WO2021254476A1 - 检测SARS-CoV-2病毒中和抗体的磁微粒化学发光试剂盒及其应用 - Google Patents

检测SARS-CoV-2病毒中和抗体的磁微粒化学发光试剂盒及其应用 Download PDF

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WO2021254476A1
WO2021254476A1 PCT/CN2021/100834 CN2021100834W WO2021254476A1 WO 2021254476 A1 WO2021254476 A1 WO 2021254476A1 CN 2021100834 W CN2021100834 W CN 2021100834W WO 2021254476 A1 WO2021254476 A1 WO 2021254476A1
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protein
ace2
variant
cov
sars
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PCT/CN2021/100834
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French (fr)
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覃喜建
汤双双
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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
    • G01N33/68—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
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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 present invention relates to the field of antibody detection, in particular to a method for detecting SARS-CoV-2 virus neutralizing antibody by means of SARS-CoV-2 virus neutralizing antibody inhibiting or blocking magnetic particle chemiluminescence technology and detecting SARS-CoV-2 virus And antibody magnetic particle chemiluminescence kit.
  • 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 epidemic has gradually turned into a global pandemic, which has caused the deaths of more than 6 million infected people and more than 300,000 patients.
  • the SARS-CoV-2 virus enters the host cell through the transmembrane spike glycoprotein (S protein) RBD domain and binds to the ACE2 receptor on the host cell surface.
  • S protein transmembrane spike glycoprotein
  • the SARS-CoV-2 virus neutralizing antibody inhibits SARS-CoV-2 virus from entering the host cell by blocking the SARS-CoV-2 RBD protein from binding to the ACE2 receptor. 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.
  • the current detection methods for neutralizing antibodies include traditional neutralization test (NT)-based detection methods and blocking ELISA detection methods.
  • the NT method is to infect the cells after the antibody sample interacts with the virus.
  • the virus titer is calculated by observing the cytopathic condition and using the cell culture half infection dose (CCID50) or half tissue cell infection dose (TCID50) method to evaluate the neutralizing antibody against the virus.
  • the ability to block infection The neutralizing antibody blocking ELISA method uses a labeled neutralizing antibody to block the binding of the neutralizing antibody in the sample with the virus or recombinant antigen coated on the ELISA plate, perform chemical enzyme-linked color development, and then use the ELISA analyzer Obtain the results, and then evaluate the neutralizing effect of the neutralizing antibody.
  • the NT method has a long experimental period and a large workload, and it is difficult to meet the rapid and high-throughput screening requirements.
  • the neutralizing antibody blocking ELISA method requires the preparation of neutralizing antibodies, which is more difficult.
  • Magnetic particle chemiluminescence analysis is a new type of analysis and detection technology that effectively combines magnetic separation technology, immunoassay technology and chemiluminescence detection technology.
  • This technology uses micron-sized magnetic particles to label antibody proteins and other biomolecules to capture the target protein in the sample.
  • This technology uses chemiluminescent substances as labels to directly label biomolecules such as antibody proteins, or uses enzymes that can catalyze chemiluminescent substances as labels to generate detection signals. After the reaction, the chemiluminescent substance generates a photon signal that can be detected by the chemiluminescence instrument in the presence of the corresponding oxidant or catalytic enzyme, so as to realize the accurate qualitative or quantitative determination of the antibody-protein complex to be tested.
  • the most commonly used markers in magnetic particle chemiluminescence immunoassay are alkaline phosphatase (AP), acridinium ester, horseradish peroxidase (HRP) and so on. Because the magnetic particle chemiluminescence technology has the advantages of high sensitivity, strong specificity, rapid separation of the magnetic separation system, short detection time, wide linear range, and easy automation. In recent years, it has been widely used in clinical diagnosis, biomedicine, food safety testing and other aspects.
  • the present invention uses the SARS-CoV-2 virus neutralizing antibody to block the SARS-CoV-2 RBD protein binding to the ACE2 receptor to develop a chemiluminescence kit for detecting the SARS-CoV-2 virus neutralizing antibody.
  • the kit is universal, easy to operate, low in use cost, and easier to popularize and apply.
  • kits for detecting neutralizing antibodies of a novel coronavirus SARS-CoV-2 or a variant thereof in a sample
  • the kit includes a novel coronavirus or a variant thereof
  • the ACE2 protein or functional fragments thereof are connected to magnetic particles; or the spike protein portion of the novel coronavirus or its variants is connected to magnetic particles, and the ACE2 protein or functional fragments thereof are connected to a label; wherein, the novel coronavirus
  • the specific binding between the spike protein part of its variant and the ACE2 protein or its functional fragment generates a detection signal.
  • the label or magnetic particles are connected by a linker or coupling, preferably by coupling. In some specific embodiments, the labels or magnetic particles are connected by coupling. In other specific embodiments, the label or magnetic particles are connected via a linker.
  • the linker can be selected from small molecule chemical linkers or peptide linkers, including but not limited to carboxyl, amino and sulfhydryl groups, etc., and the label or magnetic particles can be connected to the spike protein portion of the new coronavirus or its variants through the linker Or ACE2 protein or functional fragments thereof.
  • the coupling method is connected by forming a covalent bond, which can be connected by covalently reacting with the spike protein part of the new coronavirus or its variants or the ACE2 protein or its functional fragments by a label or magnetic particle.
  • the labels or magnetic particles are connected by covalent coupling.
  • the spike protein portion of the novel coronavirus or its variant is coupled to a label, and the ACE2 protein or functional fragment thereof is coupled to a magnetic particle. In other embodiments, the spike protein portion of the novel coronavirus or its variant is coupled to magnetic particles, and the ACE2 protein or functional fragment thereof is coupled to a label.
  • the label is selected from one or more of alkaline phosphatase, acridine ester, horseradish peroxidase, ruthenium terpyridine, isoluminol, or rare earth elements or biotin, Preferably, it is alkaline phosphatase, acridinium ester or horseradish peroxidase.
  • the label is selected from alkaline phosphatase, acridinium ester, horseradish peroxidase, or ruthenium terpyridine.
  • the label is selected from alkaline phosphatase, acridinium ester, or horseradish peroxidase.
  • the label is alkaline phosphatase.
  • the label is an acridine ester.
  • the marker is horseradish peroxidase.
  • the chemiluminescence kit is selected from the group consisting of horseradish peroxidase magnetic particle chemiluminescence kit, alkaline phosphatase magnetic particle chemiluminescence kit, acridine ester magnetic particle chemiluminescence kit or terpyridine
  • the ruthenium magnetic particle electrochemiluminescence kit is preferably an acridinium ester magnetic particle chemiluminescence kit or an alkaline phosphatase magnetic particle chemiluminescence kit.
  • the chemiluminescence kit is an acridinium ester magnetic particle chemiluminescence kit or an alkaline phosphatase magnetic particle chemiluminescence kit.
  • the acridinium ester magnetic particle chemiluminescence kit includes acridine ester-coupled new coronavirus or its variant spike protein portion and magnetic particle-coupled ACE2 protein or functional fragments thereof. In other embodiments, the acridinium ester magnetic particle chemiluminescence kit includes acridinium ester-coupled ACE2 protein or functional fragment thereof and magnetic particle-coupled novel coronavirus or its variant spike protein portion.
  • the alkaline phosphatase magnetic particle chemiluminescence kit includes alkaline phosphatase-coupled new coronavirus or its variant spike protein portion and magnetic particle-coupled ACE2 protein or its functional sheet. In other embodiments, the alkaline phosphatase magnetic particle chemiluminescence kit includes alkaline phosphatase-coupled ACE2 protein or functional fragments thereof, and magnetic particle-coupled new coronavirus or its variant spike protein.
  • the horseradish peroxidase magnetic particle chemiluminescence kit includes horseradish peroxidase-coupled new coronavirus or its variant spike protein portion and magnetic particle-coupled ACE2 protein or Its functional fragments. In other embodiments, the horseradish peroxidase magnetic particle chemiluminescence kit includes horseradish peroxidase-coupled ACE2 protein or functional fragments thereof and magnetic particle-coupled novel coronavirus or variants thereof Spike protein part.
  • the electrochemiluminescence kit of terpyridine ruthenium magnetic particles includes a novel coronavirus or its variant spike protein portion coupled with ruthenium terpyridine and ACE2 protein or functional fragments thereof coupled with magnetic particles.
  • the electrochemiluminescence kit of terpyridine ruthenium magnetic particles includes the ACE2 protein or functional fragments thereof coupled with ruthenium terpyridine and the spike protein portion of novel coronavirus or its variants coupled with magnetic particles.
  • the electrochemiluminescence kit of terpyridine ruthenium magnetic particles includes a novel coronavirus or its variant spike protein portion coupled with ruthenium terpyridine, streptavidin and biotin coupled with magnetic particles Labeled ACE2 protein or functional fragments thereof.
  • the terpyridine ruthenium magnetic particle electrochemiluminescence kit includes terpyridine ruthenium coupled ACE2 protein or functional fragments thereof, magnetic particle coupled streptavidin and biotin-labeled novel corona The spike protein portion of the virus or its variants.
  • the kit detects changes in the detection signal generated by the detection of the sample to inhibit or block the binding of the new coronavirus or its variant spike protein portion and the ACE2 protein or its functional fragments to determine the new type of coronavirus in the sample.
  • the new coronavirus or its variant spike protein portion is selected from the group consisting of a new coronavirus or its variant S protein, a new coronavirus or its variant S1 protein, a new coronavirus or its variant RBD protein Or a novel coronavirus or its variant RBD protein with a His or Fc tag, preferably a novel coronavirus or its variant RBD protein.
  • the spike protein portion of the novel coronavirus or its variant is a novel coronavirus or its variant S1 protein or a novel coronavirus or its variant virus RBD protein.
  • the spike protein portion of the novel coronavirus or its variant is a novel coronavirus or its variant RBD protein.
  • the spike protein portion of the novel coronavirus or its variants comprises the amino acid sequence shown in any one of SEQ ID NOs: 1, 2 and 4-7, or is in combination with SEQ ID NOs: 1, 2 and 4 -7 A sequence with at least 70% identity in the amino acid sequence shown in any one of them.
  • the spike protein portion of the novel coronavirus or its variants comprises at least 70%, at least 75%, or at least 80% of the amino acid sequence shown in any one of SEQ ID NOs: 1, 2 and 4-7. %, at least 83%, at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical sequences.
  • the new coronavirus spike protein portion comprises at least 70%, at least 75%, at least 80%, at least 83%, at least 85%, and amino acid sequence shown in SEQ ID NO: 1 or 2.
  • the spike protein portion of the novel coronavirus variant comprises at least 70%, at least 75%, at least 80%, at least 83%, and the amino acid sequence shown in any one of SEQ ID NO: 4-7.
  • the spike protein portion of the novel coronavirus or variants thereof includes the amino acid sequence shown in any one of SEQ ID NOs: 1, 2, and 4-7.
  • the new coronavirus spike protein portion includes the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • the spike protein portion of the novel coronavirus variant includes an amino acid sequence shown in any one of SEQ ID NOs: 4-7.
  • the amino acid sequence of the spike protein portion of the novel coronavirus or its variants is any one of the sequences shown in SEQ ID NOs: 1, 2, and 4-7.
  • the amino acid sequence of the spike protein portion of the novel coronavirus is shown in SEQ ID NO: 1 or 2.
  • the amino acid sequence of the spike protein portion of the novel coronavirus variant is shown in any one of SEQ ID NO: 4-7.
  • the concentration of the spike protein portion of the novel coronavirus or variants thereof to which the marker is coupled is 2-200 ng/mL, preferably 80 ng/mL. In some specific embodiments, the concentration of the spike protein portion of the novel coronavirus or variants thereof to which the marker is coupled is 2ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL , 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng /mL, 190ng/mL or 200ng/mL.
  • the concentration of the spike protein portion of the novel coronavirus or variants thereof to which the marker is coupled is 80 ng/mL. In some specific embodiments, the concentration of the spike protein portion of the novel coronavirus or variants thereof to which the marker is coupled is 100 ng/mL.
  • the new coronavirus or its variant spike protein portion to which the label is coupled is acridine ester-labeled new coronavirus or its variant RBD protein or alkaline phosphatase-labeled new coronavirus or Its variant RBD protein.
  • the spike protein portion of the novel coronavirus or its variant to which the label is coupled is an acridinium ester-labeled novel coronavirus or its variant RBD protein.
  • the RBD protein concentration of the acridinium ester-labeled novel coronavirus or its variant RBD is 2-200 ng/mL, preferably 10-100 ng/mL.
  • the RBD protein concentration of the acridinium ester-labeled novel coronavirus or a variant thereof is 10-100 ng/mL.
  • the acridinium ester-labeled novel coronavirus or its variant RBD protein concentration is 2ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng/ mL, 190ng/mL or 200ng/mL.
  • the spike protein portion of the novel coronavirus or its variant to which the label is coupled is a novel coronavirus or its variant RBD protein labeled with alkaline phosphatase.
  • the RBD protein concentration of the alkaline phosphatase-labeled novel coronavirus or a variant thereof is 2-200 ng/mL, preferably 50 ng/mL.
  • the alkaline phosphatase-labeled novel coronavirus or its variant RBD protein concentration is 2ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL , 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng /mL, 190ng/mL or 200ng/mL.
  • the RBD protein concentration of the alkaline phosphatase-labeled novel coronavirus or a variant thereof is 50 ng/mL.
  • the ACE2 protein or functional fragments thereof are derived from humans. In other embodiments, the ACE2 protein or functional fragment thereof is selected from the group consisting of recombinant ACE2 protein, ACE2 protein extracellular domain, or Fc-tagged ACE2 protein, ACE2 protein extracellular domain. In some embodiments, the ACE2 protein or functional fragment thereof is a recombinant human ACE2 protein. In other embodiments, the ACE2 protein or functional fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NO: 3 and 8-12, or is at least the same as the amino acid sequence shown in any one of SEQ ID NO: 3 and 8-12. 70% identical sequence.
  • the ACE2 protein or functional fragment thereof comprises at least 70%, at least 75%, at least 80%, at least 83%, at least the amino acid sequence shown in any one of SEQ ID NO: 3 and 8-12. A sequence that is 85%, at least 87%, at least 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 any one of SEQ ID NO: 3 and 8-12.
  • the amino acid sequence of the ACE2 protein or functional fragment thereof is any one of the sequences shown in SEQ ID NOs: 3 and 8-12.
  • the concentration of the ACE2 protein or functional fragment thereof in the magnetic particle-coupled ACE2 protein or functional fragment thereof is 2-15 ug/mL, preferably 5-10 ug/mL, more preferably 5 ug/mL. In some embodiments, the concentration of the ACE2 protein or functional fragments thereof in the magnetic particle-coupled ACE2 protein or functional fragments thereof is 5-10 ug/mL.
  • the concentration of the ACE2 protein or functional fragment thereof is 2ug/mL, 3ug/mL, 4ug/mL, 5ug/mL, 6ug/mL, 7ug/mL, 8ug/mL, 9ug/mL, 10ug/mL, 11ug/mL, 12ug/mL, 13ug/mL, 14ug/mL or 15ug/mL.
  • the concentration of the ACE2 protein or functional fragment thereof is 5ug/mL.
  • the concentration of the magnetic particles is 0.1-0.5 mg/mL. In some specific embodiments, the concentration of the magnetic particles is 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, or 0.5 mg/mL. In a preferred embodiment, the concentration of the magnetic particles is 0.25 mg/mL.
  • the kit further includes a sample diluent and a lotion.
  • the kit further includes a chemiluminescent substrate.
  • the chemiluminescent substrate is selected from AMPPD, APS5, CSPD, CDP-Star, luminol or PS-atto.
  • the chemiluminescence substrates in the present invention include, but are not limited to, direct chemiluminescence acid-base luminescence initiators such as acids containing H 2 O 2 (HNO 3 or HCl, etc.) and NaOH, and indirect chemiluminescence substrates include alkaline phosphatase Substrates such as AMPPD (3-(2-Spiraladamantane)-4-methoxy-4-(3-phosphooxy)-phenyl-1,2-dioxetane disodium salt), APS5 ( 9-(4'-Chlorobenzenethiophosphoroxymethylene)-10-methyl-9,10-dihydroacridine disodium salt), CSPD(3-(2-Spiraladamantane)-4- Meth
  • the kit further includes a negative control product, a sample diluent, and a lotion, and the negative control product is a sample diluent.
  • the kit further includes a positive quality control product, and the positive quality control product is a neutralizing antibody against the SARS-CoV-2 virus or a variant thereof.
  • the sample diluent includes disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, Proclin 300, sucrose, bovine serum albumin or Tween 20, and the pH is 7.0-7.5; the lotion Including Tris, sodium chloride and Tween-20, the pH is 7.5-8.0.
  • the sample diluent includes 3.0-4.0 mg/mL disodium hydrogen phosphate dodecahydrate, 0.2-0.4 mg/mL sodium dihydrogen phosphate dihydrate, 7-9 mg/mL sodium chloride, 10 -30mg/mL sucrose, 5-20mg/mL bovine serum albumin, Proclin300 and Tween 20, the pH is 7.0-7.5.
  • the sample diluent includes 3.5 mg/mL disodium hydrogen phosphate dodecahydrate, 0.26 mg/mL sodium dihydrogen phosphate dihydrate, 8.5 mg/mL sodium chloride, 20 mg/mL sucrose, 10 mg /mL bovine serum albumin, Proclin300 and Tween 20, the pH is 7.0-7.5.
  • the lotion includes 25-35 mg/mL Tris, 180-250 mg/mL sodium chloride, Tween 20, and a pH of 7.5-8.0.
  • the lotion includes 30.25 mg/ml Tris, 219.15 mg/mL sodium chloride, Tween 20, and a pH value of 7.5-8.0.
  • the novel coronavirus variant is selected from SARS-CoV-2 B.1.1.7 (GISAID login ID: EPI_ISL_1234251), B.1.351 (GISAID login ID: EPI_ISL_1191083) or B.1.617 (GISAID login ID EPI_ISL_1544002).
  • the present invention provides the application of the above-mentioned chemiluminescence kit in detecting the presence of SARS-CoV-2 virus or its variant antibodies in a sample.
  • the sample includes:
  • Another aspect of the present invention provides the application of the above-mentioned chemiluminescence kit in detecting infection or suspected infection of a subject with SARS-CoV-2 virus or a variant thereof.
  • Another aspect of the present invention provides the application of the above-mentioned chemiluminescence kit in detecting whether a subject has produced effective immunity after being vaccinated with a vaccine against SARS-CoV-2 or a variant thereof.
  • Another aspect of the present invention provides the application of the above-mentioned chemiluminescence kit for detecting neutralizing antibodies against SARS-CoV-2 or variants thereof obtained by biological methods.
  • the present invention also provides the application of the above-mentioned chemiluminescence kit in detecting whether there is SARS-CoV-2 virus or its variant antibody in a subject.
  • Another aspect of the present invention provides a method for using the above-mentioned chemiluminescence kit, which includes the following steps:
  • Another aspect of the present invention provides a method for using the above-mentioned chemiluminescence kit, which includes the following steps:
  • This application also provides a method for detecting the presence of neutralizing antibodies of the novel coronavirus or its variants in a sample, including:
  • step (1) Contacting the mixture of step (1) with the ACE2 protein or fragment thereof specifically bound to the spike protein or ACE2 binding fragment thereof;
  • This application also provides a method for detecting the presence of neutralizing antibodies of the novel coronavirus or its variants in a sample, including:
  • the above method further includes: inhibiting or blocking the binding of the spike protein part of the new coronavirus or its variants and the ACE2 protein or its functional fragments through the sample to generate changes in the detection signal to determine whether the new coronavirus or its variants are present in the sample. Neutralizing antibodies.
  • Magnetic particle chemiluminescence analysis is a new type of analysis and detection technology that effectively combines magnetic separation technology, immunoassay technology and chemiluminescence detection technology.
  • the invention discloses a SARS-CoV-2 virus neutralizing antibody magnetic particle chemiluminescence detection method, which can quickly and effectively detect the SARS-CoV-2 virus neutralizing antibody in human and other animal samples, and overcome the NT method and blocking
  • the ELISA method detects the shortcomings of neutralizing antibodies, used for the screening and identification of SARS-CoV-2 virus neutralizing antibodies, evaluating the effectiveness of vaccines, and qualitative or qualitatively targeting SARS-CoV-2 virus neutralizing antibodies in subject samples Quantitative testing.
  • the present invention provides a magnetic particle chemiluminescence detection kit for detecting neutralizing antibodies of the SARS-CoV-2 virus or its variants in a sample, including: a. SARS-Coupled with a label CoV-2 or its variant spike protein part; b. Magnetic particle-coupled ACE2 protein or its functional fragments; c. Sample diluent; d. Positive quality control; e. Negative control; f. Lotion ; And g. substrate.
  • the magnetic particle chemiluminescence detection kit of the SARS-CoV-2 virus or its variant neutralizing antibody includes: a. Marker-coupled SARS-CoV-2 or its variant spike protein Part; b. Magnetic particle-coupled ACE2 protein or functional fragments thereof; c. Sample diluent; d. Positive quality control product; e. Negative quality control product; f. Lotion; and g. Substrate.
  • the magnetic particle chemiluminescence detection kit of the SARS-CoV-2 virus or its variant neutralizing antibody includes: a. SARS-CoV-2 or its variant RBD protein coupled with a marker B. ACE2 protein coupled with magnetic particles; c. sample diluent; d. positive quality control product; e. negative quality control product; f. lotion; and g. substrate.
  • the label is selected from one or more of alkaline phosphatase, acridinium ester, horseradish peroxidase, ruthenium terpyridine, isoluminol or rare earth elements, preferably alkali Sex phosphatase, acridinium ester or horseradish peroxidase.
  • the label is selected from alkaline phosphatase, acridinium ester, horseradish peroxidase, or ruthenium terpyridine.
  • the label is selected from alkaline phosphatase, acridinium ester, or horseradish peroxidase.
  • the label is alkaline phosphatase. In another specific embodiment, the label is an acridine ester. In another specific embodiment, the marker is horseradish peroxidase. In other embodiments, the SARS-CoV-2 or its variant RBD protein to which the label is coupled is selected from the group consisting of alkaline phosphatase-coupled SARS-CoV-2 or its variant RBD protein, and acridine ester couple. SARS-CoV-2 or its variant RBD protein or horseradish peroxidase-coupled SARS-CoV-2 or its variant RBD protein.
  • the RBD protein concentration of SARS-CoV-2 or a variant thereof to which the marker is coupled is 2-200 ng/mL, preferably 80 ng/mL.
  • the SARS-CoV-2 or variant RBD protein concentration of the marker conjugated is 2ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL , 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng /mL, 190ng/mL or 200ng/mL.
  • the RBD protein concentration of SARS-CoV-2 or a variant thereof to which the marker is coupled is 50 ng/mL. In another specific embodiment, in some specific embodiments, the RBD protein concentration of SARS-CoV-2 or a variant thereof to which the marker is coupled is 100 ng/mL.
  • the SARS-CoV-2 or its variant RBD protein to which the label is coupled is selected from the group consisting of acridine ester-labeled SARS-CoV-2 or its variant RBD protein or alkaline phosphatase-labeled SARS -CoV-2 or its variant RBD protein.
  • the SARS-CoV-2 or its variant RBD protein to which the label is coupled is an acridinium ester-labeled SARS-CoV-2 or its variant RBD protein.
  • the RBD protein concentration of the acridinium ester-labeled SARS-CoV-2 or a variant thereof is 2-200 ng/mL, preferably 10-100 ng/mL.
  • the RBD protein concentration of the acridinium ester-labeled SARS-CoV-2 or a variant thereof is 10-100 ng/mL.
  • the acridinium ester-labeled SARS-CoV-2 or its variant RBD protein concentration is 2ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL, 180ng/mL, 190ng/mL or 200ng/mL.
  • the SARS-CoV-2 or its variant RBD protein to which the label is coupled is an alkaline phosphatase-labeled SARS-CoV-2 or its variant RBD protein.
  • the RBD protein concentration of the alkaline phosphatase-labeled SARS-CoV-2 or a variant thereof is 2-200 ng/mL, preferably 80 ng/mL.
  • the alkaline phosphatase-labeled SARS-CoV-2 or its variant RBD protein concentration is 2ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng /mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 110ng/mL, 120ng/mL, 130ng/mL, 140ng/mL, 150ng/mL, 160ng/mL, 170ng/mL , 180ng/mL, 190ng/mL or 200ng/mL.
  • the RBD protein concentration of the alkaline phosphatase-labeled SARS-CoV-2 or a variant thereof is 50 ng/mL.
  • the ACE2 protein or functional fragments thereof are derived from humans. In other embodiments, the ACE2 protein or functional fragment thereof is selected from the group consisting of recombinant ACE2 protein or ACE2 protein extracellular domain or Fc-tagged ACE2 protein or ACE2 protein extracellular domain. In some embodiments, the ACE2 protein or functional fragment thereof is a recombinant human ACE2 protein. In other embodiments, the ACE2 protein or functional fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NO: 3 and 8-12, or is at least the same as the amino acid sequence shown in any one of SEQ ID NO: 3 and 8-12. 70% identical sequence.
  • the ACE2 protein or functional fragment thereof comprises at least 70%, at least 75%, at least 80%, at least 83%, at least the amino acid sequence shown in any one of SEQ ID NO: 3 and 8-12. A sequence that is 85%, at least 87%, at least 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 any one of SEQ ID NO: 3 and 8-12.
  • the amino acid sequence of the ACE2 protein or functional fragment thereof is any one of the sequences shown in SEQ ID NOs: 3 and 8-12.
  • the concentration of the ACE2 protein or functional fragment thereof in the magnetic particle-coupled ACE2 protein or functional fragment thereof is 2-15 ug/mL, preferably 5-10 ug/mL, more preferably 5 ug/mL. In some embodiments, the concentration of the ACE2 protein or functional fragment thereof is 5-10 ug/mL.
  • the concentration of the ACE2 protein or functional fragment thereof is 2ug/mL, 3ug/mL, 4ug/mL, 5ug/mL, 6ug/mL, 7ug/mL, 8ug/mL, 9ug/mL, 10ug/mL, 11ug/mL, 12ug/mL, 13ug/mL, 14ug/mL or 15ug/mL.
  • the concentration of the ACE2 protein or functional fragment thereof is 5ug/mL.
  • the concentration of the magnetic particles is 0.1-0.5 mg/mL. In some specific embodiments, the concentration of the magnetic particles is 0.1 mg/mL, 0.2 mg/mL, 0.25 mg/mL, 0.3 mg/mL, 0.4 mg/mL, or 0.5 mg/mL.
  • the present invention provides a magnetic particle chemiluminescence qualitative detection kit for SARS-CoV-2 virus neutralizing antibody, comprising: a. ACE2 protein or functional fragments coupled with a marker; b. SARS- coupled with magnetic particles CoV-2 or its variant spike protein part; c. sample diluent; d. positive quality control product; e. negative quality control product; f. lotion; and g. substrate.
  • the magnetic particle chemiluminescence qualitative detection kit of the SARS-CoV-2 virus or its variant neutralizing antibody includes: a. ACE2 protein or functional fragments thereof coupled with a marker; b. magnetic particles Coupling SARS-CoV-2 spike protein part; c. sample dilution; d. positive quality control; e. negative quality control; f. lotion; and g. substrate.
  • the magnetic particle chemiluminescence qualitative detection kit of the SARS-CoV-2 virus or its variant neutralizing antibody includes: a. ACE2 protein coupled with a label; b. magnetic particle coupled SARS-CoV-2 or its variant RBD protein; c. sample diluent; d. positive quality control product; e. negative quality control product; f. lotion; and g. substrate.
  • the present invention provides a magnetic particle chemiluminescence quantitative detection kit for SARS-CoV-2 virus or its variant neutralizing antibody, comprising: a. ACE2 protein or functional fragments thereof coupled with a marker; b. magnetic particle couple Linked SARS-CoV-2 or its variant spike protein portion; c. sample diluent; d. calibrator; e. lotion; and f. substrate.
  • the magnetic particle chemiluminescence quantitative detection kit of SARS-CoV-2 virus or its variant neutralizing antibody includes: a. ACE2 protein or functional fragments thereof coupled with a marker; b. magnetic particles Coupling SARS-CoV-2 or its variant spike protein portion; c. sample diluent; d. calibrator; e. lotion; and f. substrate.
  • the magnetic particle chemiluminescence quantitative detection kit of the SARS-CoV-2 virus or its variant neutralizing antibody includes: a. ACE2 protein coupled with a label; b. magnetic particle coupled SARS-CoV-2 RBD or its variant protein; c. sample diluent; d. calibrator; e. lotion; and f. substrate.
  • the label is selected from one or more of alkaline phosphatase, acridinium ester, horseradish peroxidase, ruthenium terpyridine, isoluminol or rare earth elements, preferably alkali Sex phosphatase, acridinium ester or horseradish peroxidase.
  • the label is selected from alkaline phosphatase, acridinium ester, horseradish peroxidase, or ruthenium terpyridine.
  • the label is selected from alkaline phosphatase, acridinium ester, or horseradish peroxidase.
  • the label is alkaline phosphatase.
  • the label is an acridine ester.
  • the marker is horseradish peroxidase.
  • the ACE2 protein to which the label is coupled is selected from the group consisting of alkaline phosphatase coupled ACE2 protein, acridinium ester coupled ACE2 protein, or horseradish peroxidase coupled ACE2 protein.
  • the spike protein portion of SARS-CoV-2 or a variant thereof includes the amino acid sequence shown in any one of SEQ ID NO: 1, 2 and 4-7, or is the same as SEQ ID NO: 1, A sequence with at least 70% identity in the amino acid sequence shown in any one of 2 and 4-7.
  • the SARS-CoV-2 or its mutant spike protein portion comprises at least 70%, at least 75%, or at least the amino acid sequence shown in any one of SEQ ID NO: 1, 2 and 4-7. 80%, at least 83%, at least 85%, 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 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 sequences.
  • the SARS-CoV-2 variant spike protein portion comprises at least 70%, at least 75%, at least 80%, at least 83% of the amino acid sequence shown in any one of SEQ ID NO: 4-7 , At least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%, at least 97%, or at least 99% identical sequences.
  • the spike protein portion of SARS-CoV-2 or variants thereof includes the amino acid sequence shown in any one of SEQ ID NOs: 1, 2, and 4-7.
  • the SARS-CoV-2 variant spike protein portion includes the amino acid sequence shown in any one of SEQ ID NO: 4-7.
  • the SARS-CoV-2 spike protein portion includes the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • the amino acid sequence of the spike protein portion of SARS-CoV-2 or a variant thereof is shown in SEQ ID NO: 1, 2 and 4-7.
  • the amino acid sequence of the spike protein portion of the novel coronavirus is shown in SEQ ID NO: 1 or 2.
  • the amino acid sequence of the spike protein portion of the novel coronavirus variant is shown in any one of SEQ ID NO4-7.
  • the sample diluent in the above-mentioned chemiluminescence kit of the present invention includes disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, Proclin 300, sucrose, bovine serum albumin or Tween 20, and the pH is 7.0-7.5.
  • the sample diluent includes 3.0-4.0 mg/mL disodium hydrogen phosphate dodecahydrate, 0.2-0.4 mg/mL sodium dihydrogen phosphate dihydrate, 7-9 mg/mL sodium chloride, 10- 30mg/mL sucrose, 5-20mg/mL bovine serum albumin, Proclin300 and Tween 20, the pH is 7.0-7.5.
  • the sample diluent includes 3.5 mg/mL disodium hydrogen phosphate dodecahydrate, 0.26 mg/mL sodium dihydrogen phosphate dihydrate, 8.5 mg/mL sodium chloride, 20 mg/mL sucrose, 10 mg /mL bovine serum albumin, Proclin300 and Tween 20, the pH is 7.0-7.5.
  • the negative control substance in the above-mentioned chemiluminescence kit of the present invention is a sample diluent.
  • the negative control substance includes disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, Proclin 300, sucrose, bovine serum albumin or Tween 20, and the pH is 7.0-7.5.
  • the lotion in the above-mentioned chemiluminescence kit of the present invention includes Tris, sodium chloride and Tween-20, and the pH value is 7.5-8.0.
  • the lotion includes 25-35 mg/mL Tris, 180-250 mg/mL sodium chloride, Tween 20, and a pH value of 7.5-8.0.
  • the lotion includes 30.25 mg/ml Tris, 219.15 mg/mL sodium chloride, Tween 20, and a pH value of 7.5-8.0.
  • the positive quality control product in the above-mentioned chemiluminescence kit of the present invention is a neutralizing antibody against SARS-CoV-2 virus or a variant thereof.
  • the neutralizing antibody against the SARS-CoV-2 virus or its variants can be any neutralizing antibody against the SARS-CoV-2 virus or its variants that has been disclosed in the art, that is, the neutralizing antibody against SARS-CoV-2
  • the RBD protein binding of the virus or its variants is an antibody that can block the binding of the viral RBD to the human ACE2 receptor to prevent cell viruses from invading.
  • the present invention includes but is not limited to the clone 2B2 antibody provided in the examples.
  • the calibrator in the above-mentioned chemiluminescence kit of the present invention is anti-SARS-CoV-2 virus or its variant neutralizing antibodies with different concentrations.
  • the neutralizing antibody against the SARS-CoV-2 virus or its variants can be any neutralizing antibody against the SARS-CoV-2 virus or its variants that has been disclosed in the art, that is, the neutralizing antibody against SARS-CoV-2
  • the RBD protein binding of the virus can block the binding of the viral RBD to the human ACE2 receptor to prevent the virus from invading the cell.
  • the present invention includes but is not limited to the A02087 antibody provided in the examples.
  • the substrate in the above-mentioned chemiluminescence kit of the present invention can be a direct chemiluminescence acid-base luminescence initiator and an indirect chemiluminescence substrate, including but not limited to a direct chemiluminescence acid-base luminescence initiator such as an acid containing H 2 O 2 (HNO 3 or HCl, etc.) and NaOH, indirect chemiluminescent substrates include alkaline phosphatase substrates such as AMPPD, APS5, CSPD, CDP-Star and horseradish peroxidase substrates such as luminol and PS-atto, etc. .
  • a direct chemiluminescence acid-base luminescence initiator such as an acid containing H 2 O 2 (HNO 3 or HCl, etc.) and NaOH
  • indirect chemiluminescent substrates include alkaline phosphatase substrates such as AMPPD, APS5, CSPD, CDP-Star and horseradish peroxidas
  • the preparation method of the above-mentioned chemiluminescence kit of the present invention includes the following steps:
  • the marker is coupled to the new coronavirus or its variant spike protein part includes the SARS-CoV-2 or its variant RBD protein coupled with the marker, including the alkaline phosphatase-labeled SARS-CoV -2 RBD protein, acridinium ester labeled SARS-CoV-2 RBD protein, alkaline phosphatase labeled SARS-CoV-2 B.1.1.7 RBD protein, acridinium ester labeled SARS-CoV-2 B.1.1.7 RBD Protein, alkaline phosphatase label SARS-CoV-2 B.1.351 RBD protein or acridinium ester label SARS-CoV-2 B.1.351 RBD protein.
  • the alkaline phosphatase coupling SARS-CoV-2 or its variant RBD protein preparation method includes: (1) respectively activating SARS-CoV-2 or its variant RBD protein and alkaline phosphatase (2) The activated SARS-CoV-2 or its variant RBD protein and alkaline phosphatase were replaced by a rotating desalting column respectively; (3) The alkaline phosphatase obtained in step (2) was replaced with SARS-CoV -2 or its variant RBD protein solution was mixed and reacted, and N-ethylmaleimide solution was added and allowed to stand; (4) Pass the reactant obtained in step (3) through a rotating desalting column to replace the buffer to obtain AP Coupling SARS-CoV-2 or its variant RBD protein.
  • the preparation method of the acridinium ester-coupled SARS-CoV-2 or its variant RBD protein includes: (1) activating SARS-CoV-2 or its variant RBD protein, rotating the desalting column to replace the buffer (2) Add the acridinium ester solution to mix the reaction to avoid light; (3) Step (2) After the reaction, add glycine to the solution, and rotate the desalting column to replace the buffer to obtain the acridinium ester-coupled SARS-CoV-2 or its Variant RBD protein.
  • step (2) the preparation of magnetic particle-coupled ACE2 protein includes: (1) resuspend the magnetic particle in buffer, add ACE2 protein, mix well, and react at constant temperature; (2) remove the supernatant by magnetic separation, add blocking solution, Constant temperature mixing reaction; (3) Magnetic separation to remove the supernatant, resuspend the magnetic particles, and repeatedly wash; (4) Add sample diluent to resuspend the magnetic particles into a single particle state.
  • the principle of the neutralizing antibody magnetic particle chemiluminescence kit for detecting SARS-CoV-2 virus or its variants is: blocking magnetic particle coupling by the neutralizing antibody of SARS-CoV-2 virus or its variants SARS-CoV-2 or its variant RBD protein (or ACE2 receptor) interacts with a label-coupled ACE2 receptor (or SARS-CoV-2 or its variant RBD protein) to generate a detection signal.
  • ACE2 receptor or SARS-CoV-2 or its variant RBD protein
  • magnetic particles coupled to the SARS-CoV-2 or its variant RBD protein in the test sample and the negative control substance successively or at the same time (Or ACE2 receptor).
  • the magnetic particle-coupled SARS-CoV-2 or its variant RBD protein (or ACE2 receptor) and ACE2 coupled with the marker Receptor (or SARS-CoV-2 RBD protein) binding After washing by magnetic separation, the label bound to the magnetic particles generates a detection signal in the presence of the substrate.
  • the magnetic particle-coupled SARS-CoV-2 or its variant RBD protein (or ACE2 receptor) and the marker are coupled to the ACE2 receptor ( Or the binding amount of SARS-CoV-2 or its variant (RBD protein) is inversely proportional to the concentration of the SARS-CoV-2 virus or its variant neutralizing antibody in the sample.
  • the markers on the magnetic particles are reduced, and the signal generated is weakened.
  • the value of the sample to be tested (S) and the measured value (N) of the negative sample were read by a chemiluminescence meter, and the blocking rate (1-S/N) was used to evaluate the content of neutralizing antibodies in the sample.
  • the blocking rate is ⁇ 30%, indicating the presence of neutralizing antibodies to the SARS-CoV-2 virus or its variants in the test sample.
  • the blocking rate is less than 30%, it means that there is no neutralizing antibody of SARS-CoV-2 virus or its variants in the test sample.
  • Another aspect of the present invention provides a method for using the above-mentioned chemiluminescence kit, which includes the following steps:
  • Another aspect of the present invention provides another method of using the above-mentioned chemiluminescence kit, which includes the following steps:
  • the present invention also provides a method for using the above-mentioned chemiluminescence kit, which includes the following steps:
  • the present invention also provides another method of using the above-mentioned chemiluminescence kit, which includes the following steps:
  • the present invention provides the application of the above-mentioned chemiluminescence detection kit in detecting infection or suspected infection of a subject with SARS-CoV-2 virus or a variant thereof.
  • the body by detecting the presence or absence of the new coronavirus COVID-19 antigen 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-mentioned chemiluminescence kit in detecting whether a subject has an effective immunity after being vaccinated with a vaccine against SARS-CoV-2 or a variant thereof.
  • chemiluminescence kit 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 actively being developed clinically, 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-mentioned chemiluminescence kit for detecting SARS-CoV-2 neutralizing antibodies obtained by biological methods.
  • a large number of antibodies against SARS-CoV-2 or its variants are under development.
  • Anti-SARS-CoV-2 antibodies obtained by common methods in the field, including animal immunization, phage display library, etc., require rapid and effective screening 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.
  • the antibodies include human anti-SARS-CoV-2 virus neutralizing antibody, mouse anti-SARS-CoV-2 virus neutralizing antibody, rat anti-SARS-CoV-2 virus neutralizing antibody, rabbit anti-SARS-CoV- 2 Virus neutralizing antibody, camel anti-SARS-CoV-2 virus neutralizing antibody, anti-SARS-CoV-2 virus neutralizing antibody Fab fragment and anti-SARS-CoV-2 virus neutralizing antibody ScFv fragment, screened from phage antibody library Technology-screened SARS-CoV-2 virus neutralizing antibody, SARS-CoV-2 virus neutralizing antibody sample obtained from high-throughput DNA sequencing technology, SARS-CoV-2 virus neutralizing antibody prepared by hybridoma technology and from serum Antibodies and so on.
  • the kit of the present invention can be used to detect the presence of SARS-CoV-2 virus neutralizing antibodies in a subject.
  • the subjects include healthy subjects, subjects who have been infected with the new coronavirus and have recovered their health, or infected persons who have not shown any symptoms.
  • Samples for testing subjects include, but are not limited to, serum, plasma, or whole blood.
  • the present invention provides a method for detecting the presence of neutralizing antibodies of a novel coronavirus or its variants in a sample, including:
  • the SARS-CoV-2 virus or its variant spike protein or its ACE2 binding fragment in the step (1) is connected to biotin
  • the solid-phase carrier in the step (2) is coupled to the chain Mycoavidin
  • SARS-CoV-2 virus or its variant spike protein or its ACE2 binding fragments are connected to the solid-phase carrier through the combination of biotin and streptavidin on the solid-phase carrier.
  • the solid phase carrier is magnetic particles.
  • the present invention also provides a method for detecting the presence of neutralizing antibodies of SARS-CoV-2 virus or its variants in a sample,
  • SARS-CoV-2 virus or its variant spike protein or its ACE2 binding fragment is connected to magnetic particles, and the amount of magnetic particles or/and SARS-CoV-2 virus or its variant spike protein or its ACE2 are bound The amount of fragments controls the amount of SARS-CoV-2 virus or its variant spike protein or ACE2 binding fragments connected to magnetic particles;
  • the amount of magnetic particles can be adjusted by controlling the concentration of magnetic particles.
  • the amount of the SARS-CoV-2 virus or its variant spike protein or its ACE2 binding fragment can be controlled by controlling the added SARS-CoV-2 virus or its variant spike protein or its ACE2
  • the concentration of the binding fragment can also be adjusted by controlling the density of the SARS-CoV-2 virus or its variant spike protein or its ACE2 binding fragment per unit surface area of the magnetic particles.
  • the molar ratio achieves a better detection range of neutralizing antibodies, and improves the sensitivity and specificity of the detection method.
  • the present invention also provides a method for detecting the presence of neutralizing antibodies of the novel coronavirus or its variants in a sample, including:
  • step (1) Contacting the mixture of step (1) with the ACE2 protein or fragment thereof specifically bound to the spike protein or ACE2 binding fragment thereof;
  • the solid phase carrier is a magnetic particle.
  • the above-mentioned method for detecting the neutralizing antibody of the novel coronavirus or its variants further comprises the method of inhibiting or blocking the binding of the spike protein part of the novel coronavirus or its variants and the ACE2 protein or its functional fragments to generate a detection signal by the sample. Change to determine whether there are neutralizing antibodies for the novel coronavirus or its variants in the sample.
  • 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 SARS-CoV-2 mutant can be any mutant strain, such as B.1.1.7 (GISAID registration ID: EPI_ISL_1234251), B.1.351 (GISAID registration 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 new SARS-CoV-2 mutant strain.
  • B.1.1.7 GISAID registration ID: EPI_ISL_1234251
  • B.1.351 GISAID registration ID: EPI_ISL_1191083
  • B.1.617 EPI_ISL_1544002
  • B.1.617.1 EPI_ISL_22604608
  • B.1.617.2 EPI_ISL_2272895
  • B.1.617.3 EPI_ISL_1704535
  • 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-2 S1 subunit or SARS-CoV-2 S RBD antigen.
  • the SARS-CoV-2 S RBD antigen described in the present invention can be produced by conventional recombinant expression methods, by constructing a plasmid expressing SARS-CoV-2 S RBD, such as pFastBac1, pTT5, and transfecting expression cells with an expression vector containing the target gene , such as CHO cells and SF9 cells, express and purify SARS-CoV-2 S 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. Part of the region, such as the extracellular domain of ACE2 or the extracellular domain of ACE2 with an Fc tag.
  • new coronavirus or its variant spike protein part or “new coronavirus or its variant spike protein or its ACE2 binding fragment” refers to the new coronavirus or its variant spike protein capable of functioning Part of it can specifically bind to ACE2 protein or the extracellular domain of ACE2 protein. It can be the RBD protein of the new coronavirus or its variants, the S1 protein and S protein of the new coronavirus or its variants.
  • SARS-CoV-2 virus or its variant RBD protein Preferably it is SARS-CoV-2 virus or its variant RBD protein, SARS-CoV-2 virus or its variant S1 protein and SARS-CoV-2 virus or its variant S protein, more preferably SARS-CoV-2 Virus or its variant RBD protein or SARS-CoV-2 virus or its variant S1 protein.
  • SARS-CoV-2 virus or its variant RBD protein can be embodied as a recombinant SARS-CoV-2 virus or its variant RBD protein (as shown in SEQ ID NO: 1, 4 or 6), with SARS-CoV-2 virus or its variant RBD protein with His tag, SARS-CoV-2 virus or its variant RBD protein with Fc tag; containing SARS-CoV-2 virus or its variant RBD protein Molecules, such as SARS-CoV-2 virus or its variant S1 subunit (as shown in SEQ ID NO: 2, 5 or 7) and SARS-CoV-2 virus or its variant S protein.
  • the SARS-CoV-2 virus variant may be B.1.1.7, B.1.351, B.1.617, B.1.617.1, B.1.617.2, B.1.617.3, or any new emerging SARS-CoV-2 mutant strain.
  • magnetic particles includes, but may not be limited to, p-toluenesulfonyl modified magnetic beads (Tosyl magnetic beads), amino modified magnetic beads and carboxyl modified magnetic beads.
  • the term "marker” herein may refer to at least one of horseradish peroxidase (HRP), alkaline phosphatase (AP), acridine ester, isoluminol, or rare earth elements.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • acridine ester acridine ester
  • isoluminol or rare earth elements.
  • the label reacts with the luminescent substrate to produce chemiluminescence.
  • luminol produces chemiluminescence after being catalyzed by HRP, which can be detected by a chemiluminescence instrument.
  • the "magnetic particle conjugate” or “magnetic particle marker” in the present invention can refer to the novel coronavirus spike protein partly coupled to magnetic particles or ACE2 protein coupled magnetic particles, that is, the novel coronavirus spike protein part is covalently coupled Magnetic particles are coupled or ACE2 protein is covalently coupled to magnetic particles.
  • the present invention specifically includes the RBD protein of SARS-Cov-2 coupled with magnetic particles, the S1 protein of SARS-Cov-2 coupled with magnetic particles, or the ACE2 protein of magnetic particles coupled.
  • label conjugate or “label linker” in the present invention can refer to the new coronavirus spike protein part or ACE2 protein or functional fragments thereof coupled with a label, including but not limited to HRP-coupled new coronavirus Viral spike protein part, AP-coupled new coronavirus spike protein part, acridinium ester-coupled new coronavirus spike protein part, HRP-coupled ACE2 protein, AP-coupled ACE2 protein and acridinium ester Coupled with ACE2 protein.
  • conjugate in the present invention refers to a magnetic particle conjugate formed by covalently labeling the coronavirus spike protein part or ACE2 protein to a magnetic particle. Part or ACE2 protein is labeled to the label to form a label conjugate.
  • connecting refers to directly coupling to the C-terminus or N-terminus of the protein through a linker or a chemical bond, and can be selected from small molecule chemical linkers or peptide linkers.
  • the small molecule chemical linker includes but is not limited to carboxyl, amino, sulfhydryl, etc.
  • the peptide linker can be selected from one or more amino acid polypeptides, and a label or magnetic particle can be connected to the spike of the new coronavirus through a linker. Protein part or ACE2 protein or functional fragments thereof.
  • the coupling method is connected by forming a covalent bond, and a covalent bond can be formed by a covalent reaction between a label or a magnetic particle and a new coronavirus spike protein part or an ACE2 protein or a functional fragment thereof.
  • linker refers to a compound that can connect a protein with a label or a magnetic particle, and it can be a peptide linker, a small molecule chemical linker, or a polymer molecule linker.
  • small molecule chemical linkers include but are not limited to carboxyl, amino and sulfhydryl groups; peptide linkers can be at most 20, at most 10, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3. A polypeptide of at most 2, or 1 amino acid.
  • chemiluminescent substrate includes, but is not limited to the alkali agent luminescent promoter, such as direct chemiluminescent containing H2O2 acid (HNO 3 or HCl, etc.) and NaOH, indirect chemiluminescent alkaline phosphatase substrate comprising such as AMPPD, APS5, CSPD, CDP-Star and horseradish peroxidase substrates such as luminol and PS-atto.
  • alkali agent luminescent promoter such as direct chemiluminescent containing H2O2 acid (HNO 3 or HCl, etc.) and NaOH
  • indirect chemiluminescent alkaline phosphatase substrate comprising such as AMPPD, APS5, CSPD, CDP-Star and horseradish peroxidase substrates such as luminol and PS-atto.
  • the "chemiluminescence instrument” in the present invention is composed of two parts: an immune response system and a chemiluminescence analysis system.
  • the chemiluminescence analysis system uses a luminescence signal measuring instrument to measure the light quantum signal produced by the catalysis and oxidation of a chemiluminescence material.
  • the immune response system is to directly label chemiluminescent substances or enzymes on antigens or antibodies (immunochemiluminescence analysis). It includes chemiluminescence enzyme immunoassay and electrochemiluminescence analyzer.
  • 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 the new coronavirus; (2) plasma, serum or whole blood after inoculation with the new coronavirus vaccine; (3) the new coronavirus sting Plasma, serum or whole blood of animals immunized with spike protein; or (4) Anti-new coronavirus antibody samples obtained by biological methods.
  • Samples can be derived from humans or animals infected with or suspected of being infected with SARS-CoV-2 virus, such as plasma, serum, whole blood, pleural effusion, cerebrospinal fluid, or tissue specimens of mice, rats, and rabbits, as well as biological Scientific methods such as animal serum, plasma, whole blood or solutions of antibodies against SARS-CoV-2 coronavirus prepared by animal immunization.
  • SARS-CoV-2 virus such as plasma, serum, whole blood, pleural effusion, cerebrospinal fluid, or tissue specimens of mice, rats, and rabbits
  • biological Scientific methods such as animal serum, plasma, whole blood or solutions of antibodies against SARS-CoV-2 coronavirus prepared by animal immunization.
  • 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.
  • the SARS-CoV-2 neutralizing antibody blocking magnetic particle chemiluminescence detection kit detects the SARS-CoV-2 virus neutralizing antibody in the sample through the inhibition rate of the chemiluminescence signal.
  • the method has large detection throughput, High degree of automation, high accuracy, strong specificity, fast detection speed, simple operation and other characteristics.
  • Figure 1 is a schematic diagram of the SARS-CoV-2 virus neutralizing antibody magnetic particle chemiluminescence kit
  • Figure 2 is a diagram showing the blocking effect of four SARS-CoV-2 antibody strains analyzed by the SARS-CoV-2 virus neutralizing antibody alkaline phosphatase magnetic particle chemiluminescence detection kit.
  • Figure 3 shows the results of 30 negative samples and 20 positive samples tested by the SARS-CoV-2 virus neutralizing antibody alkaline phosphatase magnetic particle chemiluminescence detection kit.
  • Figure 4 shows the calibration curve of the SARS-CoV-2 virus and mutants B.1.1.7 and B.1.351 neutralizing antibody alkaline phosphatase magnetic particle chemiluminescence detection kit.
  • Figure 5 shows the correlation between the SARS-CoV-2 virus neutralizing antibody alkaline phosphatase magnetic particle chemiluminescence detection kit and the commercial kit cPass TM SARS-CoV-2 neutralizing antibody detection kit (FDAEUA) testing 40 samples Sex map.
  • This kit consists of the following 7 reagents:
  • step 5) Add 10mL of the diluent prepared in step 1.4, shake and mix for 1-5min, resuspend the magnetic particles to 5mg/mL (ACE2 content 100ug/mL), use an ultrasonic oscillator to aggregate the suspension into clusters of magnetic beads Disperse into a single particle state to obtain a magnetic separation reagent concentrate.
  • 5mg/mL ACE2 content 100ug/mL
  • an ultrasonic oscillator to aggregate the suspension into clusters of magnetic beads Disperse into a single particle state to obtain a magnetic separation reagent concentrate.
  • the anti-SARS-CoV-2 virus neutralizing antibody (GenScript, clone 2B2) was prepared with the sample diluent prepared by the above steps as a 200ng/mL positive quality control product.
  • the positive quality control was confirmed as neutralizing antibody by GenScript Cpass TM kit (Cat No. L00847).
  • the negative control substance is the sample diluent prepared in Example 1.4.
  • chemiluminescence substrate Beijing Aiweide Biotechnology Co., Ltd., Cat No. APSUB-1
  • N2 The detection value of the negative control 2.
  • SARS-CoV-2 virus neutralizing antibody alkaline phosphatase magnetic particle chemiluminescence kit to analyze 4 SARS-CoV-2 monoclonal antibodies (SARS-CoV-2 mAb-1/-2/-3/-4 , The antibody was prepared by GenScript).
  • N2 The detection value of the negative control 2.
  • SARS-CoV-2 mAb-1 paired with AP-conjugated SARS-CoV-2 RBD protein and The binding blocking effect of magnetic particles coupled with ACE2 is not significant.
  • SARS-CoV-2 mAb-2, SARS-CoV-2 mAb-3 and SARS-CoV-2 mAb-4 antibodies can significantly block the binding of AP coupling label SARS-CoV-2 RBD protein and magnetic particle coupled ACE2 effect.
  • SARS-CoV-2 mAb-4 can block the binding of AP-conjugated SARS-CoV-2 RBD protein and magnetic particle-coupled ACE2 at low concentrations.
  • the SARS-CoV-2 virus neutralizing antibody alkaline phosphatase magnetic particle chemiluminescence kit was used to detect 30 samples without neutralizing antibody, and the results were all negative (inhibition rate ⁇ 30% is negative), as shown in Figure 3.
  • This kit consists of the following 8 reagents:
  • step 1.4 Add 10mL of the diluent prepared in step 1.4, shake and mix for 1-5min, resuspend the magnetic particles to 5mg/mL (ACE2 content 100ug/mL), use an ultrasonic oscillator to aggregate the suspension into clusters of magnetic beads Disperse into a single particle state to obtain a magnetic separation reagent concentrate.
  • 5mg/mL ACE2 content 100ug/mL
  • an ultrasonic oscillator to aggregate the suspension into clusters of magnetic beads Disperse into a single particle state to obtain a magnetic separation reagent concentrate.
  • the preparation method and components of the lotion are the same as the preparation steps and components of Example 1 in 1.7.
  • chemiluminescence substrate Beijing Aiweide Biotechnology Co., Ltd., Cat No. APSUB-1
  • the calibration curve was fitted with four parameters using the calibrator concentration and signal, and three calibration curves were fitted with 3 groups of RBDs, as shown in Figure 4.
  • Conditions for the establishment of the curve the correlation coefficient of the calibration curve is r ⁇ 0.99, the result of the curve is established, if the result is not established, the test should be repeated.
  • the same method as the SARS-CoV-2 virus neutralizing antibody calibrator can be used for testing.
  • the kit for detecting anti-wild-type SARS-CoV-2 RBD neutralizing antibodies and the cPass TM SARS-CoV-2 neutralizing antibody detection kit (FDA EUA) (GenScript, article number L00847) were used in this example to detect 40 cases respectively
  • the sample use matrix human serum (Scantibodies, Cat No. 3SH344) to dilute the commercial SARS-CoV-2 neutralizing antibody standard (GenScript, Cat No. A02087) to different concentrations in the range of 0U/ml ⁇ 7200U/ml ), the test concentration of the embodiment and the test concentration of cPass are fitted to a straight line by the least square method, and the correlation coefficient R 2 is calculated, as shown in FIG. 5.
  • R 2 ⁇ 0.95 indicates that the kits of the Examples and the cPass kits have a good correlation.
  • This kit consists of the following 7 reagents:
  • step 5 Dilute the acridinium ester coupling labeled SARS-CoV-2 RBD protein concentrate prepared in step 4) with the sample diluent prepared in step 1.4 to 10-100 ng/mL to obtain the reagent.
  • step 6) Dilute the magnetic particle coupled ACE2 concentrate of step 5) with the sample diluent prepared in step 1.4 to a magnetic particle coupled ACE2 with a magnetic particle concentration of 0.1-0.5 mg/mL and an ACE2 concentration of 2-10ug/mL.
  • the anti-SARS-CoV-2 virus neutralizing antibody (GenScript, clone: 2B2, Cat No. A02051) was prepared as a 200ng/mL positive quality control product with the sample diluent.
  • the negative control substance is the sample diluent prepared in Example 1.4.
  • the preparation method and components of the lotion are the same as the preparation steps and components of Example 1 in 1.7.
  • N2 The detection value of the negative control 2.
  • SARS-CoV-2 spike protein RBD protein sequence SARS-CoV-2 spike protein RBD protein sequence (SEQ ID NO: 1)
  • SARS-CoV-2 spike protein S1 subunit sequence SEQ ID NO: 2
  • SARS-CoV-2 mutant strain B.1.1.7 Spike protein RBD protein sequence (SEQ ID NO: 4)
  • SARS-CoV-2 mutant strain B.1.1.7 Spike protein S1 subunit sequence (SEQ ID NO: 5)
  • SARS-CoV-2 mutant strain B.1.351
  • Spike protein RBD protein sequence SEQ ID NO: 6
  • SARS-CoV-2 mutant strain B.1.351
  • Spike protein S1 subunit sequence SEQ ID NO: 7

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Abstract

检测SARS-CoV-2病毒中和抗体的磁微粒化学发光试剂盒及其应用。试剂盒包括新型冠状病毒的刺突蛋白部分和特异性结合新型冠状病毒刺突蛋白部分的ACE2蛋白或其功能片段；其中，新型冠状病毒的刺突蛋白部分连接标记物，ACE2蛋白或其功能片段连接磁微粒；或新型冠状病毒的刺突蛋白部分连接磁微粒，ACE2蛋白或其功能片段连接标记物。该磁微粒化学发光试剂盒能用于生物样品中的中和抗体的筛选和鉴定、评价疫苗的有效性以及针对受试者样本中的SARS-CoV-2病毒中和抗体进行定性或定量检测。

Description

检测SARS-CoV-2病毒中和抗体的磁微粒化学发光试剂盒及其应用 技术领域
本发明涉及抗体检测领域，具体涉及通过SARS-CoV-2病毒中和抗体抑制或阻断磁微粒化学发光的技术检测SARS-CoV-2病毒中和抗体的方法以及检测SARS-CoV-2病毒中和抗体的磁微粒化学发光试剂盒。
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。2019冠状病毒病疫情逐渐变成一场全球性大瘟疫，已经造成600多万感染者和逾30万名患者死亡。SARS-CoV-2病毒通过跨膜刺突糖蛋白(S蛋白)RBD结构域与宿主细胞表面ACE2受体结合进入宿主细胞。SARS-CoV-2病毒中和抗体通过阻断SARS-CoV-2 RBD蛋白与ACE2受体结合抑制SARS-CoV-2病毒进入宿主细胞。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。
目前中和抗体的检测方法有基于传统中和实验(NT)的检测方法和阻断ELISA检测方法。NT法是将抗体样品与病毒作用后感染细胞，通过观察细胞病变情况并采用细胞培养半数感染量(CCID50)或半数组织细胞感染剂量(TCID50)方法计算病毒效价，进而评估中和抗体对病毒感染的阻断能力。中和抗体阻断ELISA方法是利用标记的中和抗体阻断样本中的中和抗体与包被在酶标板上的病毒或重组抗原结合，进行化学酶联显色，再用酶标分析仪获得结果，进而评估中和抗体的中和效果。但在实际应用中，NT法实验周期较长，工作量大，难于满足快速、高通量的筛选需求。中和抗体阻断ELISA方法需要制备中和抗体，困难较大。
磁性微粒化学发光分析法是将磁分离技术、免疫分析技术及化学发光检测技术三者有效结合在一起的新型的分析检测技术。该技术以微米级磁微粒标记抗体蛋白等生物分子捕获样本中的目标蛋白。该技术用化学发光物质作为标记物直接标记抗体蛋白等生物分子，或是以能催化化学发光物质的酶作为标记物，产生检测信号。在反应后，化学发光物质在相应的氧化剂或催化酶存在的环境下，产生可被化学发光仪检测到的光子信号，实现对待测抗体蛋白复合物的准确定性或定量测定。磁性微粒化学发光免疫分析法中最常用的标记物有碱性磷酸酶(AP)、吖啶酯、辣根过氧化物酶(HRP)等。由于，磁微粒化学发光技术具有高灵敏度、强特异性、磁分离系统的快速分离、检测时间短、线性范围宽、以及易实现自动化等优点。近年来被广泛应用于临床诊断、生物医学、食品安全检测等多个方面。
目前尚没有发现用磁微粒标记SARS-CoV-2 RBD蛋白(或ACE2受体)和标记物偶联ACE2受体(SARS-CoV-2 RBD蛋白)的磁性微粒化学发光分析法检测SARS-CoV-2病毒中和抗体的报道。
发明内容
本发明利用SARS-CoV-2病毒中和抗体能够阻断SARS-CoV-2 RBD蛋白与ACE2受体结合的特性开发了检测SARS-CoV-2病毒中和抗体化学发光试剂盒。该试剂盒可以通用，操作简便，使用成本低，更易推广应用。
本发明一方面提供了一种检测样品中新型冠状病毒(SARS-CoV-2)或其变体中和抗体的化学发光试剂盒，其特征在于，所述试剂盒包括新型冠状病毒或其变体的刺突蛋白部分和特异性结合所述新型冠状病毒或其变体刺突蛋白部分的ACE2蛋白或其功能片段；其中，所述新型冠状病毒或其变体的刺突蛋白部分连接标记物，所述ACE2蛋白或其功能片段连接磁微粒；或所述新型冠状病毒或其变体的刺突蛋白部分连接磁微粒，所述ACE2蛋白或其功能片段连接标记物；其中，所述新型冠状病毒或其变体的刺突蛋白部分与ACE2蛋白或其功能片段之间特异性结合产生检测信号。
在一些实施方案中，所述标记物或磁微粒是通过接头或偶联方式连接的，优选通过偶联方式连接。在一些具体实施方案中，所述标记物或磁微粒是通过偶联方式连接的。在另一些具体实施方案中，所述标记物或磁微粒是通过接头连接的。其中，所述接头可选自小分子化学接头或肽接头，包括但不限羧基、氨基和巯基等，可通过接头将标记物或磁微粒连接到新型冠状病毒或其变体的刺突蛋白部分或ACE2蛋白或其功能片段。其中，所述偶联方式连接通过形成共价键连接，可通过标记物或磁微粒与新型冠状病毒或其变体的刺突蛋白部分或ACE2蛋白或其功能片段共价反应形成共价键连接。在一个具体实施方案中，所述标记物或磁微粒是通过共价偶联方式连接的。
在一些实施方案中，所述新型冠状病毒或其变体的刺突蛋白部分偶联上标记物，所述ACE2蛋白或其功能片段偶联上磁微粒。在另一些实施方案中，所述新型冠状病毒或其变体的刺突蛋白部分偶联上磁微粒，所述ACE2蛋白或其功能片段偶联上标记物。
在一些实施方案中，所述标记物选自碱性磷酸酶、吖啶酯、辣根过氧化物酶、三联吡啶钌、异鲁米诺或稀土元素或生物素中的一种或多种，优选为碱性磷酸酶、吖啶酯或辣根过氧化物酶。在一些具体实施方案中，所述标记物选自碱性磷酸酶、吖啶酯、辣根过氧化物酶或三联吡啶钌。在另一些具体实施方案中，所述标记物选自碱性磷酸酶、吖啶酯或辣根过氧化物酶。在一个具体实施方案中，所述标记物为碱性磷酸酶。在另一个具体实施方案中，所述标记物为吖啶酯。在又一个具体实施方案中，所述标记物为辣根过氧化物酶。
在一些实施方案中，所述化学发光试剂盒选自辣根过氧化物酶磁微粒化学发光试剂盒、碱性磷酸酶磁微粒化学发光试剂盒、吖啶酯磁微粒化学发光试剂盒或三联吡啶钌磁微粒电化学发光试剂盒，优选为吖啶酯磁微粒化学发光试剂盒或碱性磷酸酶磁微粒化学发光试剂盒。在一些具体实施方案中，所述化学发光试剂盒为吖啶酯磁微粒化学发光试剂盒或碱性磷酸酶磁微粒化学发光试剂盒。
在一些实施方案中，所述吖啶酯磁微粒化学发光试剂盒包括吖啶酯偶联的新型冠状病毒或其变体刺突蛋白部分和磁微粒偶联的ACE2蛋白或其功能片段。在另一些实施方案中，所述吖啶酯磁微粒化学发光试剂盒包括吖啶酯偶联的ACE2蛋白或其功能片段和磁微粒偶联的新型冠状病毒或其变体刺突蛋白部分。
在一些实施方案中，所述碱性磷酸酶磁微粒化学发光试剂盒包括碱性磷酸酶偶联的新型冠状病毒或其变体刺突蛋白部分和磁微粒偶联的ACE2蛋白或其功能片。在另一些实施方案 中，所述碱性磷酸酶磁微粒化学发光试剂盒包括碱性磷酸酶偶联的ACE2蛋白或其功能片段和磁微粒偶联的新型冠状病毒或其变体刺突蛋白。
在一些实施方案中，所述辣根过氧化物酶磁微粒化学发光试剂盒包括辣根过氧化物酶偶联的新型冠状病毒或其变体刺突蛋白部分和磁微粒偶联的ACE2蛋白或其功能片段。在另一些实施方案中，所述辣根过氧化物酶磁微粒化学发光试剂盒包括辣根过氧化物酶偶联的ACE2蛋白或其功能片段和磁微粒偶联的新型冠状病毒或其变体刺突蛋白部分。
在一些实施方案中，所述三联吡啶钌磁微粒电化学发光试剂盒包括三联吡啶钌偶联的新型冠状病毒或其变体刺突蛋白部分和磁微粒偶联的ACE2蛋白或其功能片段。在另一些实施方案中，所述三联吡啶钌磁微粒电化学发光试剂盒包括三联吡啶钌偶联的ACE2蛋白或其功能片段和磁微粒偶联的新型冠状病毒或其变体刺突蛋白部分。
在一些实施方案中，所述三联吡啶钌磁微粒电化学发光试剂盒包括三联吡啶钌偶联的新型冠状病毒或其变体刺突蛋白部分、磁微粒偶联的链霉亲和素和生物素标记的ACE2蛋白或其功能片段。在另一些实施方案中，所述三联吡啶钌磁微粒电化学发光试剂盒包括三联吡啶钌偶联的ACE2蛋白或其功能片段、磁微粒偶联的链霉亲和素和生物素标记的新型冠状病毒或其变体刺突蛋白部分。
在一些实施方案中，所述试剂盒通过检测待测样品抑制或阻断新型冠状病毒或其变体刺突蛋白部分和ACE2蛋白或其功能片段的结合产生检测信号的变化，确定样品中新型冠状病毒或其变体中和抗体的存在情况。
在一些实施方案中，所述新型冠状病毒或其变体刺突蛋白部分选自新型冠状病毒或其变体S蛋白、新型冠状病毒或其变体S1蛋白、新型冠状病毒或其变体RBD蛋白或者带有His或者Fc标签的新型冠状病毒或其变体RBD蛋白，优选为新型冠状病毒或其变体RBD蛋白。在一些具体实施方案中，所述新型冠状病毒或其变体刺突蛋白部分为新型冠状病毒或其变体S1蛋白或新型冠状病或其变体毒RBD蛋白。在一个具体实施方案中，所述新型冠状病毒或其变体刺突蛋白部分为新型冠状病毒或其变体RBD蛋白。
在一些实施方案中，所述新型冠状病毒或其变体刺突蛋白部分包含SEQ ID NO:1、2和4-7中任一所示氨基酸序列，或与SEQ ID NO:1、2和4-7中任一所示氨基酸序列至少70％一致性的序列。在一些具体实施方案中，所述新型冠状病毒或其变体刺突蛋白部分包含与SEQ ID NO:1、2和4-7中任一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。在另一些具体实施方案中，所述新型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。在一些具体实施方案中，所述新型冠状病毒变体刺突蛋白部分包含与SEQ ID NO:4-7中任一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。在另一些具体实施方案中，所述新型冠状病毒或其变体刺突蛋白部分包括SEQ ID NO:1、2和4-7中任一所示的氨基酸序列。在一些具体实施方案中，所述新型冠状病毒刺突蛋白部分包括SEQ ID NO:1或2所示的氨基酸序列。在另一些具体实施方案中，所述新型冠状病毒变体刺突蛋白部分包括SEQ ID NO:4-7中任一所示的氨基酸序列。在一个具体实施方案中，所述新型 冠状病毒或其变体刺突蛋白部分的氨基酸序列如SEQ ID NO:1、2和4-7所示的任一序列。在另一个具体实施方案中，所述新型冠状病毒刺突蛋白部分的氨基酸序列如SEQ ID NO:1或2所示。在一个具体实施方案中，所述新型冠状病毒变体刺突蛋白部分的氨基酸序列如SEQ ID NO:4-7中任一序列所示。
在一些实施方案中，所述标记物偶联的新型冠状病毒或其变体刺突蛋白部分浓度为2-200ng/mL，优选为80ng/mL。在一些具体实施方案中，所述标记物偶联的新型冠状病毒或其变体刺突蛋白部分浓度为2ng/mL、10ng/mL、20ng/mL、30ng/mL、40ng/mL、50ng/mL、60ng/mL、70ng/mL、80ng/mL、90ng/mL、100ng/mL、110ng/mL、120ng/mL、130ng/mL、140ng/mL、150ng/mL、160ng/mL、170ng/mL、180ng/mL、190ng/mL或200ng/mL。在另一些具体实施方案中，所述标记物偶联的新型冠状病毒或其变体刺突蛋白部分浓度为80ng/mL。在一些具体实施方案中，所述标记物偶联的新型冠状病毒或其变体刺突蛋白部分浓度为100ng/mL。
在一些实施方案中，所述标记物偶联的新型冠状病毒或其变体刺突蛋白部分为吖啶酯标记的新型冠状病毒或其变体RBD蛋白或者碱性磷酸酶标记的新型冠状病毒或其变体RBD蛋白。在另一些实施方案中，所述标记物偶联的新型冠状病毒或其变体刺突蛋白部分为吖啶酯标记的新型冠状病毒或其变体RBD蛋白。在一些具体实施方案中，所述吖啶酯标记的新型冠状病毒或其变体RBD蛋白浓度为2-200ng/mL，优选为10-100ng/mL。在一些优选的实施方案中，所述吖啶酯标记的新型冠状病毒或其变体RBD蛋白浓度10-100ng/mL。在另一些具体实施方案中，所述吖啶酯标记的新型冠状病毒或其变体RBD蛋白浓度为2ng/mL、10ng/mL、20ng/mL、30ng/mL、40ng/mL、50ng/mL、60ng/mL、70ng/mL、80ng/mL、90ng/mL、100ng/mL、110ng/mL、120ng/mL、130ng/mL、140ng/mL、150ng/mL、160ng/mL、170ng/mL、180ng/mL、190ng/mL或200ng/mL。在一些实施方案中，所述标记物偶联的新型冠状病毒或其变体刺突蛋白部分为碱性磷酸酶标记的新型冠状病毒或其变体RBD蛋白。在一些具体实施方案中，所述碱性磷酸酶标记的新型冠状病毒或其变体RBD蛋白浓度为2-200ng/mL,优选为50ng/mL。在另一些具体实施方案中，所述碱性磷酸酶标记的新型冠状病毒或其变体RBD蛋白浓度为2ng/mL、10ng/mL、20ng/mL、30ng/mL、40ng/mL、50ng/mL、60ng/mL、70ng/mL、80ng/mL、90ng/mL、100ng/mL、110ng/mL、120ng/mL、130ng/mL、140ng/mL、150ng/mL、160ng/mL、170ng/mL、180ng/mL、190ng/mL或200ng/mL。在一个优选的实施方案中，所述碱性磷酸酶标记的新型冠状病毒或其变体RBD蛋白浓度为50ng/mL。
在一些实施方案中，所述ACE2蛋白或其功能片段来源于人。在另一些实施方案中，所述ACE2蛋白或其功能片段选自重组ACE2蛋白、ACE2蛋白胞外域或带Fc标签ACE2蛋白、ACE2蛋白胞外域。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。在另一些实施方案中，ACE2蛋白或其功能片段包含SEQ ID NO:3和8-12中任一所示氨基酸序列，或与SEQ ID NO:3和8-12中任一所示氨基酸序列至少70％一致性的序列。在一些具体实施方案中，所述ACE2蛋白或其功能片段包含与SEQ ID NO:3和8-12中任一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ACE2蛋白或其功能片段包含SEQ ID NO:3和8-12中任一所示的氨基酸序列。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和8-12中所示任一序列。
在一些实施方案中，所述磁微粒偶联的ACE2蛋白或其功能片段中ACE2蛋白或其功能片段的浓度为2-15ug/mL，优选为5-10ug/mL，更优选为5ug/mL。在一些实施方案中，所述磁微粒偶联的ACE2蛋白或其功能片段中ACE2蛋白或其功能片段的浓度为5-10ug/mL。在一些具体实施方案中，所述ACE2蛋白或其功能片段的浓度为2ug/mL、3ug/mL、4ug/mL、5ug/mL、6ug/mL、7ug/mL、8ug/mL、9ug/mL、10ug/mL、11ug/mL、12ug/mL、13ug/mL、14ug/mL或15ug/mL。在一个具体实施方案中，所述ACE2蛋白或其功能片段的浓度为5ug/mL。
在另一些实施方案中，所述磁微粒的浓度为0.1-0.5mg/mL。在一些具体实施方案中，所述磁微粒的浓度为0.1mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL或0.5mg/mL。在一个优选实施方案中，所述磁微粒的浓度为0.25mg/mL。
在一些实施方案中，所述试剂盒还包括样品稀释液和洗液。
在另一些实施方案中，所述试剂盒进一步包括化学发光底物。所述化学发光底物选自AMPPD、APS5、CSPD、CDP-Star、鲁米诺或PS-atto。本发明中所述化学发光底物包括但不限于直接化学发光的酸碱发光启动剂如含H 2O 2的酸(HNO 3或HCl等)和NaOH，间接化学发光底物包括碱性磷酸酶底物如AMPPD(3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷二钠盐)、APS5(9-(4'-氯苯硫代磷酰氧亚甲基)-10-甲基-9,10-二氢化吖啶二钠盐)、CSPD(3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷)、CDP-Star(3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷)和辣根过氧化物酶底物如鲁米诺和PS-atto(Lumi-Phos HRP)等。
在一些实施方案中，所述试剂盒还包括阴性质控品、样品稀释液和洗液，所述阴性质控品为样品稀释液。
在另一些实施方案中，所述试剂盒进一步包括阳性质控品，所述阳性质控品为抗SARS-CoV-2病毒或其变体的中和抗体。
在一些实施方案中，所述样品稀释液包括磷酸氢二钠、磷酸二氢钠、氯化钠、Proclin300、蔗糖、牛血清白蛋白或吐温20，pH值为7.0-7.5；所述洗液包括Tris、氯化钠和吐温-20，pH值为7.5-8.0。在一些具体实施方案中，所述样品稀释液包括3.0-4.0mg/mL十二水合磷酸氢二钠、0.2-0.4mg/mL二水合磷酸二氢钠、7-9mg/mL氯化钠、10-30mg/mL蔗糖、5-20mg/mL牛血清白蛋白、Proclin300和吐温20，pH值为7.0-7.5。在一个具体实施方案中，所述样品稀释液包括3.5mg/mL十二水合磷酸氢二钠、0.26mg/mL二水合磷酸二氢钠、8.5mg/mL氯化钠、20mg/mL蔗糖、10mg/mL牛血清白蛋白、Proclin300和吐温20，pH值为7.0-7.5。在另一些实施方案中，所述洗液包括25-35mg/mLTris、180-250mg/mL氯化钠，吐温20，pH值为7.5-8.0。在一个具体实施方案中，所述洗液包括30.25mg/ml Tris、219.15mg/mL氯化钠，吐温20，pH值为7.5-8.0。
在一些实施方案中，所述新型冠状病毒变体选自SARS-CoV-2 B.1.1.7(GISAID登录ID:EPI_ISL_1234251)、B.1.351(GISAID登录ID:EPI_ISL_1191083)或B.1.617(GISAID登录ID EPI_ISL_1544002)。
本发明提供了上述的化学发光试剂盒在检测样品中是否存在SARS-CoV-2病毒或其变体的抗体中的应用。
在一些实施方案中，所述样品包括：
(1)感染或疑似感染新型冠状病毒的血浆、血清或全血；
(2)接种新型冠状病毒疫苗后的血浆、血清或全血样品；
(3)新型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新型冠状病毒的抗体样品。
本发明另一方面提供了上述的化学发光试剂盒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。
本发明又一方面提供了上述的化学发光试剂盒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。
本发明又一方面提供了上述化学发光试剂盒用于检测通过生物学方法获得的抗SARS-CoV-2或其变体的中和抗体的应用。
本发明还提供了上述化学发光试剂盒在检测受试者中是否存在SARS-CoV-2病毒或其变体的抗体中的应用。
本发明另一方面提供了上述化学发光试剂盒的使用方法，包括如下步骤：
(1)加稀释的样品、阳性质控品或阴性质控品至检测管中；
(2)向检测管中加入标记物偶联的新型冠状病毒刺突蛋白部分，混匀，温育；
(3)加入磁微粒偶联的ACE2蛋白或其功能片段，混匀，温育，磁分离，去上清，加入洗液，反复洗涤；
(4)加入化学发光底物，混匀，检测发光强度。
本发明又一方面提供上述化学发光试剂盒的使用方法，包括如下步骤：
(1)分别加稀释的样品、阳性质控品或阴性质控品至检测管中；
(2)向检测管中加入磁微粒偶联的ACE2蛋白或其功能片段；
(3)加入标记物偶联的新型冠状病毒刺突蛋白部分，混匀，温育，磁分离，去上清，加入洗液，反复洗涤；
(4)加入化学发光底物，混匀，检测发光强度。
本申请还提供了一种检测样品中是否存在新型冠状病毒或其变体的中和抗体的方法，包括：
(1)将样品与新型冠状病毒或其变体刺突蛋白或其ACE2结合片段接触；
(2)将步骤(1)的混合物与所述刺突蛋白或其ACE2结合片段特异结合的ACE2蛋白或其片段接触；
(3)检测所述刺突蛋白或其ACE2结合片段与所述ACE2蛋白或其片段结合的水平，其中，所述新型冠状病毒或其变体刺突蛋白或其ACE2结合片段连接标记物，所述ACE2蛋白或其片段连接磁微粒。
本申请又提供了一种检测样品中是否存在新型冠状病毒或其变体的中和抗体的方法，包括：
(1)将样品与新型冠状病毒或其变体刺突蛋白或其ACE2结合片段接触；
(2)再将上述新型冠状病毒或其变体刺突蛋白或其ACE2结合片段连接磁微粒；
(3)加入与所述刺突蛋白或其ACE2结合片段特异结合的ACE2蛋白或其片段；
(4)检测所述刺突蛋白或其ACE2结合片段与所述ACE2蛋白或其片段结合的水平，其中，所述ACE2蛋白或其片段连接标记物。
以上方法进一步包括，通过样品抑制或阻断新型冠状病毒或其变体刺突蛋白部分和ACE2蛋白或其功能片段的结合产生检测信号的变化，确定样品中是否存在新型冠状病毒或其变体的中和抗体。
发明详述
磁性微粒化学发光分析法是将磁分离技术、免疫分析技术及化学发光检测技术三者有效结合在一起的新型的分析检测技术。本发明公开了一种SARS-CoV-2病毒中和抗体磁微粒化学发光检测方法，能够快速有效的检测人和其他动物样本中的SARS-CoV-2病毒中和抗体，克服NT法和阻断ELISA法检测中和抗体的缺点，用于SARS-CoV-2病毒中和抗体的筛选和鉴定、评价疫苗的有效性以及对受试者样本中针对SARS-CoV-2病毒中和抗体进行定性或定量检测。
磁微粒化学发光试剂盒及其制备方法
基于磁性微粒化学发光分析法，本发明提供的一种检测样品SARS-CoV-2病毒或其变体的中和抗体的磁微粒化学发光检测试剂盒，包括：a.标记物偶联的SARS-CoV-2或其变体刺突蛋白部分；b.磁微粒偶联的ACE2蛋白或其功能片段；c.样本稀释液；d.阳性质控品；e.阴性质控品；f.洗液；和g.底物。
在一些实施方案中，所述SARS-CoV-2病毒或其变体中和抗体的磁微粒化学发光检测试剂盒包括：a.标记物偶联的SARS-CoV-2或其变体刺突蛋白部分；b.磁微粒偶联的ACE2蛋白或其功能片段；c.样本稀释液；d.阳性质控品；e.阴性质控品；f.洗液；和g.底物。
在另一些实施方案中，所述SARS-CoV-2病毒或其变体中和抗体的磁微粒化学发光检测试剂盒包括：a.标记物偶联的SARS-CoV-2或其变体RBD蛋白；b.磁微粒偶联的ACE2蛋白；c.样本稀释液；d.阳性质控品；e.阴性质控品；f.洗液；和g.底物。
在一些实施方案中，所述标记物选自碱性磷酸酶、吖啶酯、辣根过氧化物酶、三联吡啶钌、异鲁米诺或稀土元素中的一种或多种，优选为碱性磷酸酶、吖啶酯或辣根过氧化物酶。在一些具体实施方案中，所述标记物选自碱性磷酸酶、吖啶酯、辣根过氧化物酶或三联吡啶钌。在另一些具体实施方案中，所述标记物选自碱性磷酸酶、吖啶酯或辣根过氧化物酶。在一个具体实施方案中，所述标记物为碱性磷酸酶。在另一个具体实施方案中，所述标记物为吖啶酯。在又一个具体实施方案中，所述标记物为辣根过氧化物酶。在另一些实施方案中，所述标记物偶联的SARS-CoV-2或其变体RBD蛋白选自碱性磷酸酶偶联的SARS-CoV-2或其变体RBD蛋白、吖啶酯偶联的SARS-CoV-2或其变体RBD蛋白或者辣根过氧化物酶偶联的SARS-CoV-2或其变体RBD蛋白。
在一些实施方案中，所述标记物偶联的SARS-CoV-2或其变体RBD蛋白浓度为2-200ng/mL，优选为80ng/mL。在一些具体实施方案中，所述标记物偶联的SARS-CoV-2或其变体RBD蛋白浓度为2ng/mL、10ng/mL、20ng/mL、30ng/mL、40ng/mL、50ng/mL、60ng/mL、70ng/mL、80ng/mL、90ng/mL、100ng/mL、110ng/mL、120ng/mL、130ng/mL、140ng/mL、150ng/mL、160ng/mL、170ng/mL、180ng/mL、190ng/mL或200ng/mL。在一些 具体实施方案中，所述标记物偶联的SARS-CoV-2或其变体RBD蛋白浓度为50ng/mL。在另一个具体实施方案中，在一些具体实施方案中，所述标记物偶联的SARS-CoV-2或其变体RBD蛋白浓度为100ng/mL。
在一些实施方案中，所述标记物偶联的SARS-CoV-2或其变体RBD蛋白选自吖啶酯标记的SARS-CoV-2或其变体RBD蛋白或者碱性磷酸酶标记的SARS-CoV-2或其变体RBD蛋白。在另一些实施方案中，所述标记物偶联的SARS-CoV-2或其变体RBD蛋白为吖啶酯标记的SARS-CoV-2或其变体RBD蛋白。在一些具体实施方案中，所述吖啶酯标记的SARS-CoV-2或其变体RBD蛋白浓度为2-200ng/mL，优选为10-100ng/mL。在一些优选的实施方案中，所述吖啶酯标记的SARS-CoV-2或其变体RBD蛋白浓度10-100ng/mL。在另一些具体实施方案中，所述吖啶酯标记的SARS-CoV-2或其变体RBD蛋白浓度为2ng/mL、10ng/mL、20ng/mL、30ng/mL、40ng/mL、50ng/mL、60ng/mL、70ng/mL、80ng/mL、90ng/mL、100ng/mL、110ng/mL、120ng/mL、130ng/mL、140ng/mL、150ng/mL、160ng/mL、170ng/mL、180ng/mL、190ng/mL或200ng/mL。在一些实施方案中，所述标记物偶联的SARS-CoV-2或其变体RBD蛋白为碱性磷酸酶标记的SARS-CoV-2或其变体RBD蛋白。在一些具体实施方案中，所述碱性磷酸酶标记的SARS-CoV-2或其变体RBD蛋白浓度为2-200ng/mL,优选为80ng/mL。在另一些具体实施方案中，所述碱性磷酸酶标记的SARS-CoV-2或其变体RBD蛋白浓度为2ng/mL、10ng/mL、20ng/mL、30ng/mL、40ng/mL、50ng/mL、60ng/mL、70ng/mL、80ng/mL、90ng/mL、100ng/mL、110ng/mL、120ng/mL、130ng/mL、140ng/mL、150ng/mL、160ng/mL、170ng/mL、180ng/mL、190ng/mL或200ng/mL。在一个优选的实施方案中，所述碱性磷酸酶标记的SARS-CoV-2或其变体RBD蛋白浓度为50ng/mL。
在一些实施方案中，所述ACE2蛋白或其功能片段来源于人。在另一些实施方案中，所述ACE2蛋白或其功能片段选自重组ACE2蛋白或ACE2蛋白胞外域或带Fc标签ACE2蛋白或ACE2蛋白胞外域。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。在另一些实施方案中，ACE2蛋白或其功能片段包含SEQ ID NO:3和8-12中任一所示氨基酸序列，或与SEQ ID NO:3和8-12中任一所示氨基酸序列至少70％一致性的序列。在一些具体实施方案中，所述ACE2蛋白或其功能片段包含与SEQ ID NO:3和8-12中任一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ACE2蛋白或其功能片段包含SEQ ID NO:3和8-12中任一所示的氨基酸序列。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和8-12中所示任一序列。
在一些实施方案中，所述磁微粒偶联的ACE2蛋白或其功能片段中ACE2蛋白或其功能片段的浓度为2-15ug/mL，优选为5-10ug/mL，更优选为5ug/mL。在一些实施方案中，所述ACE2蛋白或其功能片段的浓度为5-10ug/mL。在一些具体实施方案中，所述ACE2蛋白或其功能片段的浓度为2ug/mL、3ug/mL、4ug/mL、5ug/mL、6ug/mL、7ug/mL、8ug/mL、9ug/mL、10ug/mL、11ug/mL、12ug/mL、13ug/mL、14ug/mL或15ug/mL。在一个具体实施方案中，所述ACE2蛋白或其功能片段的浓度为5ug/mL。
在另一些实施方案中，所述磁微粒的浓度为0.1-0.5mg/mL。在一些具体实施方案中，所述磁微粒的浓度为0.1mg/mL、0.2mg/mL、0.25mg/mL、0.3mg/mL、0.4mg/mL或0.5mg/mL。
本发明提供了一种SARS-CoV-2病毒中和抗体的磁微粒化学发光定性检测试剂盒，包括：a.标记物偶联的ACE2蛋白或其功能片段；b.磁微粒偶联的SARS-CoV-2或其变体刺突蛋白部分；c.样本稀释液；d.阳性质控品；e.阴性质控品；f.洗液；和g.底物。
在一些实施方案中，所述SARS-CoV-2病毒或其变体中和抗体的磁微粒化学发光定性检测试剂盒包括：a.标记物偶联的ACE2蛋白或其功能片段；b.磁微粒偶联的SARS-CoV-2刺突蛋白部分；c.样本稀释液；d.阳性质控品；e.阴性质控品；f.洗液；和g.底物。
在另一些实施方案中，所述SARS-CoV-2病毒或其变体中和抗体的磁微粒化学发光定性检测试剂盒包括：a.标记物偶联的ACE2蛋白；b.磁微粒偶联的SARS-CoV-2或其变体RBD蛋白；c.样本稀释液；d.阳性质控品；e.阴性质控品；f.洗液；和g.底物。
本发明提供了一种SARS-CoV-2病毒或其变体中和抗体的磁微粒化学发光定量检测试剂盒，包括：a.标记物偶联的ACE2蛋白或其功能片段；b.磁微粒偶联的SARS-CoV-2或其变体刺突蛋白部分；c.样本稀释液；d.校准品；e.洗液；和f.底物。
在一些实施方案中，所述SARS-CoV-2病毒或其变体中和抗体的磁微粒化学发光定量检测试剂盒包括：a.标记物偶联的ACE2蛋白或其功能片段；b.磁微粒偶联的SARS-CoV-2或其变体刺突蛋白部分；c.样本稀释液；d.校准品；e.洗液；和f.底物。
在另一些实施方案中，所述SARS-CoV-2病毒或其变体中和抗体的磁微粒化学发光定量检测试剂盒包括：a.标记物偶联的ACE2蛋白；b.磁微粒偶联的SARS-CoV-2 RBD或其变体蛋白；c.样本稀释液；d.校准品；e.洗液；和f.底物。
在一些实施方案中，所述标记物选自碱性磷酸酶、吖啶酯、辣根过氧化物酶、三联吡啶钌、异鲁米诺或稀土元素中的一种或多种，优选为碱性磷酸酶、吖啶酯或辣根过氧化物酶。在一些具体实施方案中，所述标记物选自碱性磷酸酶、吖啶酯、辣根过氧化物酶或三联吡啶钌。在另一些具体实施方案中，所述标记物选自碱性磷酸酶、吖啶酯或辣根过氧化物酶。在一个具体实施方案中，所述标记物为碱性磷酸酶。在另一个具体实施方案中，所述标记物为吖啶酯。在又一个具体实施方案中，所述标记物为辣根过氧化物酶。在另一些实施方案中，所述标记物偶联的ACE2蛋白选自碱性磷酸酶偶联的ACE2蛋白、吖啶酯偶联的ACE2蛋白或者辣根过氧化物酶偶联的ACE2蛋白。
在一些实施方案中，所述SARS-CoV-2或其变体的刺突蛋白部分包含SEQ ID NO:1、2和4-7中任一所示氨基酸序列，或与SEQ ID NO:1、2和4-7中任一所示氨基酸序列至少70％一致性的序列。在一些具体实施方案中，所述SARS-CoV-2或其突变刺突蛋白部分包含与SEQ ID NO:1、2和4-7中任一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。在另一些具体实施方案中，所述SARS-CoV-2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。在一些具体实施方案中，所述SARS-CoV-2变体刺突蛋白部分包含与SEQ ID NO:4-7任一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或其变体刺突蛋白部分包括SEQ ID NO:1、2和4-7任一所示的氨基酸序列。在另一些具体实施方案中，所述SARS-CoV-2变体刺突蛋白部分包括SEQ ID NO:4-7中任一所示的氨基酸序列。在一些具体实施方案中，所述SARS-CoV-2刺突蛋白部分包括SEQ ID NO:1或2所示的氨基酸序列。在一个具体实施方案中，所述SARS-CoV-2或其变体刺突蛋白部分的氨基酸序列如SEQ ID NO:1、2和4-7所示。在另一个具体实施方案中，所述新型冠状病毒刺突蛋白部分的氨基酸序列如SEQ ID NO:1或2所示。在一个具体实施方案中，所述新型冠状病毒变体刺突蛋白部分的氨基酸序列如SEQ ID NO4-7任一序列所示。
本发明上述化学发光试剂盒中的样品稀释液包括磷酸氢二钠、磷酸二氢钠、氯化钠、Proclin300、蔗糖、牛血清白蛋白或吐温20，pH值为7.0-7.5。在一些实施方案中，所述样品稀释液包括3.0-4.0mg/mL十二水合磷酸氢二钠、0.2-0.4mg/mL二水合磷酸二氢钠、7-9mg/mL氯化钠、10-30mg/mL蔗糖、5-20mg/mL牛血清白蛋白、Proclin300和吐温20，pH值为7.0-7.5。在一个具体实施方案中，所述样品稀释液包括3.5mg/mL十二水合磷酸氢二钠、0.26mg/mL二水合磷酸二氢钠、8.5mg/mL氯化钠、20mg/mL蔗糖、10mg/mL牛血清白蛋白、Proclin300和吐温20，pH值为7.0-7.5。
本发明上述化学发光试剂盒中的阴性质控品为样品稀释液。在一些实施方案中，所述阴性质控品包括磷酸氢二钠、磷酸二氢钠、氯化钠、Proclin300、蔗糖、牛血清白蛋白或吐温20，pH值为7.0-7.5。
本发明上述化学发光试剂盒中的洗液包括Tris、氯化钠和吐温-20，pH值为7.5-8.0。在一些实施方案中，所述洗液包括25-35mg/mLTris、180-250mg/mL氯化钠，吐温20，pH值为7.5-8.0。在一个具体实施方案中，所述洗液包括30.25mg/ml Tris、219.15mg/mL氯化钠，吐温20，pH值为7.5-8.0。
本发明上述化学发光试剂盒中的阳性质控品为抗SARS-CoV-2病毒或其变体的中和抗体。所述为抗SARS-CoV-2病毒或其变体的中和抗体可以是本领域已经公开的任意一种SARS-CoV-2病毒或其变体的中和抗体，即与SARS-CoV-2病毒或其变体的RBD蛋白结合能阻断病毒RBD与人ACE2受体结合从而防止细胞病毒侵入的抗体。本发明中包括但不限于实施例提供的克隆2B2抗体。
本发明上述化学发光试剂盒中的校准品为不同浓度的抗SARS-CoV-2病毒或其变体中和抗体。所述为抗SARS-CoV-2病毒或其变体的中和抗体可以是本领域已经公开的任意一种SARS-CoV-2病毒或其变体的中和抗体，即与SARS-CoV-2病毒的RBD蛋白结合能阻断病毒RBD与人ACE2受体结合从而防止细胞病毒侵入的抗体。本发明中包括但不限于实施例提供的A02087抗体。
本发明上述化学发光试剂盒中的底物可以是直接化学发光的酸碱发光启动剂和间接化学发光底物，包括但不限于直接化学发光的酸碱发光启动剂如含H 2O 2的酸(HNO 3或HCl等)和NaOH，间接化学发光底物包括碱性磷酸酶底物如AMPPD、APS5、CSPD、CDP-Star和辣根过氧化物酶底物如鲁米诺和PS-atto等。
本发明上述化学发光试剂盒的制备方法包括如下步骤：
(1)制备标记物偶联的新型冠状病毒或其变体刺突蛋白部分；
(2)制备磁微粒偶联的ACE2蛋白；
(3)制备样品稀释液、阴阳性质控品或校准品和洗液；
(4)将上述试剂包装入试剂盒。
其中，所述步骤(1)标记物偶联新型冠状病毒或其变体刺突蛋白部分包括标记物偶联的SARS-CoV-2或其变体RBD蛋白，包括碱性磷酸酶标记SARS-CoV-2 RBD蛋白、吖啶酯标记SARS-CoV-2 RBD蛋白、碱性磷酸酶标记SARS-CoV-2 B.1.1.7 RBD蛋白、吖啶酯标记SARS-CoV-2 B.1.1.7 RBD蛋白、碱性磷酸酶标记SARS-CoV-2 B.1.351 RBD蛋白或吖啶酯标记SARS-CoV-2 B.1.351 RBD蛋白。在一些实施方案中，所述碱性磷酸酶偶联SARS-CoV-2或其变体RBD蛋白制备方法包括：(1)分别活化SARS-CoV-2或其变体RBD蛋白和碱性磷酸酶；(2)分别通过旋转脱盐柱对活化的SARS-CoV-2或其变体RBD蛋白和碱性磷酸酶置换缓冲液；(3)将步骤(2)得到的碱性磷酸酶与SARS-CoV-2或其变体RBD蛋白溶液混匀后反应，加入N-乙基马来酰亚胺溶液静置；(4)将步骤(3)得到的反应物通过旋转脱盐柱置换缓冲液，得到AP偶联的SARS-CoV-2或其变体RBD蛋白。在另一些实施方案中，所述吖啶酯偶联SARS-CoV-2或其变体RBD蛋白制备方法包括：(1)活化SARS-CoV-2或其变体RBD蛋白，旋转脱盐柱置换缓冲液；(2)加入吖啶酯溶液混匀避光反应；(3)步骤(2)反应后溶液中加入甘氨酸，旋转脱盐柱置换缓冲液得到吖啶酯偶联的SARS-CoV-2或其变体RBD蛋白。
其中，步骤(2)磁微粒偶联ACE2蛋白的制备包括：(1)用缓冲液重悬磁微粒，加入ACE2蛋白，混匀，恒温反应；(2)磁分离去上清，加入封闭液，恒温混匀反应；(3)磁分离去上清，重悬磁微粒，反复洗涤；(4)加入样品稀释液重悬磁微粒成单颗粒状态。
本发明提供的检测SARS-CoV-2病毒或其变体的中和抗体磁微粒化学发光试剂盒的原理为：通过SARS-CoV-2病毒或其变体的中和抗体阻断磁微粒偶联SARS-CoV-2或其变体RBD蛋白(或ACE2受体)和标记物偶联ACE2受体(或SARS-CoV-2或其变体RBD蛋白)相互作用产生检测信号。在待测样品和阴性质控品先后或同时加入标记物偶联ACE2受体(或SARS-CoV-2或其变体RBD蛋白)和磁微粒偶联SARS-CoV-2或其变体RBD蛋白(或ACE2受体)。当样本中不存在SARS-CoV-2病毒或其变体的中和抗体时，磁微粒偶联的SARS-CoV-2或其变体RBD蛋白(或ACE2受体)和标记物偶联的ACE2受体(或SARS-CoV-2 RBD蛋白)结合。通过磁分离洗涤后，结合在磁微粒上的标记物在底物存在下产生检测信号。当样本中存在SARS-CoV-2病毒或其变体中和抗体时，磁微粒偶联的SARS-CoV-2或其变体RBD蛋白(或ACE2受体)和标记物偶联ACE2受体(或SARS-CoV-2或其变体RBD蛋白)的结合量与样本中的SARS-CoV-2病毒或其变体中和抗体浓度呈反比。通过磁分离洗涤后磁微粒上带有的标记物减少，产生的信号减弱。通过化学发光仪分别读取待测样本值(S)和阴性样本测量值(N)，用阻断率(1-S/N)评估样本中中和抗体的含量。 但阻断率≥30％,说明检测样本中存在SARS-CoV-2病毒或其变体的中和抗体。当阻断率<30％,说明检测样本中不存在SARS-CoV-2病毒或其变体的中和抗体。
磁微粒化学发光试剂盒的使用方法
本发明另一方面提供了上述化学发光试剂盒的使用方法，包括如下步骤：
(1)分别加稀释的样品液、质控品或校准品至检测管中；
(2)向检测管中加入标记物偶联的新型冠状病毒或其变体刺突蛋白部分，混匀，温育；
(3)加入磁微粒偶联的ACE2蛋白或其功能片段，混匀，温育，磁分离，去上清，加入洗液，反复洗涤；
(4)加入化学发光底物，混匀，检测发光强度。
本发明又一方面提供上述化学发光试剂盒的另一种使用方法，包括如下步骤：
(1)分别加稀释的样品液、质控品或校准品至检测管中；
(2)向检测管中加入磁微粒偶联的ACE2蛋白或其功能片段；
(3)加入标记物偶联的新型冠状病毒或其变体刺突蛋白部分，混匀，温育，磁分离，去上清，加入洗液，反复洗涤；
(4)加入化学发光底物，混匀，检测发光强度。
本发明还提供了上述化学发光试剂盒的使用方法，包括如下步骤：
(1)分别加稀释的样品液、阳性质控品或阴性质控品至检测管中；
(2)向检测管中加入标记物偶联的ACE2蛋白；
(3)加入磁微粒偶联的新型冠状病毒或其变体刺突蛋白部分，混匀，温育，磁分离，去上清，加入洗液，反复洗涤；
(4)加入化学发光底物，混匀，检测发光强度。
本发明还提供上述化学发光试剂盒的另一种使用方法，包括如下步骤：
(1)分别加稀释的样品液、阳性质控品或阴性质控品至检测管中；
(2)向检测管中加入磁微粒偶联的新型冠状病毒或其变体的刺突蛋白部分；
(3)加入标记物偶联的ACE2蛋白，混匀，温育，磁分离，去上清，加入洗液，反复洗涤；
(4)加入化学发光底物，混匀，检测发光强度。
磁微粒化学发光试剂盒的应用
本发明提供了上述化学发光检测试剂盒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。
本发明提供了上述化学发光试剂盒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，本发明提供了上述化学发光试剂盒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。例如，所述抗体包括人抗SARS-CoV-2病毒中和抗体、小鼠抗SARS-CoV-2病毒中和抗体、大鼠抗SARS-CoV-2病毒中和抗体、兔抗SARS-CoV-2病毒中和抗体，骆驼抗SARS-CoV-2病毒中和抗体、抗SARS-CoV-2病毒中和抗体Fab片段和抗SARS-CoV-2病毒中和抗体的ScFv片段、从噬菌体抗体库筛选技术筛选的SARS-CoV-2病毒中和抗体、从高通量DNA测序技术获得的SARS-CoV-2病毒中和抗体样品、杂交瘤技术制备的SARS-CoV-2病毒中和抗体和从血清中分离出来的抗体等。
再一方面，本发明的试剂盒可用于检测受试者是否存在SARS-CoV-2病毒中和抗体。其中，所述受试者包括健康的受试者、曾经感染过新型冠状病毒已恢复健康的受试者或未表现任何症状的感染者。检测受试者的样品包括但不限于血清、血浆或全血。
本发明提供一种检测样品中是否存在新型冠状病毒或其变体的中和抗体的方法，包括：
(1)将样品与SARS-CoV-2病毒或其变体刺突蛋白或其ACE2结合片段接触；
(2)再将上述SARS-CoV-2病毒或其变体刺突蛋白或其ACE2结合片段连接至固相载体上；
(3)加入与所述刺突蛋白或其ACE2结合片段特异结合的ACE2蛋白或其片段；
(4)检测所述刺突蛋白或其ACE2结合片段与所述ACE2蛋白或其片段结合的水平，其中，所述ACE2蛋白或其片段连接标记物。
在一些实施方案中，所述步骤(1)中的SARS-CoV-2病毒或其变体刺突蛋白或其ACE2结合片段连接生物素，所述步骤(2)中固相载体上偶联链霉亲和素，SARS-CoV-2病毒或其变体刺突蛋白或其ACE2结合片段通过生物素与固相载体上链霉亲和素结合连接至固相载体上。其中，所述固相载体为磁微粒。
本发明又提供了一种检测样品中是否存在SARS-CoV-2病毒或其变体的中和抗体的方法，
包括：
(1)SARS-CoV-2病毒或其变体刺突蛋白或其ACE2结合片段连接磁微粒，通过磁微粒的量或/和SARS-CoV-2病毒或其变体刺突蛋白或其ACE2结合片段的量控制连接磁微粒的SARS-CoV-2病毒或其变体刺突蛋白或其ACE2结合片段的量；
(2)将样品与连接磁微粒的SARS-CoV-2病毒或其变体刺突蛋白或其ACE2结合片段接触；
(3)加入与所述刺突蛋白或其ACE2结合片段特异结合的ACE2蛋白或其片段；
(4)检测所述刺突蛋白或其ACE2结合片段与所述ACE2蛋白或其片段结合的水平，其中，所述ACE2蛋白或其片段连接标记物。
在一些实施方案中，所述磁微粒的量可以通过控制磁微粒的浓度来调节。在另一些实施方案中，所述SARS-CoV-2病毒或其变体刺突蛋白或其ACE2结合片段的量可以通过控制加入的SARS-CoV-2病毒或其变体刺突蛋白或其ACE2结合片段的浓度来调节，也可以通过控制磁微粒单位表面积上连接SARS-CoV-2病毒或其变体刺突蛋白或其ACE2结合片段的密度来调节。通过控制磁微粒或SARS-CoV-2病毒或其变体刺突蛋白或其ACE2结合片段的量控制SARS-CoV-2病毒或其变体刺突蛋白或其ACE2结合片段相对于样本中抗体的摩尔比，达到较佳的中和抗体检测范围，提高检测方法的灵敏度和特异性。
本发明还提供了一种检测样品中是否存在新型冠状病毒或其变体的中和抗体的方法，包括：
(1)将样品与新型冠状病毒或其变体刺突蛋白或其ACE2结合片段接触；
(2)将步骤(1)的混合物与所述刺突蛋白或其ACE2结合片段特异结合的ACE2蛋白或其片段接触；
(3)检测所述刺突蛋白或其ACE2结合片段与所述ACE2蛋白或其片段结合的水平，其中，所述新型冠状病毒或其变体刺突蛋白或其ACE2结合片段连接标记物，所述ACE2蛋白或其片段连接固相载体。
在一些实施方案中，所述固相载体为磁微粒。
以上所述检测新型冠状病毒或其变体的中和抗体的方法，进一步包括通过样品抑制或阻断新型冠状病毒或其变体刺突蛋白部分和ACE2蛋白或其功能片段的结合产生检测信号的变化，确定样品中是否存在新型冠状病毒或其变体的中和抗体。
术语解释
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。SARS-CoV-2突变体可以为任何突变株，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SARS-CoV-2突变株。
“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-2 S1亚基或SARS-CoV-2 S RBD抗原。本发明中所述SARS-CoV-2 S 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-2 S 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-2 S 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，如ACE2的胞外域或带Fc标签的ACE2胞外域。
本发明中“新型冠状病毒或其变体刺突蛋白部分”或“新型冠状病毒或其变体刺突蛋白或其ACE2结合片段”是指包括新型冠状病毒或其变体能发挥功能的刺突蛋白部分，能特异性结合ACE2蛋白或ACE2蛋白胞外域。可以是新型冠状病毒或其变体的RBD蛋白，新型冠状病毒或其变体的S1蛋白和S蛋白。优选地为SARS-CoV-2病毒或其变体RBD蛋白，SARS-CoV-2病毒或其变体S1蛋白和SARS-CoV-2病毒或其变体S蛋白，更优选为SARS-CoV-2病毒或其变体RBD蛋白或SARS-CoV-2病毒或其变体S1蛋白。
本文术语“SARS-CoV-2病毒或其变体RBD蛋白”，可以体现为重组SARS-CoV-2病毒或其变体RBD蛋白(如SEQ ID NO:1、4或6所示序列)，带有His标签的SARS-CoV-2病毒或其变体RBD蛋白，带有Fc标签的SARS-CoV-2病毒或其变体RBD蛋白；包含有SARS-CoV-2病毒或其变体RBD蛋白的分子，比如SARS-CoV-2病毒或其变体S1亚基(如SEQ ID NO:2，5或7所示序列)和SARS-CoV-2病毒或其变体S蛋白。其中，所述SARS-CoV-2病毒变体可以是B.1.1.7、B.1.351、B.1.617、B.1.617.1、B.1.617.2、B.1.617.3、或任何新出现的SARS-CoV-2突变株。
术语“磁微粒”包括可不限于对甲苯磺酰基修饰的磁珠(Tosyl磁珠)、氨基修饰的磁珠和羧基修饰的磁珠。
本文术语“标记物”可以指辣根过氧化物酶(HRP)、碱性磷酸酶(AP)、吖啶酯、异鲁米诺或稀土元素至少一种。标记物与发光底物作用后产生化学发光，如鲁米诺受HRP催化反应后发生化学发光，可用化学发光仪进行检测。
本发明中的“磁微粒偶联物”或“磁微粒标记物”可以指新型冠状病毒刺突蛋白部分偶联磁微粒或ACE2蛋白偶联磁微粒，也即新型冠状病毒刺突蛋白部分共价偶联了磁微粒或ACE2蛋白共价偶联了磁微粒。在本发明中具体包括磁微粒偶联的SARS-Cov-2的RBD蛋白、磁微粒偶联的SARS-Cov-2的S1蛋白或磁微粒偶联的ACE2蛋白。
本发明中的“标记物偶联物”或“标记物连接物”可以指新型冠状病毒刺突蛋白部分或ACE2蛋白或其功能片段偶联上标记物，包括但不限于HRP偶联的新型冠状病毒刺突蛋白部分、AP偶联的新型冠状病毒刺突蛋白部分、吖啶酯偶联的新型冠状病毒刺突蛋白部分、HRP偶联的ACE2蛋白、AP偶联的ACE2蛋白和吖啶酯的偶联ACE2蛋白。
本发明中的“偶联物”是指通过共价键将冠状病毒刺突蛋白部分或ACE2蛋白标记到磁微粒上形成的磁微粒偶联物，可以指通过共价键将冠状病毒刺突蛋白部分或ACE2蛋白标记到标记物上形成的标记物偶联物。
本发明“连接标记物或磁微粒”中“连接”是指通过接头或化学键直接偶联连接在蛋白的C端或N端，可选自小分子化学接头或肽接头。所述小分子化学接头包括但不限羧基、氨基和巯基等，所述肽接头可选自1个或多个氨基酸的多肽，可通过接头将标记物或磁微粒连接到新型冠状病毒的刺突蛋白部分或ACE2蛋白或其功能片段。其中，所述偶联方式连接通过形成共价键连接，可通过标记物或磁微粒与新型冠状病毒刺突蛋白部分或ACE2蛋白或其功能片段共价反应形成共价键连接。
本发明中“接头”是指能将蛋白与标记物或磁微粒连接起来的化合物，可以是肽接头、小分子化学接头或多聚物分子接头。其中，小分子化学接头包括但不限于羧基、氨基和巯基；肽接头可以是至多20个、至多10个、至多8个、至多7个、至多6个、至多5个、至多4个、至多3个、至多2个或1个氨基酸的多肽。
术语“化学发光底物”包括但不限于直接化学发光的酸碱发光启动剂如含H2O2的酸(HNO 3或HCl等)和NaOH，间接化学发光包括碱性磷酸酶底物如AMPPD、APS5、CSPD、CDP-Star和辣根过氧化物酶底物如鲁米诺和PS-atto等。
本发明中的“化学发光仪”由免疫反应系统和化学发光分析系统两个部分组成。化学发光分析系统是利用发光信号测量仪器测量化学发光物质经催化和氧化产生的光量子信号。免疫反应系统是将化学发光物质或酶直接标记在抗原或抗体(免疫化学发光分析)上。它包括化学发光酶免疫分析和电化学发光分析仪等。
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)感染或疑似感染新型冠状病毒的血浆、血清或全血；(2)接种新型冠状病毒疫苗后的血浆、血清或全血；(3)新型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新型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，如小鼠、大鼠、兔的血浆、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
目前市场上没有SARS-CoV-2中和抗体磁微粒化学发光检测试剂盒产品出售。利用中和抗体能够阻断新型冠状病毒刺突蛋白部分与ACE2蛋白或其功能片段结合的特性，开发用于检测SARS-CoV-2病毒中和抗体的磁微粒化学发光试剂盒的方法比较新颖，尚为被开发利用。磁微粒化学发光检测SARS-CoV-2中和抗体在SARS-CoV-2病毒辅助诊断、抗体药物筛选和疫苗评价的价值尚未被开发。本发明提供的SARS-CoV-2中和抗体阻断磁微粒化学发光检测试剂盒通过化学发光信号的抑制率检测样本中的SARS-CoV-2病毒中和抗体，该方法具有检测通量大、自动化程度高、准确性高、特异性强，检测速度快、操作简便等特点。
附图说明
图1为SARS-CoV-2病毒中和抗体磁微粒化学发光试剂盒盒的原理图；
图2为SARS-CoV-2病毒中和抗体碱性磷酸酶磁微粒化学发光检测试剂盒分析4株SARS-CoV-2抗体阻断效果图。
图3为SARS-CoV-2病毒中和抗体碱性磷酸酶磁微粒化学发光检测试剂盒测试30例阴性样本和20例阳性样本的结果图。
图4为SARS-CoV-2病毒及突变体B.1.1.7和B.1.351中和抗体碱性磷酸酶磁微粒化学发光检测试剂盒的校准曲线图。
图5为SARS-CoV-2病毒中和抗体碱性磷酸酶磁微粒化学发光检测试剂盒与商品化试剂盒cPass TMSARS-CoV-2中和抗体检测试剂盒(FDAEUA)测试40例样本的相关性图。
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病毒中和抗体碱性磷酸酶磁微粒化学发光试剂盒制备及性能评估
1.1试剂盒组成
本试剂盒由以下7个试剂组成:
a.AP偶联SARS-CoV-2 RBD蛋白；
b.磁微粒偶联ACE2；
c.样本稀释液；
d.阳性质控品；
e.阴性质控品；
f.洗液；
g.化学发光底物；
1.2 AP偶联SARS-CoV-2 RBD蛋白的制备
1)向2mLEP管中加入1mL的1mg/mL重组SARS-CoV-2 RBD蛋白(金斯瑞，Cat No.T80302)溶液。
2)向上述EP管中加入60ul的1.376mg/mL的Traut's Reagent(Thermo，Cat No.26101)溶液，混合均匀后室温静置20min。
3)向另一支2mL的EP管中加入600ul的浓度为10mg/mL的碱性磷酸酶溶液。
4)向上述装有碱性磷酸酶溶液的EP管中加入11ul的17.5mg/mL的(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐(Thermo，Cat No.22322)溶液，混匀后室温静置20min。
5)向活化后的重组SARS-CoV-2 RBD蛋白溶液加入l M的甘氨酸溶液6-24uL，混匀后室温静置10min。
6)向活化后的碱性磷酸酶溶液加入lM的甘氨酸溶液4.4-8.8uL，混匀后室温静置10min。
7)用Zeba TM旋转脱盐柱(Thermo，Cat No.89891)将步骤5)所得蛋白置换缓冲液 (0.1M三乙醇胺；5mM Mg 2+；1mM Zn 2+；pH7.2-7.4)，收集活化后的重组SARS-CoV-2 RBD蛋白溶液。
8)用Zeba TM旋转脱盐柱(Thermo，Cat No.89882)将步骤6)所得碱性磷酸酶置换缓冲液(0.1M三乙醇胺；5mM Mg 2+；1mM Zn 2+；pH7.2-7.4)，收集活化后的碱性磷酸酶溶液。
9)取4-6mg的上述碱性磷酸酶(AP)溶液，加入到1mg的重组SARS-CoV-2 RBD蛋白溶液中，混匀后置于2-8℃静置反应18-24h；
10)向AP偶联SARS-CoV-2 RBD蛋白反应物中加入10-15ul的12.5mg/mL的N-乙基马来酰亚胺溶液，室温静置30min。
11)用Zeba TM旋转脱盐柱(Thermo，Cat No.89891)置换缓冲液纯化(0.1M三乙醇胺；5mM Mg 2+；1mM Zn 2+；0.05％ProClin300(v/v)；pH7.2-7.6)纯化，获得AP偶联SARS-CoV-2 RBD浓缩液。
12)用步骤1.4制备的稀释液稀释上述步骤得到的AP偶联SARS-CoV-2 RBD浓缩液至2-200ng/mL，得到AP偶联SARS-CoV-2 RBD蛋白。
1.3磁微粒偶联ACE2的制备
1)取500ul浓度为100mg/mL的磁微粒(JSR，MS300/Tosyl)放入10mL的离心管中，磁分离去上清。
2)用5mL的硼酸缓冲液(0.1mol/LpH 9.2)重悬磁微粒，向离心管中加入1mg的ACE2蛋白(金斯瑞，Cat No.Z03484)，37℃恒温混匀反应16-24h。
3)磁分离去上清，向离心管中加入50ul封闭液(10％(w/v)BSA)，37℃恒温混匀反应6h以上。
4)磁分离去上清，用5mL的1*TBST(10mM Tris；150mMNaCl；0.05％Tween-20(v/v)；pH 7.4-7.5)重悬磁微粒，混匀1-5min，磁分离去上清。重复洗涤步骤2-3次。
5)加入10mL步骤1.4制备的稀释液，震荡混匀1-5min，将磁微粒重悬到5mg/mL(ACE2含量100ug/mL)，使用超声振荡仪将混悬液中聚集成团的磁珠分散成单颗粒状态，得到磁分离试剂浓缩液。
6)用磁分离试剂稀释液将上述液体稀释成磁微粒浓度0.1-0.5mg/mL，ACE2浓度2-10ug/mL的磁微粒偶联ACE2。
1.4样本稀释液的制备
加800mL纯化水、3.5g的十二水合磷酸氢二钠和0.26g的二水合磷酸二氢钠到容器中，搅拌混合均匀，再加8.5g的氯化钠和0.3mL的Proclin300，搅拌至完全溶解，pH值为7.0-7.5，加20g的蔗糖搅拌至完全溶解，再加0.5mL的吐温20和10g的牛血清白蛋白到 容器中，搅拌至完全溶解，用纯化水定容至1L，使用0.22μm滤器过滤，将获得样本稀释液保存在2-8℃待用。
1.5阳性质控品制备
用上述步骤制备的样本稀释液将抗SARS-CoV-2病毒中和抗体(金斯瑞，克隆2B2)配制为200ng/mL的阳性质控品。阳性质控品由金斯瑞Cpass TM试剂盒(Cat No.L00847)确认为中和抗体。
1.6阴性质控品制备
阴性质控品为本实施例1.4中制备的样本稀释液。
1.7洗液的制备
加800mL纯化水、30.25g的Tris和219.15g的NaCl于1L容器中，充分搅拌至完全溶解；再加入12.5mL的吐温-20，充分搅拌，直至完全混匀，调pH值控制在7.5-8.0，定容1L，用0.2μm过滤器过滤，室温进行保存，使用前将所述清洗液按照每40mL加纯化水至1L混合混匀。
2试剂盒的操作
1)加50uL阳性质控品、50uL阴性质控品或50uL的1:10稀释后的血清或血浆样本检测管中。
2)加50uL的AP偶联SARS-CoV-2 RBD蛋白所述检测管中，混匀后，37℃温育30min。
3)加50uL的磁微粒偶联ACE2蛋白到检测管中，混匀后，37℃温育15min，进行磁分离，去上清。
4)加300uL的清洗液至检测管中，混匀，进行磁分离去上清。重复洗涤步骤两遍。
5)加150uL的化学发光底物(北京爱维德生物技术有限公司，Cat No.APSUB-1)至检测管中，混匀，用化学发光仪检测发光强度。
6)结果计算[见式(1)-式(3)]：
Figure PCTCN2021100834-appb-000001
N2：阴性对照2的检测值。
Figure PCTCN2021100834-appb-000002
P2：阳性对照2的检测值：
Figure PCTCN2021100834-appb-000003
S：样本检测值
7)试验成立条件：标准阳性对照的抑制率≥30％,该实验结果成立，若试验不成立，应进行重复检测。
8)如果被检测样本的抑制率≥30％，则该样本可以判为阳性。如被检测样本的抑制率＜30％，则该样本可以判断为阴性。
3 SARS-CoV-2抗体阻断效果评估
1)用SARS-CoV-2病毒中和抗体碱性磷酸酶磁微粒化学发光试剂盒分析4株SARS-CoV-2单抗(SARS-CoV-2 mAb-1/-2/-3/-4，抗体由金斯瑞制备)。
2)用样本稀释液将SARS-CoV-2 mAb-1稀释成“50000pM”、“16667pM”、“5556pM”、“1852pM”、“617pM”、“206pM”和“23pM”7个浓度梯度样品。
3)按3.2)操作步骤，制备SARS-CoV-2 mAb -2/-3/-4抗体不同浓度梯度样品。
4)加50uL阳性质控品、50uL阴性质控品或50uL步骤3.1-3.2制备的样品至检测管中。
5)加50uL的AP偶联SARS-CoV-2 RBD蛋白所述检测管中，混匀后，37℃温育30min。
6)加50uL的磁微粒偶联ACE2到检测管中，混匀后，37℃温育15min，进行磁分离，去上清。
7)加300uL的清洗液至检测管中，混匀，进行磁分离去上清。重复洗涤步骤两遍。
8)加150uL的化学发光底物(北京爱维德生物技术有限公司，Cat No.APSUB-1)至检测管中，混匀，用化学发光仪检测发光强度。
9)根据下列公式计算步骤3.2-3.3制备的每个样本的抑制率。[见式(1)-式(3)]：
Figure PCTCN2021100834-appb-000004
N2：阴性对照2的检测值。
Figure PCTCN2021100834-appb-000005
P2：阳性对照2的检测值：
Figure PCTCN2021100834-appb-000006
S：样本检测值
使用GraphPad Prism 6，以抗体浓度对数为横坐标，以抑制率为纵坐标作图，见图2，结果表明SARS-CoV-2 mAb-1对AP偶联标记SARS-CoV-2 RBD蛋白和磁微粒偶联ACE2的结合阻断作用不显著。SARS-CoV-2 mAb-2、SARS-CoV-2 mAb-3和SARS-CoV-2 mAb-4抗体能够显著阻断AP偶联标记SARS-CoV-2 RBD蛋白和磁微粒偶联ACE2的结合作用。其中SARS-CoV-2 mAb-4在低浓度下就可以阻断AP偶联标记SARS-CoV-2 RBD蛋白和磁微粒偶联ACE2的结合作用。
4试剂盒的性能评估
4.1)重复性评价
取本实施例中的试剂盒重复检测浓度为200±40ng/mL和500±100ng/mL的样品各10次，计算10次测量结果的平均值Μ和标准差SD，根据公式CV＝SD/M×100％得出变异系数CV，本方法变异系数(CV)不大于10％。
4.2)批间差评价
将实施例的试剂盒取三批，每批试剂盒均测定浓度在200±40ng/mL和500±100mg/mL范围内的样品，每批重复测定10次，计算30次测定结果的平均值(Μ)和标准差(SD)，根据公式CV＝SD/M×100％计算变异系数(CV)，本方法变异系数(CV)不大于15％。
4.3)特异性评估
通过SARS-CoV-2病毒中和抗体碱性磷酸酶磁微粒化学发光试剂盒检测30例不含中和抗体的样本，结果均为阴性(抑制率<30％为阴性)，见图3。
4.4)灵敏性评估
通过SARS-CoV-2病毒中和抗体碱性磷酸酶磁微粒化学发光试剂盒检测20例含不同浓度抗SARS-CoV-2中和抗体的阳性样本(由金斯瑞Cpass TM试剂盒(Cat No.L00847)检测确认为阳性的)，该样本是由5B7D7(金斯瑞，货号A02056)、6D11F2(金斯瑞，货号A02055)、R2B2(金斯瑞，货号A02051)、11D5D3(金斯瑞制备)用样本稀释液配制成39、78、156、313和625ng/ml的溶液。磁微粒化学发光试剂盒的检测结果均为阳性(抑制率≥30％为阳性)，见图3。
实施例2抗SARS-CoV-2病毒及突变株B.1.1.7和B.1.351中和抗体碱性磷酸酶磁微粒化学发光定量试剂盒制备及性能评估
1.1试剂盒组成
本试剂盒由以下8个试剂组成:
a.AP偶联SARS-CoV-2 RBD重组蛋白；
b.AP偶联SARS-CoV-2突变株(B.1.1.7)RBD重组蛋白
c.AP偶联SARS-CoV-2突变株(B.1.351)RBD重组蛋白
d.磁微粒偶联重组人ACE2；
e.样本稀释液；
f.校准品；
g.洗液；
h.化学发光底物；
1.2 AP偶联SARS-CoV-2 RBD及其变体的制备
同实施例1中1.2的方法，制备AP偶联SARS-CoV-2 RBD及其变体，具体方法如下：
1)向2mLEP管中加入1mL的1mg/mL重组SARS-CoV-2 RBD蛋白(金斯瑞，Cat No.T80302)、SARS-CoV-2突变株(B.1.1.7)RBD重组蛋白(金斯瑞，Cat No.Z03533)或SARS-CoV-2突变株(B.1.351)RBD重组蛋白(金斯瑞，Cat No.Z03537)溶液。
2)向上述EP管中加入60ul的1.376mg/mL的Traut's Reagent(Thermo，Cat No.26101)溶液，混合均匀后室温静置20min。
3)向另一支2mL的EP管中加入600ul的浓度为10mg/mL的碱性磷酸酶溶液。
4)向上述装有碱性磷酸酶溶液的EP管中加入11ul的17.5mg/mL的(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐(Thermo，Cat No.22322)溶液，混匀后室温静置20min。
5)向活化后的重组SARS-CoV-2及其突变体的RBD蛋白溶液加入lM的甘氨酸溶液6-24uL，混匀后室温静置10min。
6)向活化后的碱性磷酸酶溶液加入lM的甘氨酸溶液4.4-8.8uL，混匀后室温静置10min。
7)用Zeba TM旋转脱盐柱(Thermo，Cat No.89891)将步骤5)所得蛋白置换缓冲液(0.1M三乙醇胺；5mM Mg 2+；1mM Zn 2+；pH7.2-7.4)，收集活化后的重组SARS-CoV-2 RBD、SARS-CoV-2突变株(B.1.1.7)RBD重组蛋白或SARS-CoV-2突变株(B.1.351)RBD重组蛋白蛋白溶液。
8)用Zeba TM旋转脱盐柱(Thermo，Cat No.89882)将步骤6)所得碱性磷酸酶置换缓冲液(0.1M三乙醇胺；5mM Mg 2+；1mM Zn 2+；pH7.2-7.4)，收集活化后的碱性磷酸酶溶液。
9)取4-6mg的上述碱性磷酸酶(AP)溶液，加入到1mg的重组SARS-CoV-2 RBD、SARS-CoV-2 B.1.1.7 RBD或SARS-CoV-2 B.1.351 RBD重组蛋白溶液中，混匀后置于2-8℃静置反应18-24h；
10)向AP偶联SARS-CoV-2 RBD、SARS-CoV-2 B.1.1.7 RBD或SARS-CoV-2 B.1.351 RBD重组蛋白反应物中加入10-15ul的12.5mg/mL的N-乙基马来酰亚胺溶液，室温静置30min。
11)用Zeba TM旋转脱盐柱(Thermo，Cat No.89891)置换缓冲液纯化(0.1M三乙醇胺；5mM Mg 2+；1mM Zn 2+；0.05％ProClin300(v/v)；pH7.2-7.6)纯化，获得AP偶联SARS-CoV-2 RBD、SARS-CoV-2 B.1.1.7 RBD或SARS-CoV-2 B.1.351 RBD重组蛋白浓缩液。
12)用步骤1.6制备的稀释液稀释上述步骤得到的AP偶联SARS-CoV-2 RBD或其变体的浓缩液至50ng/mL，得到试剂盒中的AP偶联SARS-CoV-2或其变体的RBD蛋白。
1.3磁微粒偶联人ACE2的制备
1)取500ul浓度为100mg/mL的磁微粒(JSR，MS160/Carboxyl)放入10mL的离心管中，磁分离去上清。
2)用5mL的MES缓冲液(0.1mol/LpH 5.0)重悬磁微粒，混匀1-5min，磁分离去上清。重复洗涤步骤1次。
3)向离心管中加入3.2mLMES缓冲液(0.1mol/LpH 5.0)重悬磁微粒，混匀1-5min。
4)向上述离心管中加入10mg/ml的NHS(Thermo，货号24500)溶液1.15ml和10mg/ml的EDC(Thermo，货号22980)溶液0.5ml，室温混匀反应30min。
5)反应结束后，磁分离弃上清，加入4.35mLMES缓冲液(0.1mol/L pH 5.0)重悬磁微粒，混匀1-5min。
6)向上述离心管中加入1mg的ACE2胞外域-Fc蛋白(金斯瑞，Cat No.Z03484)，室温混匀反应3-20h。
7)向离心管中加入250ul封闭液(JSR，CE210)，室温混匀反应18-24h。
8)磁分离去上清，用5mL的洗液(25mM Tris；150mM NaCl；0.1％BSA；0.05％Tween-20(v/v)；0.1％ProClin300；pH 7.4-7.5)重悬磁微粒，混匀1-5min，磁分离去上清。重复洗涤步骤2-3次。
9)加入10mL步骤1.4制备的稀释液，震荡混匀1-5min，将磁微粒重悬到5mg/mL(ACE2含量100ug/mL)，使用超声振荡仪将混悬液中聚集成团的磁珠分散成单颗粒状态，得到磁分离试剂浓缩液。
10)用磁分离试剂稀释液将上述液体稀释成磁微粒浓度0.25mg/mL，ACE2浓度5ug/mL的磁微粒偶联ACE2。
1.4样本稀释液的制备
制备方法和组分同实施例1的1.4制备步骤和组分。
1.5校准品制备
用上述步骤制备的样本稀释液将抗SARS-CoV-2病毒中和抗体标准品(金斯瑞，货号A02087)配制为系列浓度点：6000U/ml、3000U/ml、1000U/ml、400U/ml、150U/ml和0U/ml。
1.6洗液的制备
洗液的制备方法和组分同实施例1的1.7制备步骤和组分。
2试剂盒的操作
准备3组25uLSARS-CoV-2病毒中和抗体校准品到检测管中，再加入25uL样本稀释液。向3组检测管中分别加50uL上述制备的AP偶联的SARS-CoV-2 RBD、SARS-CoV-2突变株(B.1.1.7)RBD和SARS-CoV-2突变株(B.1.351)RBD重组蛋白。再分别加50uL的磁微粒偶联ACE2蛋白至检测管中，37℃混匀反应30min，进行磁分离，去上清。加300uL的洗液至检测管中，混匀，进行磁分离去上清。重复洗涤步骤两遍。加150uL的化学发光底物(北京爱维德生物技术有限公司，Cat No.APSUB-1)至检测管中，混匀，用化学发光仪检测发光强度。
用校准品浓度和信号用四参数拟合校准曲线，3组RBD拟合3条校准曲线，见图4。曲线成立条件：校准曲线相关系数r≥0.99,该曲线结果成立，若结果不成立，应进行重复检测。
若要检测样本可以采用检测SARS-CoV-2病毒中和抗体校准品相同的操作方法，进行检测。
3试剂盒的性能评估
3.1)检测限评价
取本实施例中的2批试剂盒，连续3天，检测5个空白样本和5个低值样本，每个样本重复4次，通过2批次空白样本的60个结果，计算空白检测限LOB；通过2批次低值样本的60个结果，计算最低检测限LOD；通过2批低值样本的60个结果，计算CV＝20％时的定量限LOQ。
RBD类型 LOB LOD LOQ
野生型 146.32U/ml 259.32U/ml 326U/ml
B.1.1.7突变型 266.24U/ml 452.06U/ml 523U/ml
B.1.351突变型 446.12U/ml 537.17U/ml  
3.2)线性评价
用接近线性范围上限的高值样本(接近线性范围上限(6000U/ml))按一定比例稀释成5个浓度点，每个浓度重复3次，计算其浓度均值，将结果的均值和稀释比例按最小二乘法进行直线拟合，计算线性相关系数r，在规定的线性范围内，线性r应不小于0.99。
3.3)回收率评估
用低中高三个浓度的样本进行检测，每个样本重复3次，计算其浓度均值，每个浓度的回收率应在(80％，120％)范围内。
3.4)重复性评估
用浓度为750±150U/mL和1500±300U/mL两个浓度的样本进行检测，每个样本重复10次，计算10次测量结果的平均值Μ和标准差SD，根据公式CV＝SD/M×100％得出变异系数CV，本方法变异系数(CV)不大于10％。
3.5)批间差评估
将实施例的试剂盒取三批，每批试剂盒均测定浓度在750±150U/mL和1500±300U/mL范围内的样品，每批重复测定10次，计算30次测定结果的平均值(Μ)和标准差(SD)，根据公式CV＝SD/M×100％计算变异系数(CV)，本方法变异系数(CV)不大于15％。
3.6)方法学比对评估
用本实施例检测抗野生型SARS-CoV-2 RBD中和抗体的试剂盒和cPass TMSARS-CoV-2中和抗体检测试剂盒(FDA EUA)(金斯瑞，货号L00847)分别检测40例样本(用基质人血清(Scantibodies，Cat No.3SH344)稀释商品化SARS-CoV-2中和抗体标准品(金斯瑞，Cat No.A02087)成0U/ml～7200U/ml范围内的不同浓度)，将实施例测试浓度和cPass测试浓度用最小二乘法进行直线拟合，计算相关系数R 2，见图5。R 2≥0.95表明实施例试剂盒和cPass试剂盒有良好的相关性。
实施例3 SARS-CoV-2病毒中和抗体吖啶酯磁微粒化学发光试剂盒制备及性能评估
1.1试剂盒组成
本试剂盒由以下7个试剂组成:
a.吖啶酯偶联SARS-CoV-2 RBD蛋白；
b.磁微粒标记ACE2；
c.样本稀释液；
d.阳性质控品；
e.阴性质控品；
f.洗液；
g.底物(预激发液A和激发液B)；
1.2吖啶酯偶联SARS-CoV-2 RBD蛋白的制备
1)将1mL的1mg/mL重组SARS-CoV-2 RBD蛋白(金斯瑞，Cat No.T80302)溶液用Zeba TM旋转脱盐柱置换至20mM磷酸盐缓冲液(pH7.4)中，放入2mL的EP管中。
2)向EP管中加入80uL的5mM的吖啶酯溶液(深圳美凯特，Cat No.Intlus-B05)，震荡混匀30-60s后，置于2-8℃避光静置反应18-24h。
3)向吖啶酯偶联标记SARS-CoV-2 RBD蛋白反应产物中加入10ul的50mM的甘氨酸溶液，室温避光静置30min。
4)用Zeba TM旋转脱盐柱置换缓冲液(20mM PB；150mM NaCl；pH7.4)纯化，获得吖啶酯偶联SARS-CoV-2 RBD蛋白浓缩液。
5)将步骤4)制备的吖啶酯偶联标记SARS-CoV-2 RBD蛋白浓缩液用步骤1.4制备的样本稀释液稀释到10-100ng/mL，得到所述的试剂。
1.3磁微粒偶联ACE2的制备
1)取500ul浓度为100mg/mL的磁微粒(JSR，MS300/Tosyl)放入10mL的离心管中，磁分离去上清。
2)用5mL的硼酸缓冲液(0.1mol/LpH 9.2)重悬磁微粒，向离心管中加入1mg的ACE2胞外域-Fc蛋白(金斯瑞，Cat No.Z03484)，37℃恒温混匀反应16-24h。
3)磁分离去上清，向离心管中加入加入50ul封闭液(10％(w/v)BSA)37℃恒温混匀反应6h以上。
4)磁分离去上清，用5mL的1*TBST(10mM Tris；150mM NaCl；0.05％Tween-20(v/v)；pH 7.4-7.5)重悬磁微粒，混匀1-5min，磁分离去上清。重复洗涤步骤2-3次。
5)加入10mL磁分离试剂稀释液，震荡混匀1-5min，将磁微粒重悬到5mg/mL(ACE2含量100ug/mL)，使用超声振荡仪将混悬液中聚集成团的磁珠分散成单颗粒状态，得到磁分离试剂浓缩液。
6)用步骤1.4制备的样本稀释液稀释将步骤5)的磁微粒偶联ACE2浓缩液稀释成磁微粒浓度0.1-0.5mg/mL，ACE2浓度2-10ug/mL的磁微粒偶联ACE2。
1.4样本稀释液的制备
制备方法和组分同实施例1的1.4制备步骤和组分。
1.5阳性质控品制备
用样本稀释液将抗SARS-CoV-2病毒中和抗体(金斯瑞，克隆：2B2，Cat No.A02051)配制为200ng/mL的阳性质控品。
1.6阴性质控品制备
阴性质控品为本实施例1.4中制备的样本稀释液。
1.7洗液的制备
洗液的制备方法和组分同实施例1的1.7制备步骤和组分。
2试剂盒的操作
1)加50uL阳性质控品、50uL阴性质控品或50uL的1:10稀释后的血清或血浆标本至检测管中。
2)加50uL的吖啶酯偶联SARS-CoV-2 RBD蛋白所述检测管中，混匀后，37℃温育30min。
3)加50uL的磁微粒偶联ACE2到检测管中，混匀后，37℃温育15min，进行磁分离，去上清。
4)加300uL的清洗液至检测管中，混匀，进行磁分离去上清。重复洗涤步骤两遍。
5)分别加50uL的预激发液A(0.1M HCl；1.32％(w/v)H 2O 2)和50uL的激发液B(0.35M NaOH；0.025％(w/v)CTAC(十六烷基三甲基氯化铵))至检测管中，混匀，化学发光仪检测发光强度。
6)结果计算[见式(1)-式(3)]：
Figure PCTCN2021100834-appb-000007
N2：阴性对照2的检测值。
Figure PCTCN2021100834-appb-000008
P2：阳性对照2的检测值：
Figure PCTCN2021100834-appb-000009
S：样本检测值
7)试验成立条件：标准阳性对照的抑制率≥30％,该实验结果成立，若试验不成立，应进行重复检测。
8)如果被检测样本的抑制率≥30％，则该样本可以判为阳性。如被检测样本的抑制率＜30％，则该样本可以判断为阴性。
3试剂盒的性能评估
3.1重复性评价
取本实施例的试剂盒重复检测中和抗体浓度为200±40ng/mL和500±100ng/mL的样品各10次，计算10次测量结果的平均值Μ和标准差SD，根据公式CV＝SD/M×100％得出变异系数CV，本方法变异系数(CV)不大于10％。
3.2批间差评价
将实施例的试剂盒取三批，每批试剂盒均测定中和抗体浓度在200±40ng/mL和500±100mg/mL范围内的样品，每批重复测定10次，计算30次测定结果的平均值(Μ)和标准差(SD)，根据公式CV＝SD/M×100％计算变异系数(CV)，本方法变异系数(CV)不大于15％。
3.3特异性评估
通过SARS-CoV-2病毒中和抗体阻断阻断吖啶酯磁微粒化学发光检测试剂盒检测20例不含SARS-CoV-2中和抗体的样本，结果应为阴性。
3.4灵敏性评估
通过SARS-CoV-2病毒中和抗体阻断吖啶酯磁微粒化学发光检测试剂盒检测20例含不同浓度的SARS-CoV-2中和抗体阳性样本(由金斯瑞Cpass TM试剂盒(Cat No.L00847)确认为中和抗体)，结果应为阳性。
序列信息：
SARS-CoV-2刺突蛋白RBD蛋白序列(SEQ ID NO:1)
Figure PCTCN2021100834-appb-000010
SARS-CoV-2刺突蛋白S1亚基序列(SEQ ID NO:2)
Figure PCTCN2021100834-appb-000011
人ACE2蛋白序列(SEQ ID NO:3)
Figure PCTCN2021100834-appb-000012
SARS-CoV-2突变株(B.1.1.7)刺突蛋白RBD蛋白序列(SEQ ID NO:4)
Figure PCTCN2021100834-appb-000013
SARS-CoV-2突变株(B.1.1.7)刺突蛋白S1亚基序列(SEQ ID NO:5)
Figure PCTCN2021100834-appb-000014
SARS-CoV-2突变株(B.1.351)刺突蛋白RBD蛋白序列(SEQ ID NO:6)
Figure PCTCN2021100834-appb-000015
SARS-CoV-2突变株(B.1.351)刺突蛋白S1亚基序列(SEQ ID NO:7)
Figure PCTCN2021100834-appb-000016
人ACE21型异构体(SEQ ID NO:8)
Figure PCTCN2021100834-appb-000017
人ACE21型异构体胞外域(SEQ ID NO:9)
Figure PCTCN2021100834-appb-000018
人ACE22型异构体(SEQ ID NO:10)
Figure PCTCN2021100834-appb-000019
人ACE2(SEQ ID NO:11)
Figure PCTCN2021100834-appb-000020
人ACE2胞外域(SEQ ID NO:12)
Figure PCTCN2021100834-appb-000021

Claims (28)

  1. 一种检测样品中新型冠状病毒或其变体的中和抗体的化学发光试剂盒，其特征在于，所述试剂盒包括新型冠状病毒或其变体的刺突蛋白部分和特异性结合所述新型冠状病毒或其变体刺突蛋白部分的ACE2蛋白或其功能片段；
    其中，所述新型冠状病毒或其变体的刺突蛋白部分连接标记物，所述ACE2蛋白或其功能片段连接磁微粒；或所述新型冠状病毒或其变体的刺突蛋白部分连接磁微粒，所述ACE2蛋白或其功能片段连接标记物；
    其中，所述新型冠状病毒或其变体的刺突蛋白部分与ACE2蛋白或其功能片段之间特异性结合产生检测信号。
  2. 根据权利要求1所述的化学发光试剂盒，其中所述标记物或磁微粒是通过接头或偶联方式连接，优选通过偶联方式连接。
  3. 根据权利要求1或2所述的化学发光试剂盒，所述新型冠状病毒或其变体的刺突蛋白部分偶联上标记物，所述ACE2蛋白或其功能片段偶联上磁微粒。
  4. 根据权利要求1-3中任一项所述的化学发光试剂盒，所述标记物选自碱性磷酸酶、吖啶酯、辣根过氧化物酶、三联吡啶钌、生物素、异鲁米诺或稀土元素中的一种或多种，优选为碱性磷酸酶、吖啶酯或辣根过氧化物酶。
  5. 根据权利要求1所述的化学发光试剂盒，其包括：
    吖啶酯偶联的新型冠状病毒或其变体的刺突蛋白部分和磁微粒偶联的ACE2蛋白或其功能片段，或吖啶酯偶联的ACE2蛋白或其功能片段和磁微粒偶联的新型冠状病毒或其变体的刺突蛋白部分。
  6. 根据权利要求1所述的化学发光试剂盒，其包括：
    碱性磷酸酶偶联的新型冠状病毒或其变体刺突蛋白部分和磁微粒偶联的ACE2蛋白或其功能片段，或碱性磷酸酶偶联的ACE2蛋白或其功能片段和磁微粒偶联的新型冠状病毒或其变体刺突蛋白。
  7. 根据权利要求1-6中任一项所述的化学发光试剂盒，其特征在于，通过检测待测样品抑制或阻断所述新型冠状病毒或其变体刺突蛋白部分和ACE2蛋白或其功能片段之间的结合产生检测信号的变化，确定样品中是否存在新型冠状病毒或其变体的中和抗体。
  8. 根据权利要求1-7中任一项所述的化学发光试剂盒，所述新型冠状病毒或其变体刺突蛋白部分选自新型冠状病毒或其变体S蛋白、新型冠状病毒或其变体S1蛋白、新型冠状病毒或其变体RBD蛋白或者带有His或Fc标签的新型冠状病毒或其变体RBD蛋白，优选为新型冠状病毒或其变体RBD蛋白。
  9. 根据权利要求1-8中任一项所述的化学发光试剂盒，所述新型冠状病毒或其变体的刺突蛋白部分包含SEQ ID NO:1、2和4-7中任一所示的氨基酸序列，或与SEQ ID NO:1、2和4-7中任一所示的氨基酸序列至少70％一致性的序列。
  10. 根据权利要求1-9中任一项所述的化学发光试剂盒，所述标记物偶联的新型冠状病毒或其变体刺突蛋白部分浓度为2-200ng/mL。
  11. 根据权利要求10所述的化学发光试剂盒，所述偶联标记物的新型冠状病毒或其变体刺突蛋白部分为吖啶酯标记或者碱性磷酸酶标记的新型冠状病毒或其变体RBD蛋白。
  12. 根据权利要求11所述的化学发光试剂盒，所述吖啶酯或碱性磷酸酶标记的新型冠状病毒或其变体RBD蛋白浓度为2-200ng/mL，优选为10-100ng/mL。
  13. 根据权利要求1-12中任一项所述的化学发光试剂盒，所述ACE2蛋白或其功能片段来源于人。
  14. 根据权利要求1-13中任一项所述的化学发光试剂盒，所述ACE2蛋白或其功能片段选自重组ACE2蛋白或ACE2蛋白胞外域或带Fc标签ACE2蛋白或ACE2蛋白胞外域。
  15. 根据权利要求13或14所述的化学发光试剂盒，所述ACE2蛋白或其功能片段包含SEQ ID NO:3和8-12中任一所示氨基酸序列，或与SEQ ID NO:3和8-12中任一所示氨基酸序列至少70％一致性的序列。
  16. 根据权利要求1-15中任一项所述的化学发光试剂盒，所述磁微粒偶联的ACE2蛋白或其功能片段中ACE2蛋白或其功能片段的浓度为2-15ug/mL，优选为5-10ug/mL。
  17. 根据权利要求16所述的化学发光试剂盒，所述磁微粒的浓度为0.1-0.5mg/mL。
  18. 根据权利要求1-17中任一项所述的化学发光试剂盒，所述试剂盒进一步包括化学发光底物，所述化学发光底物选自AMPPD、APS5、CSPD、CDP-Star、鲁米诺或PS-atto。
  19. 根据权利要求1-18中任一项所述的化学发光试剂盒，所述试剂盒还包括阴性质控品、样品稀释液和洗液，所述阴性质控品为样本稀释液。
  20. 根据权利要求1-19中任一项所述的化学发光试剂盒，所述试剂盒进一步包括阳性质控品，所述阳性质控品为抗SARS-CoV-2病毒或其变体的中和抗体。
  21. 根据权利要求1-20中任一项所述的化学发光试剂盒，所述新型冠状病毒变体选自SARS-CoV-2 B.1.1.7、SARS-CoV-2 B.1.351或SARS-CoV-2 B.1.617。
  22. 根据权利要求1-21中任一项所述的化学发光试剂盒在检测样品中是否存在新型冠状病毒或其变体的抗体中的应用。
  23. 根据权利要求1-21中任一项所述的化学发光试剂盒或权利要求22所述化学发光试剂盒的应用，其中，所述样品包括：
    (1)感染或疑似感染新型冠状病毒或其变体的血浆、血清或全血；
    (2)接种新型冠状病毒或其变体疫苗后的血浆、血清或全血样品；
    (3)新型冠状病毒或其变体刺突蛋白免疫动物后的血浆、血清或全血；或
    (4)生物学方法获得抗新型冠状病毒或其变体的抗体样品。
  24. 根据权利要求1-21中任一项所述的化学发光试剂盒的使用方法，包括如下步骤：
    (1)加稀释的样品、阳性质控品或阴性质控品至检测管中；
    (2)向检测管中加入标记物偶联的新型冠状病毒或其变体刺突蛋白部分，混匀，温育；
    (3)加入磁微粒偶联的ACE2蛋白或其功能片段，混匀，温育，磁分离，去上清，加入洗液，反复洗涤；
    (4)加入化学发光底物，混匀，检测发光强度。
  25. 根据权利要求1-21中任一项所述的化学发光试剂盒的使用方法，包括如下步骤：
    (1)分别加稀释的样品、阳性质控品或阴性质控品至检测管中；
    (2)向检测管中加入磁微粒偶联的ACE2蛋白或其功能片段；
    (3)加入标记物偶联的新型冠状病毒或其变体刺突蛋白部分，混匀，温育，磁分离，去上清，加入洗液，反复洗涤；
    (4)加入化学发光底物，混匀，检测发光强度。
  26. 一种检测样品中是否存在新型冠状病毒或其变体的中和抗体的方法，包括：
    (1)将样品与新型冠状病毒或其变体刺突蛋白或其ACE2结合片段接触；
    (2)将步骤(1)的混合物与所述刺突蛋白或其ACE2结合片段特异结合的ACE2蛋白或其片段接触；
    (3)检测所述刺突蛋白或其ACE2结合片段与所述ACE2蛋白或其片段之间结合的水平，
    其中，所述新型冠状病毒或其变体刺突蛋白或其ACE2结合片段连接标记物，所述ACE2蛋白或其片段连接磁微粒。
  27. 一种检测样品中是否存在新型冠状病毒或其变体的中和抗体的方法，包括：
    (1)将样品与新型冠状病毒或其变体刺突蛋白或其ACE2结合片段接触；
    (2)再将上述新型冠状病毒或其变体刺突蛋白或其ACE2结合片段连接磁微粒；
    (3)加入与所述刺突蛋白或其ACE2结合片段特异结合的ACE2蛋白或其片段；
    (4)检测所述刺突蛋白或其ACE2结合片段与所述ACE2蛋白或其片段之间结合的水平，
    其中，所述ACE2蛋白或其片段连接标记物。
  28. 根据权利要求26或27所述的方法，进一步包括通过样品抑制或阻断新型冠状病毒或其变体刺突蛋白或其ACE2结合片段和ACE2蛋白或其功能片段的结合产生检测信号的变化，确定样品中是否存在新型冠状病毒或其变体的中和抗体。
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