  • a second concentration determination point can be formed by comparing the color depth of the detection line and the capture line: when the neutralizing antibody exceeds a certain concentration, the detection line is lighter than the capture line. At higher concentrations of neutralizing antibody, the capture line disappeared.
  • the kit provided by the present invention adopts the method of immunochromatography, which has simple operation and fast detection speed.
  • Fig. 1 is the structural representation of the kit of the present invention
  • the present invention relates to a highly sensitive SARS-CoV-2 neutralizing antibody detection method, which realizes the detection of SARS-CoV-2 neutralizing antibodies based on a sandwich method combined with a blocking method.
  • the specificity of neutralizing antibodies was detected by sandwich method.
  • the high-sensitivity SARS-CoV-2 neutralizing antibody detection method uses ACE2 protein as a capture material, blocks non-neutralizing antibodies, and forms a coated RBD protein-neutralizing antibody-RBD protein- The marker complex enables the detection of SARS-CoV-2 neutralizing antibodies.
  • the detection method of the highly sensitive SARS-CoV-2 neutralizing antibody comprises:
  • the sample is contacted with an RBD protein marker, which captures non-neutralizing antibodies and/or neutralizing antibodies targeting RBD in the sample to form non-neutralizing antibodies and/or neutralizing antibodies-RBD protein- marker complex;
  • step (2) the non-neutralizing antibody and/or the neutralizing antibody-RBD protein-marker complex is brought into contact with a capture line comprising the ACE2 protein, and the ACE2 protein in the capture line captures the RBD protein, so that the The non-neutralizing antibody-RBD protein-label is immobilized on the capture line, while the neutralizing antibody-RBD protein-label complex cannot be captured by the capture line;
  • step (3) (4) the neutralizing antibody-RBD protein-marker complex not captured in step (3) is contacted with the detection line comprising the RBD protein to form a coated RBD protein-neutralizing antibody-RBD protein-marker complex, Make the corresponding signal appear on the detection line.
  • the method for determining the detection result of the highly sensitive SARS-CoV-2 neutralizing antibody detection method directly observes the reaction results of the capture line, the detection line and the quality control line to determine SARS-CoV-2 Neutralizing antibody content; or the instrument reads the reaction results of the capture line, detection line and quality control line, and judges the SARS-CoV-2 neutralizing antibody content by the ratio of the detection line and the quality control line.
  • the above-mentioned method steps (1) to (4) can realize the interpretation method of strips under certain conditions, and the interpretation method is more intuitive and easier than the conventional method of comparing the color depth of the strips;
  • the presence of neutralizing antibodies in the sample can be determined by the presence or absence of the detection line, and a second concentration determination point can be formed by comparing the color of the detection line and the capture line: when the neutralizing antibody exceeds a certain concentration, the color of the detection line is stronger than that of the capture line. Lines are shallow. At higher concentrations of neutralizing antibody, the capture line disappeared.
  • the detection principle of the method composed of steps (1) to (4) of the above method is as follows: the present invention detects the neutralizing antibody by using the blocking method and the sandwich method, and adds the capture line to detect the non-neutralizing antibody targeting RBD. Capture in advance to ensure the specificity of subsequent detection of neutralizing antibodies by sandwich method: Through the design of capture lines, detection lines and RBD protein markers, RBD protein markers are used to capture non-neutralizing antibodies targeting RBD in the sample to form antibody- The RBD protein-labeled carrier complexes and captures the RBD protein through the ACE2 protein in the capture line, thereby immobilizing the RBD-targeting non-neutralizing antibody on the capture line.
  • the neutralizing antibody-RBD protein-labeled carrier complex formed after the neutralizing antibody is captured by the RBD protein marker cannot be captured by the capture line. As the chromatography reaches the detection line, it is captured by the RBD at the detection line to form a packet.
  • the RBD protein-neutralizing antibody-RBD protein-labeled carrier complex causes a corresponding signal to appear on the detection line, and the neutralizing antibody concentration in the sample is judged by this signal.
  • the role of the ACE2 protein coated on the capture line is to capture the RBD protein
  • the role of the RBD protein coated on the detection line is to capture the neutralizing antibody in the sample
  • the amount of the test can be determined by pre-detecting the concentration range of the substance to be detected in the sample, or by conventional experiments such as concentration gradient experiments.
  • the ACE2 protein can be coated with a coating solution with a concentration of 0.1 mg/mL to 0.3 mg/mL (eg, 0.15 mg/mL, 0.2 mg/mL, 0.25 mg/mL) to obtain a capture line
  • the RBD protein can be 0.1 mg/mL.
  • the detection line is obtained by coating the coating solution with a concentration of ⁇ 0.3 mg/mL (eg 0.15 mg/mL, 0.2 mg/mL, 0.25 mg/mL); of course, the ACE2 protein coated on the capture line is relative to the RBD to be bound.
  • the protein content can be excessive; the RBD coated on the detection line can also be excessive relative to the neutralizing antibody in the sample; the specific method of coating the protein can be spraying or streaking to immobilize the corresponding protein on the capture line.
  • the specific coating amount can be specifically adjusted according to the cost and requirements for sensitivity and specificity, etc., which is easy for those skilled in the art.
  • the "ACE2 protein" coated on the capture line and the "RBD protein” coated on the detection line should be interpreted within the range known to those skilled in the art; for example, the ACE2 protein can be Selected from natural or recombinant ACE2 protein and ACE2 protein extracellular domain, or their conjugates with tags; its amino acid sequence can also be mutated, as long as it can bind to the RBD protein in the sample, and the binding region can be detected. Blocked by neutralizing antibodies.
  • RBD protein can be understood as, for example, the native or recombinant SARS-CoV-2 spike protein, S1 protein or RBD protein, or their conjugates with tags, the amino acid sequence of which can also be mutated, as long as the It can be combined with the SARS-CoV-2 neutralizing antibody and non-neutralizing antibody to be detected in the sample.
  • the term "tag” refers to a modified fragment or substance that does not affect the mutual binding of the ACE2 protein to the RBD, and preferably is detectable (eg, recognized using its antibody), which is familiar to those skilled in the art
  • it can be one or more of His tag, Strep tag, Flag tag, T7 tag, V5-peptide tag, GST tag, c-Myc tag, biotin and Fc tag, preferably Fc tag; Fc tag;
  • the species source of the tag can be rat, mouse, sheep, horse, donkey, rabbit, chicken, duck, goose, fish, pigeon, dog, human, etc.; further, for viviparous mammals, it is preferably Fc of IgA and IgG, For avians, it can be the Fc of IgY.
  • SARS-CoV-2 in the present invention is a new type of coronavirus, also known as 2019-nCoV; it shall include virus variants that mutate within a moderate range.
  • virus variant refers to a virus with a high degree of sequence homology of the currently discovered novel coronavirus, which can be isolated from any host of SARS-CoV-2.
  • a coronavirus isolated from Malayan pangolin by a research team has shown 100%, 98.2%, 96.7% and 90.4% amino acids with SARS-CoV-2 in E, M, N and S genes, respectively consistency.
  • 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 virus variant in the present invention has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the virus variant can be any known mutant strain, such as B.1.1.7 (GISAID accession ID: EPI_ISL_1234251), B.1.351 (GISAID accession ID: EPI_ISL_1191083), B.1.617 (EPI_ISL_1544002), B.1.617.1 ( EPI_ISL_2260468), B.1.617.2 (EPI_ISL_2272895), B.1.617.3 (EPI_ISL_1704535), or any other emerging mutants of SARS-CoV-2.
  • sample in the present invention generally refers to a material suspected of containing an analyte (eg a sample collected in an indoor environment, especially a hospital sample), more preferably a biological material.
  • samples of non-biological origin are, for example, water, food, etc., used to perform environmental (especially public places such as hospitals, schools, canteens, or factories such as food processing plants) assays.
  • Biologically derived samples can be from any biological source, such as physiological fluids, including blood, tissue fluid, saliva, ocular lens fluid, cerebrospinal fluid, sweat, urine, emulsion, ascites, mucus, nasal fluid, sputum, synovial fluid, peritoneal fluid, Vaginal fluid, menstruation, amniotic fluid, semen, etc; , bronchoalveolar lavage fluid, deep cough sputum, etc.), eye conjunctival swabs, fecal specimens, anticoagulation and serum specimens, etc.; clinical specimens should try to collect respiratory specimens (especially lower respiratory tract specimens) in the early stage of the case and the incidence of 7 The acute phase serum within the day and the convalescent phase serum from the 3rd to 4th week after the onset of the disease.
  • physiological fluids including blood, tissue fluid, saliva, ocular lens fluid, cerebrospinal fluid, sweat, urine, emulsion, ascites, mucus, nasal fluid,
  • Solid materials suspected of containing analytes can also be used as samples.
  • Samples can be used directly after being obtained from biological sources, or they can be used after pretreatment to improve the properties of the sample.
  • pretreatment may include preparing plasma from blood, diluting viscous fluids, and the like.
  • Pretreatment methods also include filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, and addition of reagents, lysis, and the like.
  • the sample can be fingertip blood, whole blood, serum, plasma, and the like.
  • a sample can be collected from a subject.
  • subject can be understood as a patient or an animal suspected of carrying the SARS-CoV-2 virus, or an animal that has been vaccinated with the new crown vaccine, especially a mammal, more preferably a natural host of SARS-CoV-2, For example, bats, civet cats; preferably primates, more preferably humans.
  • the RBD protein label refers to the connection between the RBD protein and the label, and the connection mode can be direct coupling, linker connection and the like.
  • the marker 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.
  • the RBD protein marker comprises a marker carrier and an RBD protein bound to the marker carrier.
  • the label carrier is used as a visual or instrument-detectable indicator substance, and its shape can be granular (sometimes called “beads” or “microbeads”), typically such as spheres, near spheres, plates Shape, rod, disc, strip, tube, cube, polyhedron or irregular shape; its surface can be modified with one or more active functional groups in one step, such as -OH, -COOH, -NH 2 , -CHO, -SO 3 H, aldehyde group, bromoacetyl group, iodoacetyl group, thiol group, epoxy group.
  • active functional groups such as -OH, -COOH, -NH 2 , -CHO, -SO 3 H, aldehyde group, bromoacetyl group, iodoacetyl group, thiol group, epoxy group.
  • the size of the label carrier can also vary, for example, the particles can range in average size (eg, diameter) from about 0.1 nanometers to about 100 micrometers, in some embodiments, from about 1 nanometer to about 10 micrometers, and in some implementations In the scheme, it is about 10 to about 100 nanometers.
  • average size eg, diameter
  • the labeling carrier comprises one or more of colloidal gold, latex microspheres, nanocarbon, magnetic microspheres or fluorescent microspheres.
  • the particle size of the colloidal gold is preferably between 1 nm and 200 nm, such as 10 nm, 20 nm, 50 nm, 70 nm, 100 nm, and 150 nm.
  • Colloidal gold can be prepared by any suitable method, such as white phosphorus reduction, ascorbic acid reduction, trisodium citrate reduction, and tannic acid-trisodium citrate reduction.
  • the particle size of the latex microspheres is preferably between 10nm and 500nm, such as 20nm, 50nm, 70nm, 100nm, 150nm, 200nm, 300nm, 400nm, although any latex microspheres can be used in the present invention, the latex microspheres are typically Consists of the following: polystyrene, butadiene styrene, styrene acrylic-vinyl terpolymer, polymethyl methacrylate, polyethyl methacrylate, styrene-maleic anhydride copolymer, polyacetic acid Vinyl ester, polyvinylpyrimidine, polydivinylbenzene, polybutylene terephthalate, acrylonitrile, vinyl chloride-acrylate, etc., or their aldehyde, carboxyl, amino, hydroxyl, or hydrazide derivatives.
  • the material thereof is preferably polystyrene. It can be any color that
  • the particle size of the nanocarbon is preferably between 10 nm and 200 nm, such as 20 nm, 50 nm, 70 nm, 100 nm, and 150 nm.
  • the magnetic microspheres may contain iron element, or a complex formed by iron element and other metals or non-metals. Other metals such as AlNiCo metal, etc.; composites formed by metals and non-metals such as NdFeB rubber magnetic composites.
  • the magnetic microspheres are preferably prepared from water-soluble salts of Fe 2+ and Fe 3+ , and the water-soluble salts can be one of chlorides, sulfates, nitrates and their hydrates that can hydrolyze Fe 2+ and Fe 3+ . one or more mixtures.
  • the fluorescent microspheres may contain rare earth elements such as europium (Eu) ions, samarium (Sm) ions, terbium (Tb) ions, samarium (Sm) ions, neodymium (Nd) ions, dysprosium (Dy) ions, technetium (Te) ions
  • rare earth elements such as europium (Eu) ions, samarium (Sm) ions, terbium (Tb) ions, samarium (Sm) ions, neodymium (Nd) ions, dysprosium (Dy) ions, technetium (Te) ions
  • Eu europium
  • Sm samarium
  • Tb terbium
  • Sm samarium
  • Nd neodymium
  • Dy dysprosium
  • Te technetium
  • Fluorescent microspheres can also be understood as particles labeled with fluorophores, which can be selected from fluorescein-based dyes, rhodamine-based dyes, and cyanine dyes.
  • fluorescein dyes include standard fluorescein and derivatives thereof, such as fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein ( TET) and the like;
  • the rhodamine dyes include R101, tetraethylrhodamine (RB200) and carboxytetramethylrhodamine (TAMRA) and the like.
  • the cyanine dyes are mainly selected from two categories, one is thiazole orange (TO), oxazole orange (YO) series and their dimer dyes, and the other is polymethine series cyanine dyes; fluorescence;
  • the group can also choose the following dyes: stilbene, naphthalimide, coumarins, acridines, pyrenes and the like.
  • suitable fluorescent microspheres include fluorescent carboxylated microspheres sold by Molecular Probes, Inc.
  • the present invention also relates to a high-sensitivity SARS-CoV-2 neutralizing antibody detection kit based on the above-mentioned detection method, which comprises a capture line coated with ACE2 protein.
  • the present invention also relates to a high-sensitivity SARS-CoV-2 neutralizing antibody detection kit, which includes a sample pad, a binding pad, an NC membrane and a water-absorbing pad, and a capture line is arranged on the NC membrane, and the capture line is coated with ACE2 protein.
  • a detection line, a quality control line, and a water-absorbing pad are arranged on one side (sequentially) of the capture line, and a binding pad and a sample pad are arranged on the other side of the capture line in sequence;
  • the capture line, detection line and quality control line are all set on the NC membrane;
  • the detection line is coated with RBD protein, and the binding pad is provided with an RBD protein marker, and the RBD protein marker includes a marker carrier and the RBD protein bound to the marker carrier.
  • NC membrane refers to a nitrocellulose membrane, which may be nitrocellulose alone, or a mixed ester of nitric acid with other acids, such as aliphatic carboxylic acids having 1-7 carbon atoms.
  • the size and shape of NC films can generally vary, as is readily understood by those skilled in the art.
  • the length of the NC film strips may be from about 10 to about 100 millimeters, in some embodiments about 20 to about 80 millimeters, and in some embodiments, about 40 to about 60 millimeters.
  • the width of the film strip may also range from about 0.5 to about 20 millimeters, in some embodiments, about 1 to about 15 millimeters, and in some embodiments, about 2 to about 10 millimeters.
  • the membrane strip thickness is usually small enough to allow transmission-based detection.
  • the thickness of the film strip may be less than about 500 microns, in some embodiments, less than about 250 microns, and in some embodiments, less than about 150 microns.
  • NC films can be cast on a carrier, where the resulting laminate can be die cut to the desired size and shape.
  • the NC film may be laminated to the carrier using only means such as an adhesive.
  • the carrier may be formed of a light transmissive material, such as a transparent or light scattering (eg translucent) material.
  • the carrier be liquid impermeable so that liquid does not leak through the carrier as it flows through the NC membrane.
  • carrier materials include, but are not limited to, glass; polymeric materials such as polystyrene, polypropylene, polyester, polybutadiene, polyvinyl chloride, polyamide, polycarbonate, epoxies, methacrylates And polymelamine (polymelamine) and so on.
  • 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 film.
  • the binding pad is a glass fiber membrane or non-woven fabric or filter paper impregnated with a treatment liquid, and the treatment liquid contains the following components: Tween 20, Triton X-405, Casein, BSA, PEG- 20000, PVP and NaCl.
  • the absorbent pad of the present invention can be made of any material capable of absorbing liquid, but the absorbent capacity should be large enough. Materials that can be used include, but are not limited to, absorbent cotton absorbent pads, silicone absorbent pads, or sponge absorbent pads.
  • the bottom plate of the present invention is used to carry the sample pad, the binding pad, the reaction pad (such as NC film) and the water-absorbing pad; the bottom plate can be various non-absorbent sheets with a supporting function.
  • the bottom plate It can be polyvinyl chloride (PVC) board, polypropylene (PP) board, polyethylene (PE) board or polyurethane (PU) board, preferably PVC board.
  • the "quality control line” is set on the reaction pad (such as NC membrane), and is a line formed by spraying or marking control compounds, such as protein, biotin, biotin, ligand, etc., usually set Downstream of the detection line in the direction of sample flow and parallel to the detection line. First, select the downstream position of the detection line along the flow direction of the sample on the reaction pad, and then immobilize molecules such as protein, biotin or avidin at the position of the quality control line by spraying or scribing to form a quality control line.
  • the reaction pad such as NC membrane
  • the quality control line includes a first molecule
  • the sample loading pad includes a second molecule
  • a second molecule is added to the sample loading pad during detection
  • the second molecule can bind to the first molecule
  • a detection signal is generated.
  • the first molecule/second molecule combination is exemplarily selected from the following molecule pairs: biotin or a derivative thereof/streptavidin, biotin or a derivative thereof/avidin, Biotin or its derivatives / NeutrAvidin, Biotin or its derivatives / Biotin or its derivatives antibody), peptide/antibody, receptor/ligand, digoxigenin/digoxigenin, carbohydrate/lectin or polynucleotide/complementary polynucleotide;
  • the derivative of biotin is any one of D-biotin, activated biotin, biocytin, ethylenediamine biotin, cadaverine biotin and desthiobiotin.
  • the biotin-binding protein family includes the aforementioned streptavidin, avidin, and NeutrAvidin proteins, each capable of binding four biotin molecules with high affinity and specificity.
  • streptavidin which is not glycosylated and has very low levels of nonspecific binding.
  • Avidin is a highly cationized glycoprotein with an isoelectric point around 10.5, and its positively charged residues and oligosaccharide components can mediate nonspecific binding, resulting in high background in some applications The problem.
  • NeutrAvidin proteins are deglycosylated and their equipotential point lowered, thereby reducing their background staining.
  • biotin can also be replaced by its equivalent derivatives, such as D-biotin, activated biotin, biocytin, ethylenediamine biotin, cadaverine biotin or desthiobiotin.
  • the first molecule is selected from anti-immunoglobulin antibodies, ligands or biotin, and correspondingly the second molecule is selected from signal-labeled immunoglobulins, signal-labeled receptors, or Avidin.
  • the first molecule is selected from immunoglobulins, receptors or avidins, and correspondingly the second molecule is selected from signal-labeled anti-immunoglobulin antibodies, signal-labeled ligands or biotin.
  • the first molecule is an anti-immunoglobulin antibody
  • the second molecule is a signal-labeled immunoglobulin.
  • the first molecule is an immunoglobulin
  • the second molecule is a signal-labeled anti-immunoglobulin antibody.
  • the first molecule is a ligand and the second molecule is a signal-labeled receptor.
  • the first molecule is a receptor and the second molecule is a signal-labeled ligand.
  • the first molecule is biotin and the second molecule is avidin.
  • the first molecule is avidin and the second molecule is biotin.
  • the sample pad is further provided with a blood filter pad or an anti-RBC antibody.
  • the detection result determination method determines the SARS-CoV-2 neutralizing antibody content by directly observing the reaction results of the capture line, the detection line and the quality control line.
  • the detection result determination method uses an instrument to read the reaction results of the capture line, the detection line and the quality control line, and determines the SARS-CoV-2 neutralizing antibody content by the ratio of the detection line and the quality control line.
  • a SARS-CoV-2 neutralizing antibody detection kit comprising a chromatography test paper, a cartridge and a sample diluent, the chromatography test paper comprising a bottom plate, a sample pad 1, a binding pad 2, an NC membrane 3 and a water-absorbing pad 4, The sample pad 1, the binding pad 2, the NC film 3 and the water absorbing pad 4 are sequentially attached to one side of the bottom plate; the NC film 3 is provided with a capture line 5, a detection line 6 and a quality control line 7 in sequence, and the capture line 5 is arranged on the NC film 3 in sequence.
  • Line 5 is close to the binding pad 2
  • the quality control line 7 is close to the water absorption pad 4
  • the capture line 5 is coated with ACE2 protein
  • the detection line 6 is coated with RBD protein
  • the binding pad is provided with RBD protein markers and anti-chicken IgY marker
  • the marker carrier is colloidal gold
  • the quality control line is coated with a quality control test substance
  • the quality control test substance is chicken IgY.
  • NC membrane coating using 1XPBS + 4% sucrose as solvent, respectively prepare the capture line coating solution with ACE2 protein concentration of 0.2 mg/mL, the detection line coating solution with RBD protein concentration of 0.2 mg/mL, and 0.5 mg/mL of RBD protein concentration.
  • mg/mL chicken IgY protein concentration quality control line coating solution the coating solution prepared above was scratched on the NC membrane using a gold-spraying membrane device, dried at 35°C for 12 hours, and coated with the NC membrane;
  • the bottom plate is made of PVC material
  • the sample pad 1 is glass fiber cut into a width of 18mm
  • the absorbent pad is absorbent paper cut into a width of 25mm.
  • the pads 4 are overlapped in sequence and pasted on one side of the bottom plate to obtain a large plate;
  • sample diluent of the present invention is prepared by ultrapure water, and the formula is 0.02moL/L PB+0.3% Triton 100.
  • a detection kit for SARS-CoV-2 neutralizing antibody comprising a chromatography test paper, a cartridge and a sample diluent, the chromatography test paper comprising a bottom plate, a sample pad, a binding pad, an NC membrane and a water-absorbing pad, the sample pad , the binding pad, the NC film and the water absorbing pad are sequentially attached to one side of the bottom plate;
  • the NC film is sequentially provided with a capture line, a detection line and a quality control line, the capture line is close to the binding pad, and the quality control line is close to the water absorption line pad, the capture line is coated with ACE2 protein, and the detection line is coated with RBD protein;
  • the binding pad is provided with an RBD protein marker, and the RBD protein marker includes a marker carrier and an RBD protein bound to the marker carrier , the labeling carrier is red latex microspheres.
  • the quality control line is coated with a quality control detection substance, and the quality control detection substance is an anti-RBD antibody.
  • the labeled carrier is red latex microspheres
  • the labeled protein is only RBD protein
  • the coating solution of the quality control line is 0.5 mg/mL anti-RBD monoclonal antibody
  • a sample addition hole and a diluent hole are set on the cartridge, which are used to add sample and diluent respectively.
  • a detection kit for SARS-CoV-2 neutralizing antibody comprising a chromatography test paper, a cartridge and a sample diluent, the chromatography test paper comprising a bottom plate, a sample pad, a binding pad, an NC membrane and a water-absorbing pad, the sample pad , the binding pad, the NC film and the water absorbing pad are sequentially attached to one side of the bottom plate;
  • the NC film is sequentially provided with a capture line, a detection line and a quality control line, the capture line is close to the binding pad, and the quality control line is close to the water absorption line pad, the capture line is coated with ACE2 protein, and the detection line is coated with RBD protein;
  • the binding pad is provided with an RBD protein marker, and the RBD protein marker includes a marker carrier and an RBD protein bound to the marker carrier , the labeled carrier time-resolved fluorescent microspheres.
  • the quality control line is coated with a quality control detection substance, and the quality control detection substance is an anti-RBD
  • the labeled carrier is a time-resolved fluorescent microsphere
  • the labeled protein is only RBD protein
  • the coating solution of the quality control line is 0.5 mg/mL anti-RBD monoclonal Antibody.
  • Example 2 The difference between this example and Example 1, Example 2, and Example 3 is that the sample pad includes a blood filter pad or an anti-RBC antibody is added to the sample pad, which can directly detect fingertip blood and whole blood samples.
  • the sample pad includes a blood filter pad or an anti-RBC antibody is added to the sample pad, which can directly detect fingertip blood and whole blood samples.
  • Example 3 Collect the serum samples of the subjects before the injection of the vaccine and the serum samples of the 1st, 2nd, 3rd, 4th, 5th and 6th weeks after the vaccine injection, and use the test kit of Example 3 for detection.
  • the collected serum is added dropwise to the sample addition hole of the detection card of the present invention, and then the diluent is added dropwise to the diluent hole, and the reaction is carried out for 15 minutes; then the matching fluorescence detector is used to read the reaction results of the capture line, the detection line and the quality control line. , and the SARS-CoV-2 neutralizing antibody content was judged by the ratio of the detection line and the capture line.
  • the detection kit of the present invention has high sensitivity, strong specificity, simple detection operation and fast detection speed; it is suitable for the detection of SARS-CoV-2 neutralizing antibodies.
  • T1/C refers to the ratio of the non-neutralizing antibody detection line to the quality control line
  • T2/C refers to the ratio of the neutralizing antibody detection line to the quality control line.

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Abstract

本发明公开了一种高灵敏SARS-CoV-2中和抗体的检测方法、检测试剂盒，属于生物医学检测技术领域，本发明试剂盒包括层析试纸、卡壳和样本稀释液，所述层析试纸包括底板、样品垫、结合垫、NC膜和吸水垫，所述NC膜上依次设置有捕获线、检测线和质控线，所述捕获线包被有ACE2蛋白，所述检测线包被有RBD蛋白，所述结合垫设置有RBD蛋白标记物；本发明采用阻断法加夹心法原理提高检测中和抗体的灵敏度，通过添加捕获线的方式，将靶向RBD的非中和抗体提前捕获，保证后续通过夹心法检测中和抗体的特异性。

Description

一种高灵敏SARS-CoV-2中和抗体的检测方法、检测试剂盒
相关申请的交叉引用
本申请要求于2021年03月31日提交中国专利局的申请号为202110344480.3、名称为“一种高灵敏SARS-CoV-2中和抗体的检测方法、检测试剂盒”的中国专利申请的优先权，并将其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物医学检测技术领域，尤其涉及一种高灵敏SARS-CoV-2中和抗体的检测方法、检测试剂盒。
背景技术
新型冠状病毒肺炎(新冠肺炎，COVID-19)的病原为新型冠状病毒，属于β属的冠状病毒，有包膜，颗粒呈圆形或椭圆形，直径60～140nm。具有5个必需基因，分别针对核蛋白(N)、病毒包膜(E)、基质蛋白(M)和刺突蛋白(S)4种结构蛋白及RNA依赖性的RNA聚合酶(RdRp)。核蛋白(N)包裹RNA基因组构成核衣壳，外面围绕着病毒包膜(E)，病毒包膜包埋有基质蛋白(M)和刺突蛋白(S)等蛋白。刺突蛋白(S)通过受体结合位点(RBD)结合宿主的血管紧张素转化酶2(ACE-2)进入细胞。S蛋白是病毒进入人体的关键，是疫苗和治疗性中和抗体的主要靶点。S蛋白的受体结合(RBD)结构域可与宿主ACE2受体结合，介导病毒入侵宿主细胞，也是大多数有效的中和抗体的结合部位。
中和抗体与RBD结构域结合后，可使病毒失去吸附、入侵宿主细胞能力，从而抑制病毒在宿主体内复制传播的抗体，病毒中和抗体在清除宿主体内的病毒中起着重要作用。
传统中和抗体检测的“金标准”是感染抑制试验，主要是先将病毒与待检测抗体进行混合，再将混合物接种到敏感动物、胚胎或细胞，通过敏感动物、胚胎或细胞的病变情况来测定残存的病毒感染力，从而间接确定待检测抗体对病毒的中和效果。其中在体外进行利用活病毒进行蚀斑减少中和试验(plaque reduction neutralizationtest，PRNT)和通过检测细胞病变(cytopathic effct，CPE)量来进行分析的微量细胞中和试验是常用方法。
对于新型冠状病毒烈性病毒性传染病来讲，即使是体外实验也面临活病毒株 来源和需要在高等级生物安全实验室操作的限制，杜克-新加坡国立大学医学院新发传染病重点研究项目主任王林发教授带领的研究团队，基于中和抗体可阻断RBD和ACE2结合的原理开发了阻断ELISA的cPass血清检测盒，由金斯瑞生物进行商业化推广，并获FDA授权用于恢复期血浆筛查。但仍存在检测操作繁琐、耗时长的缺点。之后，多家公司基于此原理开发了侧向层析试纸条，主要方法是将ACE2划到膜上，并用胶体金标记RBD蛋白。当样本中无中和抗体时候，胶体金标记的RBD和ACE2结合形成检测线。样本中有中和抗体时，中和抗体阻断RBD和ACE2的结合，从而降低检测线的显色强度。通过机器判读，或者检测线显色强度的深浅变化来判断中和抗体存在情况。肉眼判读时候需要检测线深浅有较大变化才能做判定，此形式检测卡的灵敏度有待提高。
发明内容
本发明的目的在于解决现有技术中SARS-CoV-2中和抗体检测操作繁琐、耗时长，灵敏度低的技术问题，提供一种简便、快速、灵敏度高的SARS-CoV-2中和抗体的检测方法、检测试剂盒。本发明的技术方案如下：
一种高灵敏SARS-CoV-2中和抗体的检测方法，基于夹心法结合阻断法实现SARS-CoV-2中和抗体的检测。优选的，高灵敏SARS-CoV-2中和抗体的检测方法通过提前捕获非中和抗体，增强后续通过夹心法检测中和抗体的特异性。
优选的，高灵敏SARS-CoV-2中和抗体的检测方法以ACE2蛋白为捕获物，阻断非中和抗体，通过形成包被RBD蛋白-中和抗体-RBD蛋白-标记载体复合物，实现SARS-CoV-2中和抗体的检测。
一种基于上述检测方法的高灵敏SARS-CoV-2中和抗体检测试剂盒，包括捕获线，所述捕获线包被有ACE2蛋白。优选的，在所述捕获线的一侧依次设有检测线、质控线、吸水垫，在所述捕获线的另一侧依次设有结合垫、样品垫；
所述捕获线、检测线、质控线均设置在NC膜上；
所述检测线上包被有RBD蛋白，所述结合垫设置有RBD蛋白标记物，所述RBD蛋白标记物包括标记载体和结合于所述标记载体上的RBD蛋白。
优选的，所述标记载体包括胶体金、乳胶微球、纳米碳、磁微球或荧光微球中的一种或多种。
优选的，所述样品垫上还设有滤血垫或抗RBC抗体。
优选的，所述检测试剂盒，其检测结果判定方法通过直接观测捕获线、检测线和质控线的反应结果，来判断SARS-CoV-2中和抗体含量。
优选的，所述检测试剂盒，其检测结果判定方法通过采用仪器读取捕获线、检测线和质控线的反应结果，并通过检测线和质控线的比值判断SARS-CoV-2中和抗体含量。
本发明的检测原理为：本发明通过采用阻断法加夹心法原理检测中和抗体，通过添加捕获线的方式，将靶向RBD的非中和抗体提前捕获，保证后续通过夹心法检测中和抗体的特异性：通过捕获线、检测线和RBD蛋白标记物的设计，利用RBD蛋白标记物捕获样品中靶向RBD的非中和抗体，形成抗体-RBD蛋白-标记载体复合物，并通过捕获线中的ACE2蛋白捕捉RBD蛋白，从而将靶向RBD的非中和抗体固定在捕获线上。而中和抗体被RBD蛋白标记物捕获后形成的中和抗体-RBD蛋白-标记载体复合物不能被捕获线被捕获，随着层析到达检测线处，被检测线处的RBD捕获，形成包被RBD蛋白-中和抗体-RBD蛋白-标记载体复合物，使检测线出现相应的信号，并通过此信号判断样本中的中和抗体浓度。
通过采用上述技术方案，达到的技术效果如下：
(1)本发明通过添加捕获线的方式，将靶向RBD的非中和抗体提前捕获，保证后续通过夹心法检测中和抗体的特异性，通过检测线的有无来判定样本中是否有中和抗体，灵敏度较通过阻断式竞争法的更好。
(2)条带有无的判读方式，较常规通过对比条带显色深浅的方式，判读方法更加直观、容易一些。
(3)除了通过检测线出现与否来判定样品中和抗体存在情况，还能通过检测线和捕获线颜色深浅对比形成第二个浓度判定点：在中和抗体超过特定浓度的时候，检测线的颜色较捕获线浅。当中和抗体浓度更高时，捕获线消失。
(4)本发明所提供的试剂盒采用免疫层析的方法，操作简单，检测速度快。
(5)在样本中的中和抗体浓度极低时，通过捕获线、检测线的系统设计，增加了ACE2的筛选，保证了检测的特异性；在样本中的中和抗体浓度较高时，还可以通过捕获线的抑制信号判断中和抗体的浓度，极大的提升了检测范围。
附图说明
图1是本发明试剂盒的结构示意图；
图中：1、样品垫；2、结合垫；3、NC膜；4、吸水垫；5、捕获线；6、检测线；7、质控线。
具体实施方式
本发明涉及一种高灵敏SARS-CoV-2中和抗体的检测方法，其基于夹心法结合阻断法实现SARS-CoV-2中和抗体的检测，通过提前捕获非中和抗体，增强后续通过夹心法检测中和抗体的特异性。
在一些实施方式中，所述的高灵敏SARS-CoV-2中和抗体的检测方法以ACE2蛋白为捕获物，阻断非中和抗体，通过形成包被RBD蛋白-中和抗体-RBD蛋白-标记物复合物，实现SARS-CoV-2中和抗体的检测。
在一些实施方式中，所述的高灵敏SARS-CoV-2中和抗体的检测方法包括：
(1)设计RBD蛋白标记物、捕获线和检测线，所述捕获线包含ACE2蛋白，所述检测线包含RBD蛋白；
(2)样本与RBD蛋白标记物接触，所述RBD蛋白标记物捕获样品中靶向RBD的非中和抗体和/或中和抗体，形成非中和抗体和/或中和抗体-RBD蛋白-标记物复合物；
(3)步骤(2)中非中和抗体和/或中和抗体-RBD蛋白-标记物复合物与包含ACE2蛋白的捕获线接触，所述捕获线中的ACE2蛋白捕捉RBD蛋白，从而将其中非中和抗体-RBD蛋白-标记物固定在捕获线上，而中和抗体-RBD蛋白-标记物复合物不能被捕获线捕获；
(4)步骤(3)中未被捕获的中和抗体-RBD蛋白-标记物复合物与包含RBD蛋白的检测线接触，形成包被RBD蛋白-中和抗体-RBD蛋白-标记物复合物，使检测线出现相应的信号。
在一些实施方式中，所述的高灵敏SARS-CoV-2中和抗体的检测方法的检测结果判定方法通过直接观测捕获线、检测线和质控线的反应结果，来判断SARS-CoV-2中和抗体含量；或仪器读取捕获线、检测线和质控线的反应结果，并通过检测线和质控线的比值判断SARS-CoV-2中和抗体含量。
上述方法步骤(1)～(4)所组成方法在某些条件下，能够实现条带有无的判读方式，较常规通过对比条带显色深浅的方式，判读方法更加直观、容易一些； 除了通过检测线出现与否来判定样品中和抗体存在情况，还能通过检测线和捕获线颜色深浅对比形成第二个浓度判定点：在中和抗体超过特定浓度的时候，检测线的颜色较捕获线浅。当中和抗体浓度更高时，捕获线消失。
上述方法步骤(1)～(4)所组成方法的检测原理为：本发明通过采用阻断法加夹心法原理检测中和抗体，通过添加捕获线的方式，将靶向RBD的非中和抗体提前捕获，保证后续通过夹心法检测中和抗体的特异性：通过捕获线、检测线和RBD蛋白标记物的设计，利用RBD蛋白标记物捕获样品中靶向RBD的非中和抗体，形成抗体-RBD蛋白-标记载体复合物，并通过捕获线中的ACE2蛋白捕捉RBD蛋白，从而将靶向RBD的非中和抗体固定在捕获线上。而中和抗体被RBD蛋白标记物捕获后形成的中和抗体-RBD蛋白-标记载体复合物不能被捕获线被捕获，随着层析到达检测线处，被检测线处的RBD捕获，形成包被RBD蛋白-中和抗体-RBD蛋白-标记载体复合物，使检测线出现相应的信号，并通过此信号判断样本中的中和抗体浓度。
根据上述原理，本领域技术人员可确定捕获线上所包被的ACE2蛋白的作用是捕获RBD蛋白，而检测线上所包被的RBD蛋白的作用是捕获样本中的中和抗体，因此其包被量可通过预先检测样本中待检测物的浓度范围，或者通过常规实验例如浓度梯度实验以进行确定。作为示例，ACE2蛋白可以0.1mg/mL～0.3mg/mL(例如0.15mg/mL、0.2mg/mL、0.25mg/mL)浓度的包被液包被获得捕获线，RBD蛋白可以0.1mg/mL～0.3mg/mL(例如0.15mg/mL、0.2mg/mL、0.25mg/mL)浓度的包被液包被获得检测线；当然，捕获线上所包被的ACE2蛋白相对于待结合的RBD蛋白含量可以为过量的；检测线上所包被的RBD相对于样本中的中和抗体也可以为过量的；具体的包被蛋白的方法可以是喷涂或划线方法将相应的蛋白固定在捕获线或检测线上，具体的包被量可以根据成本及对灵敏度和特异性的要求等进行具体调节，这对本领域技术人员是容易的。
本发明中捕获线上所包被的“ACE2蛋白”和检测线上所包被的“RBD蛋白”应当是在本领域技术人员所公知的范围内所做出的解释；例如所述ACE2蛋白可选自天然或重组的ACE2蛋白以及ACE2蛋白胞外域，或者它们与标签的偶联物；其氨基酸序列亦可以发生突变，只要其能够与样本中的RBD蛋白结合，并且结 合区域能够被需要检测的中和抗体所阻断即可。同理，“RBD蛋白”可以理解为例如天然或重组的SARS-CoV-2的刺突蛋白、S1蛋白或RBD蛋白，或者它们与标签的偶联物，其氨基酸序列亦可以发生突变，只要其能够与样本中需要检测的SARS-CoV-2中和抗体和非中和抗体所结合即可。
根据本发明，术语“标签”是指不影响ACE2蛋白与RBD的互相结合的修饰性片段或物质，并优选其是可被检测的(例如使用其抗体进行识别)，这对本领域技术人员是熟悉的；例如其可以为His标签、Strep标签、Flag标签、T7标签、V5-肽标签、GST标签、c-Myc标签、生物素和Fc标签中的一种或多种，优选为Fc标签；Fc标签的种属来源可以为大鼠、小鼠、羊、马、驴、兔、鸡、鸭、鹅、鱼、鸽子、犬、人等；进一步对于胎生哺乳类动物优选为IgA和IgG的Fc，对于禽类而言，可以为IgY的Fc。
本发明中术语“SARS-CoV-2”即新型冠状病毒，亦称为2019-nCoV；其应包含在适度范围内发生突变的病毒变体。具体的，本发明中“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，其可分离自SARS-CoV-2的任何宿主。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SARS-CoV-2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)。在一些实施方式中，本发明中所述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。病毒变体可以为任何已知的突变株，例如B.1.1.7(GISAID登录ID:EPI_ISL_1234251)、B.1.351(GISAID登录ID:EPI_ISL_1191083)、B.1.617(EPI_ISL_1544002)、B.1.617.1(EPI_ISL_2260468)、B.1.617.2(EPI_ISL_2272895)、B.1.617.3(EPI_ISL_1704535)，或任何新出现的其他SARS-CoV-2突变株。
本发明中术语“样本”通常是指被怀疑含有分析物的材料(例如室内环境所采集的样本，特别是医院内样本)，更优选为生物材料。非生物源的样本例如用于实施环境(特别是公共场所如医院、学校、食堂，或工厂例如食品加工厂)测定 的水、食品等。生物源样品可以来自任何生物源，例如生理流体，包括血液、组织液、唾液、眼晶状体液、脑脊髓液、汗液、尿液、乳液、腹水、粘液、鼻液、痰、滑液、腹膜液、阴道液、月经、羊膜液、精液等等；更为优选的，为受试者的上呼吸道标本(如咽拭子、鼻拭子等)、下呼吸道标本(如呼吸道吸取物、支气管灌洗液、肺泡灌洗液、深咳痰液等)、眼结膜拭子、粪便标本、抗凝血和血清标本等；临床标本应尽量采集病例发病早期的呼吸道标本(尤其是下呼吸道标本)和发病7天内急性期血清以及发病后第3～4周的恢复期血清。怀疑含有分析物的固体材料也可用作样本。样本可以从生物源获得后直接使用，或者在预处理后使用以改良样品的特性。例如预处理可包括用血液制备血浆、稀释粘稠液等等。预处理方法还包括过滤、沉淀、稀释、蒸馏、混合、浓缩、干扰成分的灭活、以及添加试剂、溶菌等等。而且，有益的是，将固体测试样品改变成液态介质或者释放分析物。在一些实施方案中，样本可以是指尖血、全血、血清、血浆等。
样本可采集自受试者。本发明中“受试者”可理解为患者或怀疑携带有SARS-CoV-2病毒的动物，或者接种过新冠疫苗的动物，特别是哺乳动物，更优选为SARS-CoV-2的天然宿主，例如蝙蝠、果子狸；优选为灵长类动物，更优选为人。
在本发明中，RBD蛋白标记物是指RBD蛋白与标记物连接，连接的方式可以是直接偶联、连接子连接等。其中标记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。RBD蛋白与标记物连接的方法也是本领域容易获得的。
在一些实施方式中，所述RBD蛋白标记物包括标记载体和结合于所述标记载体上的RBD蛋白。
在本发明中，标记载体为用作可视或可被仪器检测的指示物质，其形状可以为颗粒状(有时称为“珠”或“微珠”)，典型的例如球体、近球体、板状、棒状、盘状、条状、管状、立方体、多面体或不规则形状；其表面可一步修饰有一种或多种活性功能基团，所述活性功能基团例如-OH、-COOH、-NH 2、-CHO、-SO 3H、醛基、溴乙酰基、碘乙酰基、硫醇基、环氧基。标记载体的大小也可以有所不同，例如，颗粒的平均尺寸(例如直径)范围可以在大约0.1纳米到大约100微米，在一些实施方案中，为大约1纳米到大约10微米，而在一些实施方案中，为大约 10到大约100纳米。
在一些实施方式中，所述标记载体包括胶体金、乳胶微球、纳米碳、磁微球或荧光微球中的一种或多种。
胶体金的粒径优选1nm～200nm之间，例如10nm、20nm、50nm、70nm、100nm、150nm。胶体金可采用任何合适的方法制备得到，例如白磷还原法、抗坏血酸还原法、柠檬酸三钠还原法和鞣酸-柠檬酸三钠还原法。
乳胶微球的粒径优选10nm～500nm之间，例如20nm、50nm、70nm、100nm、150nm、200nm、300nm、400nm，尽管本发明中可以使用任何乳胶微球，但所述乳胶微球典型地是由以下物质构成：聚苯乙烯、丁二烯苯乙烯、苯乙烯丙烯酸-乙烯基三聚物、聚甲基丙烯酸甲酯、聚甲基丙烯酸乙酯、苯乙烯-马来酐共聚物、聚乙酸乙烯酯、聚乙烯基嘧啶、聚二乙烯苯、聚对苯二甲酸丁二醇酯、丙烯腈、氯乙烯-丙烯酸酯等等，或者其醛、羧基、氨基、羟基、或者酰肼衍生物。其材质优选为聚苯乙烯。其可以为任何能够实现指示作用而被检测到的颜色。市场上可买到的合适的有色乳胶微粒实例包括Bang’s Laboratory，Inc.出售的羧化乳胶微珠。
纳米碳的粒径优选10nm～200nm之间，例如20nm、50nm、70nm、100nm、150nm。
磁微球可以包含铁元素、或铁元素与其他金属或非金属等所形成的复合物。其他金属例如铝镍钴金属等；金属与非金属所形成的复合物例如钕铁硼橡胶磁复合材料。磁微球优选由Fe 2+和Fe 3+的水溶性盐制备得到，水溶性盐可以为能够水解出Fe 2+和Fe 3+的氯化物、硫酸物、硝酸物及其水合物中的一种或多种的混合物。
荧光微球可以包含稀土元素，例如含有铕(Eu)离子、钐(Sm)离子、铽(Tb)离子、钐(Sm)离子、钕(Nd)离子、镝(Dy)离子、锝(Te)离子等各种镧系元素的荧光材料中的一种或者几种。每个微球中可负载几万到几十万个稀土元素离子螯合物，检测过程中并不需要解离增强过程，便可发出很强的荧光。荧光微球也可以理解为标记有荧光团的颗粒，所述荧光团可选自荧光素类染料、罗丹明类染料以及菁染料。作为非限制的说明，在一些实施方式中，所述荧光素类染料包括标准荧光素及其衍生物，如异硫氰酸荧光素(FITC)、羟基荧光素(FAM)、四氯荧光素(TET)等；所述罗丹明类染料包括R101、四乙基罗丹明(RB200)和羧 基四甲基罗丹明(TAMRA)等。所述菁染料主要选自两类，一类是噻唑橙(thiazole orange,TO)、噁唑橙(oxazole orange,YO)系列及其二聚体染料，另一类是多甲川系列菁染料；荧光团还可以选择下述染料：二苯乙烯、萘酰亚胺、香豆素类、吖啶类、芘类等。市场上可买到的合适荧光微球的实例包括Molecular Probes，Inc.出售的商品名为“FluoSphere”(Red 580/605)和“TransfluoSphere”(543/620)的荧光羧化微球，以及同样由Molecular Probes，Inc.出售的“Texas Red”，和5-、6-羧基四甲基罗丹明。
本发明还涉及一种基于如上所述的检测方法的高灵敏SARS-CoV-2中和抗体检测试剂盒，其包括捕获线，所述捕获线包被有ACE2蛋白。
本发明还涉及一种高灵敏SARS-CoV-2中和抗体检测试剂盒，其包括样品垫、结合垫、NC膜和吸水垫，在NC膜上设置有捕获线，所述捕获线包被有ACE2蛋白。
在一些实施方式中，在所述捕获线的一侧(依次)设有检测线、质控线、吸水垫，在所述捕获线的另一侧依次设有结合垫、样品垫；
所述捕获线、检测线、质控线均设置在NC膜上；
所述检测线上包被有RBD蛋白，所述结合垫设置有RBD蛋白标记物，所述RBD蛋白标记物包括标记载体和结合于所述标记载体上的RBD蛋白。
应当理解，术语“NC膜”即为硝酸纤维素膜，其可以是单独的硝化纤维素，或者是硝酸与其它酸，例如具有1-7个碳原子的脂肪族羧酸的混合酯。NC膜的尺寸和形状通常可以有所不同，如本领域技术人员很容易理解的那样。例如，NC膜条带的长度可以是从大约10到大约100毫米，在一些实施方式中为大约20到大约80毫米，而在一些实施方式中，为大约40到大约60毫米。膜条带的宽度范围也可以在大约0.5到大约20毫米，在一些实施方式中，为大约1到大约15毫米，而在一些实施方式中，为大约2到大约10毫米。同样，膜条带厚度通常足够小以允许基于透过的检测。例如，所述膜条带的厚度可以小于大约500微米，在一些实施方式中，小于大约250微米，而在一些实施方式中，小于大约150微米。
此外，正如本领域所公知，NC膜可以浇注在载体上，其中可以将得到的叠层冲切成预期的大小和形状。或者，所述NC膜可以仅利用诸如粘合剂之类的方 式层压到载体上。载体可以由可透光的材料形成，例如透明的或光散射(例如半透明)材料。而且，通常希望载体是不透液体的，这样液体流过NC膜时不会泄漏通过载体。载体材料的实例包括但不限于，玻璃；聚合物材料，例如聚苯乙烯、聚丙烯、聚酯、聚丁二烯、聚氯乙烯、聚酰胺、聚碳酸酯、环氧化物、甲基丙烯酸脂和聚密胺(polymelamine)等等。
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(如NC膜)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X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“质控线”是设在反应垫(如NC膜)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。
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。所述第一分子/第二分子的组合示例性地选自如下分子对：生物素或其衍生物/链霉亲和素(streptavidin)，生物素或其衍生物/亲和素(avidin)，生物素或其衍生物/中性抗生物素蛋白(NeutrAvidin)，生物素或其衍生物/生物素或其衍生物抗体，半抗原/抗体，抗原/抗体(例如免疫球蛋白/抗免疫球蛋白抗体)，肽/抗体，受体/配体，地高辛/地高辛配基，碳水化合物/凝集素或多核苷酸/互补的多核苷酸；
其中生物素的衍生物为D-生物素、活化生物素、生物胞素、乙二胺生物素、尸胺生物素及脱硫生物素中的任一种。
生物素结合的蛋白家族包括上述的链霉亲和素(streptavidin)、亲和素(avidin)和NeutrAvidin蛋白，每个蛋白都能够以高度的亲和力和特异性结合四个生物素分子。其中最常使用的是链霉亲和素，它未经糖基化且具有很低的非特异性结合水平。亲和素则是一种高度阳离子化的糖蛋白，等电点在10.5左右，它的正电荷残基和低聚糖成份能够介导非特异性结合，从而在某些应用中导致本底过高的问题。NeutrAvidin蛋白经过去糖基化处理和降低等电势点，从而减少了其背景着色。
此外，本领域技术人员公知生物素也可用其同功能衍生物所替代，例如D-生物素、活化生物素、生物胞素、乙二胺生物素、尸胺生物素或脱硫生物素。
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。
在一些实施方式中，所述样品垫上还设有滤血垫或抗RBC抗体。
在一些实施方式中，其检测结果判定方法通过直接观测捕获线、检测线和质控线的反应结果，来判断SARS-CoV-2中和抗体含量。
在一些实施方式中，其检测结果判定方法通过采用仪器读取捕获线、检测线和质控线的反应结果，并通过检测线和质控线的比值判断SARS-CoV-2中和抗体含量。
下面结合附图对本发明作进一步的说明。
下述的实施例是为了进一步说明本发明的一些优选实施例，并非全部实施例。本领域专业人员在没有进行创造性劳动的前提下做出的基于本发明的其他实施例，都属于本发明的权利保护范围。
实施例1胶体金检测试剂盒
一种SARS-CoV-2中和抗体的检测试剂盒，包括层析试纸、卡壳和样本稀释液，所述层析试纸包括底板、样品垫1、结合垫2、NC膜3和吸水垫4，所述样品垫1、结合垫2、NC膜3和吸水垫4依次黏贴与底板一侧；所述NC膜3上依次设置有捕获线5、检测线6和质控线7，所述捕获线5靠近结合垫2，所述质控线7靠近吸水垫4，所述捕获线5包被有ACE2蛋白，所述检测线6包被有RBD蛋白；所述结合垫设置有RBD蛋白标记物和抗鸡IgY标记物，所述标记载体为胶体金，所述质控线包被有质控检测物，所述质控检测物为鸡IgY。
本实施例胶体金测试试剂盒的制备方法：
(1)胶体金标记，采用万分之一浓度的胶体金分别标记RBD蛋白和抗鸡IgY，离心去上清，浓缩十倍得到RBD蛋白标记物和抗鸡IgY标记物；
(2)结合垫制备，将(1)得到的RBD蛋白标记物和抗鸡IgY标记物按照1：1比例混合后，再稀释3倍后均匀涂抹于玻璃纤维垫片上，35℃烘干30分钟，然后采用斩切机，切割成5mm宽，得到结合垫；
(3)NC膜包被，采用1XPBS+4％蔗糖作为溶剂，分别配制0.2mg/mL的ACE2蛋白浓度的捕获线包被液、0.2mg/mL的RBD蛋白浓度的检测线包被液、0.5mg/mL的鸡IgY蛋白浓度质控线包被液，采用喷金划膜仪器将上述配制的包被液划在NC膜上，35℃烘干12小时，包被NC膜；
(4)大板组装，底板采用PVC材质底板，样品垫1为切割成宽度18mm的玻璃纤维，吸水垫为切割成宽度25mm的吸水纸，将样品垫1、结合垫2、NC膜3和吸水垫4依次重叠，黏贴于底板一侧，得到大板；
(5)样本稀释液配制，本发明样本稀释液采用超纯水配制，配方为0.02moL/L PB+0.3％曲拉通100。
(6)切条、组装，封装，采用切条机将步骤(4)得到的大板切成3mm宽度的试纸条，放置于所述卡壳的内部完成组装，然后同干燥剂一起封装于铝箔袋 内，再同配套的样本稀释液一起放置于包装盒内，完成本发明试剂盒的制备。
使用时，收集待检测的血清并滴加在所述卡壳的加样孔，然后向加样孔滴加稀释液，反应15分钟；通过直接观测捕获线、检测线和质控线的反应结果，并与配套使用的浓度显色卡进行比对，来判断SARS-CoV-2中和抗体含量。
实施例2乳胶微球检测试剂盒
一种SARS-CoV-2中和抗体的检测试剂盒，包括层析试纸、卡壳和样本稀释液，所述层析试纸包括底板、样品垫、结合垫、NC膜和吸水垫，所述样品垫、结合垫、NC膜和吸水垫依次黏贴与底板一侧；所述NC膜上依次设置有捕获线、检测线和质控线，所述捕获线靠近结合垫，所述质控线靠近吸水垫，所述捕获线包被有ACE2蛋白，所述检测线包被有RBD蛋白；所述结合垫设置有RBD蛋白标记物，所述RBD蛋白标记物包括标记载体和结合于标记载体的RBD蛋白，所述标记载体为红色乳胶微球。所述质控线包被有质控检测物，所述质控检测物为抗RBD抗体。
本发明试剂盒的制备方法与实施例1的不同之处在于，所述标记载体为红色乳胶微球，标记蛋白只有RBD蛋白，质控线包被液为0.5mg/mL的抗RBD单克隆抗体，另外，卡壳上设置加样孔和稀释液孔，分别用于添加样本和稀释液。
使用时，收集待检测的血清并滴加在所述卡壳的加样孔，然后向稀释液孔滴加稀释液，反应15分钟；通过直接观测捕获线、检测线和质控线的反应结果，并与配套使用的浓度显色卡进行比对，来判断SARS-CoV-2中和抗体含量。
实施例3荧光检测试剂盒
一种SARS-CoV-2中和抗体的检测试剂盒，包括层析试纸、卡壳和样本稀释液，所述层析试纸包括底板、样品垫、结合垫、NC膜和吸水垫，所述样品垫、结合垫、NC膜和吸水垫依次黏贴与底板一侧；所述NC膜上依次设置有捕获线、检测线和质控线，所述捕获线靠近结合垫，所述质控线靠近吸水垫，所述捕获线包被有ACE2蛋白，所述检测线包被有RBD蛋白；所述结合垫设置有RBD蛋白标记物，所述RBD蛋白标记物包括标记载体和结合于标记载体的RBD蛋白，所述标记载体时间分辨荧光微球。所述质控线包被有质控检测物，所述质控检测物为抗RBD抗体。
本发明试剂盒的制备方法与实施例1的不同之处在于，所述标记载体为时间 分辨荧光微球，标记蛋白只有RBD蛋白，质控线包被液为0.5mg/mL的抗RBD单克隆抗体。
使用时，收集待检测的血清并滴加在所述卡壳的加样孔，然后向加样孔滴加稀释液，反应15分钟；然后采用仪器读取捕获线、检测线和质控线的反应结果，并通过检测线和质控线的比值判断SARS-CoV-2中和抗体含量。
实施例4
本实施例与实施例1、实施例2、实施例3不同之处在于所述样品垫包括滤血垫或所述样品垫上添加有抗RBC抗体，能够直接检测指尖血和全血样本。
实施例5中和抗体检测
收集受测试者注射疫苗前血清及注射疫苗后第1周、第2周、第3周、第4周、第5周和第6周的血清样本，采用实施例3检测试剂盒进行检测，将收集的血清并滴加在本发明检测卡的加样孔，然后向稀释液孔滴加稀释液，反应15分钟；然后采用配套荧光检测仪读取捕获线、检测线和质控线的反应结果，并通过检测线和捕获线的比值判断SARS-CoV-2中和抗体含量。
检测结果如下表：
Figure PCTCN2022081424-appb-000001
从以实施例可以看出，本发明检测试剂盒灵敏度高，特异性强，检测操作简单，检测速度快；适合于SARS-CoV-2中和抗体的检测。
实施例6实施例3的试剂盒中和抗体检测
收集待检测的血清15ul并滴加在所述壳体的加样孔，然后向加样孔滴加稀释液70ul，反应15分钟；然后采用相应的仪器读取非中和抗体检测线、中和抗体检测线和质控线的反应结果，并通过检测线和质控线的比值判断SARS-CoV-2 中和抗体含量。
检测结果如下表所示：
Figure PCTCN2022081424-appb-000002
T1/C指非中和抗体检测线与质控线的比值，T2/C指中和抗体检测线与质控线的比值。判断结果：阴性(-)、弱阳性(+)、阳性(++)。在接种过疫苗的样本中，“金标准”病毒中和试验方法判定的阴性的样本，实施例3中和抗体检测线的结果均为阴性；病毒中和试验方法判定为阳性的样本，实施例3的试剂盒检测结果均为阳性，说明本发明的试剂盒有较高的准确性和灵敏度。
以上所述仅为本发明的优选实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的简单修改或变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。以上实施例仅用以说明本发明而并非限制本发明所描述的技术方案；因此，尽管本说明书参照上述的实施例 对本发明已进行了详细的说明，但是，本领域的普通技术人员应当理解，仍然可以对本发明进行修改或等同替换；而一切不脱离本发明的精神和范围的技术方案及其改进，其均应涵盖在本发明的权利要求范围中。

Claims (12)

  1. 一种高灵敏SARS-CoV-2中和抗体的检测方法，其特征在于，基于夹心法结合阻断法实现SARS-CoV-2中和抗体的检测，通过提前捕获非中和抗体，增强后续通过夹心法检测中和抗体的特异性。
  2. 根据权利要求1所述的高灵敏SARS-CoV-2中和抗体的检测方法，其特征在于，以ACE2蛋白为捕获物，阻断非中和抗体，通过形成包被RBD蛋白-中和抗体-RBD蛋白-标记物复合物，实现SARS-CoV-2中和抗体的检测。
  3. 根据权利要求1所述的高灵敏SARS-CoV-2中和抗体的检测方法，其特征在于，包括：
    (1)设计RBD蛋白标记物、捕获线和检测线，所述捕获线包含ACE2蛋白，所述检测线包含RBD蛋白；
    (2)样本与RBD蛋白标记物接触，所述RBD蛋白标记物捕获样品中靶向RBD的非中和抗体和/或中和抗体，形成非中和抗体和/或中和抗体-RBD蛋白-标记物复合物；
    (3)步骤(2)中非中和抗体和/或中和抗体-RBD蛋白-标记物复合物与包含ACE2蛋白的捕获线接触，所述捕获线中的ACE2蛋白捕捉RBD蛋白，从而将其中非中和抗体-RBD蛋白-标记物固定在捕获线上，而中和抗体-RBD蛋白-标记物复合物不能被捕获线捕获；
    (4)步骤(3)中未被捕获的中和抗体-RBD蛋白-标记物复合物与包含RBD蛋白的检测线接触，形成包被RBD蛋白-中和抗体-RBD蛋白-标记物复合物，使检测线出现相应的信号。
  4. 根据权利要求3所述的高灵敏SARS-CoV-2中和抗体的检测方法，其检测结果判定方法通过直接观测捕获线、检测线和质控线的反应结果，来判断SARS-CoV-2中和抗体含量；或仪器读取捕获线、检测线和质控线的反应结果，并通过检测线和质控线的比值判断SARS-CoV-2中和抗体含量。
  5. 根据权利要求3或4中任一项所述的高灵敏SARS-CoV-2中和抗体的检测方法，所述RBD蛋白标记物包括标记载体和结合于所述标记载体上的RBD蛋白。
  6. 根据权利要求5所述的高灵敏SARS-CoV-2中和抗体的检测方法，所述标记载体包括胶体金、乳胶微球、纳米碳、磁微球或荧光微球中的一种或多种。
  7. 一种高灵敏SARS-CoV-2中和抗体检测试剂盒，其包括样品垫、结合垫、NC膜和吸水垫，其特征在于，在NC膜上设置有捕获线，所述捕获线包被有ACE2蛋白。
  8. 根据权利要求7所述的检测试剂盒，其特征在于，在所述捕获线的一侧设有检测线、质控线、吸水垫，在所述捕获线的另一侧依次设有结合垫、样品垫；所述捕获线、检测线、质控线均设置在NC膜上；
    所述检测线上包被有RBD蛋白，所述结合垫设置有RBD蛋白标记物，所述RBD蛋白标记物包括标记载体和结合于所述标记载体上的RBD蛋白。
  9. 根据权利要求7或8所述的检测试剂盒，其特征在于，所述标记载体包括胶体金、乳胶微球、纳米碳、磁微球或荧光微球中的一种或多种。
  10. 根据权利要求7或8所述的检测试剂盒，其特征在于，所述样品垫上还设有滤血垫或抗RBC抗体。
  11. 根据权利要求7或8所述的检测试剂盒，其特征在于，其检测结果判定方法通过直接观测捕获线、检测线和质控线的反应结果，来判断SARS-CoV-2中和抗体含量。
  12. 根据权利要求7或8所述的检测试剂盒，其特征在于，其检测结果判定方法通过采用仪器读取捕获线、检测线和质控线的反应结果，并通过检测线和质控线的比值判断SARS-CoV-2中和抗体含量。
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