WO2022100303A1 - Kit et méthode de détection d'anticorps neutralisant le sars-cov-2 - Google Patents

Kit et méthode de détection d'anticorps neutralisant le sars-cov-2 Download PDF

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WO2022100303A1
WO2022100303A1 PCT/CN2021/120953 CN2021120953W WO2022100303A1 WO 2022100303 A1 WO2022100303 A1 WO 2022100303A1 CN 2021120953 W CN2021120953 W CN 2021120953W WO 2022100303 A1 WO2022100303 A1 WO 2022100303A1
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cov
sars
seq
amino acid
functionally active
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PCT/CN2021/120953
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时亚斌
陈志才
马可
徐晓昱
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南京诺唯赞生物科技股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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 application relates to the field of biomedicine, in particular to a kit and method for detecting SARS-CoV-2 neutralizing antibodies.
  • the spike protein on the surface of SARS-CoV-2 is an important receptor binding site on the surface of the coronavirus, which can bind to virus-specific receptors on the cell surface, mediate the fusion of the outer membrane of the virus with the cell, and the adsorption and penetration of the virus.
  • Membrane; the receptor binding domain (RBD) of the coronavirus S protein can bind to angiotensin-converting enzyme 2 (ACE2) on the surface of the host cell and allow the virus to enter the host cell.
  • ACE2 angiotensin-converting enzyme 2
  • Neutralizing antibody is a soluble protein secreted by adaptive immune response cells, which has the function of recognizing viral surface proteins and blocking their binding to specific receptors on the surface of human cells, thereby exerting antiviral effects.
  • the amount of neutralizing antibodies is an important indicator of the protective effect of vaccine immunity and an important basis for vaccine evaluation and quality control.
  • the existing detection methods for SARS-CoV-2 mainly include nucleic acid detection and antibody detection.
  • the main methods are fluorescent PCR, enzyme-linked immunosorbent assay, colloidal gold chromatography, and chemiluminescence.
  • nucleic acid detection has high sensitivity and specificity. It has strong performance and high accuracy, but requires relatively high operation and long detection time.
  • Antibody detection has high sensitivity and specificity, but IgM/IgG antibodies can only appear 7-14 days after infection.
  • these methods can only detect whether they are in the infection stage, and cannot confirm whether there is a risk of re-infection in cured patients, and whether there is an exposure and infection risk in the general public. Therefore, new methods to detect SARS-CoV-2 are urgently needed.
  • the application provides a kit and method for detecting SARS-CoV-2 neutralizing antibody, which has one or more of the following properties: 1) After fusion of RBD, ACE2 receptor protein or its functionally active fragment with Fc , compared with the protein of natural structure, it has better stability and reactivity; 2) The detection sensitivity is improved; 3) The clinical efficacy of the COVID-19 vaccine can be judged, and the effect of individual autoimmunity can be evaluated; 4) It can be judged whether There is a risk of re-infection with SARS-CoV-2; and 5) the reference antibody has a good neutralizing effect and can well mimic the natural form of neutralizing antibodies in humans.
  • the application provides a detection kit for SARS-CoV-2 neutralizing antibodies, comprising:
  • SARS-CoV-2 spike protein or a functionally active fragment thereof
  • a competitive binding agent which comprises a substance capable of competing with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or a functionally active fragment thereof.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a receptor binding region RBD.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a full-length SARS-CoV-2 spike protein.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a trimeric full-length SARS-CoV-2 spike protein.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a receptor binding region RBD, and the RBD is directly or indirectly linked to the Fc region.
  • the RBD is fused in-frame to the Fc.
  • the Fc region is selected from the group consisting of rFc, mFc, and hFc.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 6-11.
  • the concentration of the SARS-CoV-2 spike protein or functionally active fragment thereof is 50ng/mL-4000ng/mL.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof is immobilized on a solid support.
  • the competitive binding agent comprises an ACE2 receptor protein or a functionally active fragment thereof.
  • the ACE2 receptor protein or functionally active fragment thereof comprises a human ACE2 receptor protein or functionally active fragment thereof.
  • the ACE2 receptor protein or functionally active fragment thereof comprises an extracellular domain ECD.
  • the ACE2 receptor protein or functionally active fragment thereof is linked directly or indirectly to the Fc region.
  • the ACE2 receptor protein or functionally active fragment thereof is fused in-frame to the Fc region.
  • the Fc region is selected from the group consisting of rFc, mFc, and hFc.
  • the competitive binding agent comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-5.
  • the competing binding agent comprises one or more labels capable of directly or indirectly generating a signal indicative of the presence and/or amount of the competing binding agent.
  • the marker comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
  • HRP horseradish peroxidase
  • the concentration of the competing binding agent is between 100 ng/mL and 2000 ng/mL.
  • the kit further comprises a calibrator comprising one or more reference antibody molecules that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof or antigen-binding fragments thereof.
  • the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof .
  • the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof .
  • the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises the HCDR1, HCDR2 and HCDR3, the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in SEQ ID NO: 33-35; the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NO: 36-38, the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in SEQ ID NO:39-41; or the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NO:42-44, and the VL comprises SEQ ID NO:42-44 LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in ID NO: 45-47.
  • the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises the amino acid sequence shown in SEQ ID NO: 21, and the VL comprises SEQ ID The amino acid sequence shown in NO: 22; the VH comprises the amino acid sequence shown in SEQ ID NO: 24, the VL comprises the amino acid sequence shown in SEQ ID NO: 25; or the VH comprises the amino acid sequence shown in SEQ ID NO: 27 The amino acid sequence shown in the VL comprises the amino acid sequence shown in SEQ ID NO: 28.
  • the reference antibodies include antibodies 32-1, 12-5 and 12-8.
  • the concentration of the reference antibody is between 50 ng/mL and 1000 ng/mL.
  • the SARS-CoV-2 spike protein, or functionally active fragment thereof comprises one or more markers capable of directly or indirectly producing a spike that is indicative of the SARS-CoV-2 A signal of the presence and/or amount of a protein or functionally active fragment thereof.
  • the marker comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
  • HRP horseradish peroxidase
  • the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising: using the kit.
  • the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising:
  • step 2) adding the sample to be tested in step 1) to react with the SARS-CoV-2 spike protein or its functionally active fragment;
  • the competitive binding agent comprises a substance capable of competing with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or its functionally active fragment; the SARS-CoV-2 spike protein or a functionally active fragment thereof comprising a receptor binding region RBD; the competitive binding agent comprises an ACE2 receptor protein or a functionally active fragment thereof.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a full-length SARS-CoV-2 spike protein.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a trimeric full-length SARS-CoV-2 spike protein.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a receptor binding region RBD, and the RBD is directly or indirectly linked to the Fc region.
  • the RBD is fused in-frame to the Fc.
  • the Fc region is selected from the group consisting of rFc, mFc, and hFc.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 6-11.
  • the concentration of the SARS-CoV-2 spike protein or functionally active fragment thereof is 50ng/mL-4000ng/mL.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof is immobilized on a solid support.
  • the competitive binding agent comprises an ACE2 receptor protein or a functionally active fragment thereof.
  • the ACE2 receptor protein or functionally active fragment thereof comprises a human ACE2 receptor protein or functionally active fragment thereof.
  • the ACE2 receptor protein or functionally active fragment thereof comprises an extracellular domain ECD.
  • the ACE2 receptor protein or functionally active fragment thereof is linked directly or indirectly to the Fc region.
  • the ACE2 receptor protein or functionally active fragment thereof is fused in-frame to the Fc region.
  • the Fc region is selected from the group consisting of rFc, mFc, and hFc.
  • the competitive binding agent comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-5.
  • the competing binding agent comprises one or more labels capable of directly or indirectly generating a signal indicative of the presence and/or amount of the competing binding agent.
  • the marker comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
  • HRP horseradish peroxidase
  • the concentration of the competing binding agent is between 100 ng/mL and 2000 ng/mL.
  • the kit further comprises a calibrator comprising one or more reference antibody molecules that specifically recognize the SARS-CoV-2 spike protein or a functionally active fragment thereof or antigen-binding fragments thereof.
  • the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof .
  • the one or more reference antibody molecules or antigen-binding fragments thereof specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof .
  • the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises the HCDR1, HCDR2 and HCDR3, the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in SEQ ID NO: 33-35; the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NO: 36-38, the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in SEQ ID NO:39-41; or the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NO:42-44, and the VL comprises SEQ ID NO:42-44 LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in ID NO: 45-47.
  • the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises the amino acid sequence shown in SEQ ID NO: 21, and the VL comprises SEQ ID The amino acid sequence shown in NO: 22; the VH comprises the amino acid sequence shown in SEQ ID NO: 24, the VL comprises the amino acid sequence shown in SEQ ID NO: 25; or the VH comprises the amino acid sequence shown in SEQ ID NO: 27 The amino acid sequence shown in the VL comprises the amino acid sequence shown in SEQ ID NO: 28.
  • the reference antibodies include antibodies 32-1, 12-5 and 12-8.
  • the concentration of the reference antibody is between 50 ng/mL and 1000 ng/mL.
  • the sample is selected from the group consisting of urine, saliva, serum, plasma, nasopharyngeal aspirate, and bronchial lavage fluid.
  • the sample is derived from a subject.
  • the subject is a recovered COVID-19 patient, a healthy subject, and/or a subject who has received a COVID-19 vaccine.
  • the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, comprising:
  • step 3 adding the incubated product described in step 2) to step 1);
  • the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a receptor binding region RBD
  • the RBD comprises one or more markers capable of directly or indirectly producing a protein indicative of the RBD presence and/or content signals.
  • the marker comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
  • HRP horseradish peroxidase
  • the RBD is directly or indirectly linked to the Fc region.
  • the RBD is fused in-frame to the Fc.
  • the Fc region is selected from the group consisting of rFc, mFc, and hFc.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:7.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:8.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:9.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:10.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 11.
  • the concentration of the SARS-CoV-2 spike protein or functionally active fragment thereof is 50ng/mL-4000ng/mL.
  • the ACE2 receptor protein or functionally active fragment thereof is linked directly or indirectly to the Fc region.
  • the ACE2 receptor protein or functionally active fragment thereof is fused in-frame to the Fc region.
  • the Fc region is selected from the group consisting of rFc, mFc, and hFc.
  • the ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:2.
  • the ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:3.
  • the ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:4.
  • the concentration of the ACE2 receptor protein or functionally active fragment thereof is 100 ng/mL-2000 ng/mL.
  • the method includes a sample diluent having a pH of 6.0-9.6.
  • the detection is obtained by comparison to a calibrator comprising one or more references that specifically recognize the SARS-CoV-2 spike protein or functionally active fragment thereof An antibody molecule or an antigen-binding fragment thereof;
  • the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein: the VH comprises the HCDR1, HCDR2 of the amino acid sequences shown in SEQ ID NOs: 30-32 and HCDR3, the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in SEQ ID NO: 33-35; the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NO: 36-38, The VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 39-41; or the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 42-44, the VL comprises LCDR1, LCDR2 and
  • the detection is obtained by comparison to a calibrator comprising one or more references that specifically recognize the SARS-CoV-2 spike protein or functionally active fragment thereof Antibody molecule or antigen-binding fragment thereof;
  • the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL; wherein: the VH comprises the amino acid sequence shown in SEQ ID NO: 21, and the VL comprises SEQ ID NO: 21 The amino acid sequence set forth in ID NO: 22; the VH comprises the amino acid sequence set forth in SEQ ID NO: 24, the VL comprises the amino acid sequence set forth in SEQ ID NO: 25; or the VH comprises the amino acid sequence set forth in SEQ ID NO: 27 The amino acid sequence shown, the VL comprises the amino acid sequence shown in SEQ ID NO: 28.
  • the reference antibodies include antibodies 32-1, 12-5 and 12-8.
  • the concentration of the reference antibody is between 50 ng/mL and 1000 ng/mL.
  • the sample to be tested is selected from the group consisting of urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirate, and bronchial lavage fluid.
  • the test sample is derived from a subject.
  • the subject is a recovered COVID-19 patient, a healthy subject, and/or a subject who has been treated with a COVID-19 vaccine.
  • Figure 1 shows the trend of changes in the content of neutralizing antibodies in the human body simulated by the calibrator described in this application.
  • Figure 2 shows the ROC curve of the Cut-off values described in this application.
  • sample generally refers to any substance that contains or is supposed to contain antibodies, especially SARS-CoV-2 neutralizing antibodies.
  • a “sample” may be of natural or synthetic origin, and may be obtained by any means known to those skilled in the art.
  • sample can be a tissue or fluid sample isolated from a subject, including but not limited to urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirate, and bronchial lavage fluid.
  • the samples can be research samples and clinical samples.
  • the sample can also be a blood sample for transfusion or treatment. Samples can also be synthetic and include, but are not limited to, in vitro cell culture components, including but not limited to conditioned media, recombinant cells, and cell components. In this application, the terms “sample” and “sample” are used interchangeably.
  • detecting generally refers to finding the presence or presence of something.
  • the term "antigen” generally refers to a substance that promotes the production of antibodies and can elicit an immune response. They can also be used for diagnostic or patient selection or characterization purposes.
  • an antibody generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen.
  • an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof.
  • the term “antibody” includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single chain antibodies (eg, scFvs), and antibodies with antigens Bound antibody fragments (eg, Fab, Fab' and (Fab)2 fragments).
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • the VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL can consist of three CDRs and four FR regions, which can be arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the term "antigen-binding fragment” generally refers to one or more fragments of an antibody that function to specifically bind an antigen.
  • the antigen-binding function of an antibody can be achieved by full-length fragments of the antibody.
  • Antigen-binding functions of antibodies can also be achieved by fragments including heavy chains of fragments of Fv, ScFv, dsFv, Fab, Fab' or F(ab')2, or alternatively, including Fv, ScFv, dsFv, Fab, Fab' or a light chain of a fragment of F(ab')2.
  • Fab fragment i.e.
  • a monovalent fragment consisting of VL, VH, CL and CH domains (2) F(ab')2 fragment, comprising two Fab fragments linked by a disulfide bond at the hinge region Bivalent fragments; (3) Fd fragments composed of VH and CH domains; (4) Fv fragments composed of VL and VH domains of the antibody one-arm; (5) dAb fragments composed of VH domains (Ward et al. , (1989) Nature 341:544-546); (6) a combination of separate complementarity determining regions (CDRs) and (7) two or more separate CDRs optionally linked by a linker.
  • CDRs complementarity determining regions
  • the "antigen-binding portion” may also include an immunoglobulin fusion protein comprising a binding domain selected from the group consisting of: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) with an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
  • the term "subject” generally refers to any organism, including but not limited to mammals such as mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates.
  • a subject may be a human, pet or livestock.
  • the subject can be a recovered patient from COVID-19, a healthy subject and/or a subject who has been treated with a COVID-19 vaccine.
  • the term "RBD” refers to the receptor binding domain of the SARS-CoV-2 spike protein (S protein).
  • the RBD may be a mutant or truncated form of the SARS-CoV-2 spike protein (S protein).
  • the truncation generally refers to anything less than the whole.
  • a truncate is at least 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than the amino acid sequence of the reference sequence.
  • “mutant” generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.
  • truncations or mutants may or may not occur naturally. Non-naturally occurring truncations or mutants can be generated using techniques known in the art.
  • SARS-CoV-2 spike protein or a functionally active fragment thereof generally refers to the capsid surface glycoprotein of the coronavirus.
  • SARS-COV-2 binds to the ACE2 receptor and invades cells through the SARS-CoV-2 spike protein.
  • the SARS-CoV-2 spike protein contains a transmembrane region comprising a fragment of at least 1213 amino acids from the N-terminus or from amino acid 14 of the capsid surface glycoprotein from coronaviruses, or corresponding regions from other SARS viruses.
  • the SARS-CoV-2 spike protein may be a trimeric full-length SARS-CoV-2 spike protein.
  • the SARS-CoV-2 spike protein may be the full-length SARS-CoV-2 spike protein.
  • the term "ACE2 receptor protein or a functionally active fragment thereof" generally refers to the functional receptor of the coronavirus spike protein.
  • the ACE2 receptor protein can be a human ACE2 receptor protein.
  • the ACE2 receptor protein sequence can be found in Uniprot Accession No. Q9BYF1.
  • a functionally active fragment generally refers to a fragment of a larger polypeptide or polynucleotide that retains the same activity or ability as its larger counterpart.
  • the level of activity of a functionally active fragment may be the same as, or less or greater than, the activity of the larger counterpart.
  • a functionally active fragment of the SARS-CoV-2 spike protein can be a polypeptide that consists of fewer amino acids than the full-length SARS-CoV-2 spike protein, but still retains the full-length SARS-CoV-2 spike protein protein activity.
  • a functionally active fragment of a human ACE2 receptor protein can be a polypeptide that consists of fewer amino acids than a full-length human ACE2 receptor protein, but still retains the activity of the full-length human ACE2 receptor protein.
  • Fc region generally refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the constant region.
  • Fc regions include native sequence Fc regions and variant Fc regions.
  • the Fc region can be directly or indirectly linked to the SARS-CoV-2 spike protein.
  • the Fc region can be fused in-frame to the SARS-CoV-2 spike protein.
  • the Fc region can be linked directly or indirectly to the ACE2 receptor protein.
  • the Fc region can be fused in-frame to the ACE2 receptor protein.
  • Fc regions include human or non-human (eg, rat, mouse, rabbit) Fc regions.
  • the Fc region can include a rabbit Fc region (rFc).
  • the Fc region can include a mouse Fc region (mFc).
  • the Fc region can include a human Fc region (hFc).
  • in-frame fusion generally refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF in a manner that maintains the correct reading frame of the original ORF .
  • ORFs open reading frames
  • the resulting "fusion polypeptide” is a single protein comprising two or more fragments corresponding to the polypeptide encoded by the original ORF (the fragments are not normally linked as such in nature).
  • a "fusion site” refers to a sequence where two or more fragments are joined together. In some cases, the fusion site may be the same sequence as the sequences in the two or more fragments to be joined. In some cases, the fusion site may further comprise a gapped segment that is not identical to either of the sequences of the two or more segments being ligated.
  • the present application provides a SARS-CoV-2 neutralizing antibody detection kit, which may comprise:
  • SARS-CoV-2 spike protein or a functionally active fragment thereof
  • a competitive binding agent which comprises a substance capable of competing with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or a functionally active fragment thereof.
  • the SARS-CoV-2 spike protein or its functionally active fragment may comprise a receptor binding region RBD.
  • the SARS-CoV-2 spike protein or its functionally active fragment may comprise the full-length SARS-CoV-2 spike protein.
  • the SARS-CoV-2 spike protein or its functionally active fragment may comprise a trimeric full-length SARS-CoV-2 spike protein.
  • the SARS-CoV-2 spike protein or its functionally active fragment may comprise the amino acid sequence shown in SEQ ID NO: 10.
  • the SARS-CoV-2 spike protein or its functionally active fragment may comprise the amino acid sequence shown in SEQ ID NO: 11.
  • the SARS-CoV-2 spike protein or its functionally active fragment may comprise a receptor binding region RBD, and the RBD may be directly or indirectly linked to the Fc region.
  • the RBD can be fused in-frame with the Fc.
  • the Fc may be selected from the group consisting of rFc, mFc and hFc.
  • the RBD can be directly or indirectly linked to the Fc region.
  • the Fc can be rFc.
  • the RBD can be directly or indirectly linked to the Fc region.
  • the Fc may be mFc.
  • the RBD can be directly or indirectly linked to the Fc region.
  • the Fc may be hFc.
  • the SARS-CoV-2 spike protein or its functionally active fragment may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the SARS-CoV-2 spike protein or its functionally active fragment can comprise the amino acid sequence shown in SEQ ID NO:8.
  • the SARS-CoV-2 spike protein or its functionally active fragment may comprise the amino acid sequence shown in SEQ ID NO:9.
  • the concentration of the SARS-CoV-2 spike protein or its functionally active fragment may be 50ng/mL-4000ng/mL.
  • the concentration of the SARS-CoV-2 spike protein or its functionally active fragment may be 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL, 800ng/mL, 900ng/mL, 1000ng /mL, 1500ng/mL, 2000ng/mL, 2500ng/mL, 3000ng/mL, 3500ng/mL or 4000ng/mL.
  • the SARS-CoV-2 spike protein or its functionally active fragment can be immobilized on a solid support.
  • the solid support may be a reaction plate or an ELISA plate (a reaction plate or an ELISA plate suitable for ELISA assay).
  • the competitive binding agent may comprise an ACE2 receptor protein or a functionally active fragment thereof.
  • the competitive binding agent may comprise a known RBD neutralizing antibody.
  • the ACE2 receptor protein may comprise human ACE2 receptor protein or a functionally active fragment thereof.
  • the ACE2 receptor protein or functionally active fragment thereof may comprise an extracellular domain ECD.
  • the ACE2 receptor protein or its functionally active fragment can be directly or indirectly linked to the Fc region.
  • the ACE2 receptor protein or a functionally active fragment thereof may be fused in-frame with the Fc region.
  • the Fc region may be selected from the group consisting of rFc, mFc and hFc.
  • the Fc region may be rFc. In the present application, the Fc region may be mFc. In the present application, the Fc region may be hFc.
  • the competitive binding agent may comprise the amino acid sequence shown in any one of SEQ ID NOs: 1-5.
  • the competitive binding agent can comprise the amino acid sequence shown in SEQ ID NO:2.
  • the competitive binding agent may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the competitive binding agent may comprise the amino acid sequence shown in SEQ ID NO:4.
  • the competing binding agent may comprise one or more labels capable of directly or indirectly generating a signal indicative of the presence and/or amount of the competing binding agent.
  • the label may comprise horseradish peroxidase (HRP) or an HRP-like enzyme.
  • the marker may be horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase or beta-galactosidase beta-Galactosidase.
  • the marker may be horseradish peroxidase (HRP).
  • the competitive binding agent may include horseradish peroxidase (HRP) or an HRP-like enzyme and one or more labels selected from the group consisting of AVI tags, biotin, fluorescein and chain Mycovidin.
  • HRP horseradish peroxidase
  • HRP-like enzyme one or more labels selected from the group consisting of AVI tags, biotin, fluorescein and chain Mycovidin.
  • the competitive binding agent may comprise HRP, an AVI tag, streptavidin.
  • the concentration of the competing binding agent may be 100 ng/mL-2000 ng/mL.
  • the concentration of the competing binding agent may be 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL, 500ng/mL, 600ng/mL, 700ng/mL, 800ng/mL, 1000ng/mL, 1500ng/mL , 1800ng/mL, 1900ng/mL or 2000ng/mL.
  • the kit may include a sample diluent, and the pH of the sample diluent may be 6.0-9.6.
  • the pH of the sample diluent may be 6.0, 6.1, 6.2, 6.3, 6.5, 7.0, 7.5, 7.8, 8.0, 8.5, 9.0, 9.5, or 9.6.
  • the pH of the sample diluent may be 6.0.
  • the kit further comprises a calibrator, which may comprise one or more reference antibody molecules or their functionally active fragments that specifically recognize the SARS-CoV-2 spike protein or its functionally active fragment. Antigen-binding fragments.
  • the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or functionally active fragments thereof.
  • the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or functionally active fragments thereof.
  • the present application provides a detection kit for SARS-CoV-2 neutralizing antibody, which is different from the aforementioned kit in that the SARS-CoV-2 spike protein or its functionally active fragment may contain one or more A variety of markers that can directly or indirectly generate a signal indicative of the presence and/or amount of the competing binding agent.
  • the competitive binding agent may be label-free.
  • the reference antibodies may include antibodies 32-1, 12-5 and 12-8.
  • a reference antibody or antigen-binding fragment thereof described herein may comprise a heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region VL which may comprise LCDR1, LCDR2 and LCDR3.
  • the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 35 or a variant thereof.
  • the reference antibody or antigen-binding fragment thereof may include antibody 32-1 or an antibody having the same HCDR1-3 and LCDR1-3.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 21 or a variant thereof; and the VL may comprise the amino acid sequence shown in SEQ ID NO: 22 or a variant thereof.
  • the nucleic acid sequence encoding VH may comprise the sequence shown in SEQ ID NO: 12; and the nucleic acid sequence encoding VL may comprise the sequence shown in SEQ ID NO: 13.
  • the reference antibody or antigen-binding fragment thereof may include antibody 32-1 or an antibody having the same light chain variable region and heavy chain variable region.
  • a reference antibody or antigen-binding fragment thereof described herein may comprise a heavy chain constant region CH and a light chain constant region CL.
  • the CH may comprise the amino acid sequence shown in SEQ ID NO: 29 or a variant thereof; and the CL may comprise the amino acid sequence shown in SEQ ID NO: 23 or a variant thereof.
  • the reference antibody or antigen-binding fragment thereof may include antibody 32-1 or an antibody having the same heavy and light chain constant regions.
  • the antibody described herein may be 32-1.
  • HCDR1, HCDR2 and HCDR3 of antibody 32-1 may comprise the amino acid sequence shown in SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, respectively;
  • VH may comprise the amino acid sequence shown in SEQ ID NO:21;
  • LCDR1, LCDR2 and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively;
  • VL may comprise the amino acid sequence shown in SEQ ID NO:22.
  • a reference antibody or antigen-binding fragment thereof described herein may comprise a heavy chain variable region VH and a light chain variable region VL, the VH may comprise HCDR1, HCDR2 and HCDR3, and the VL may comprise LCDR1, LCDR2 and LCDR3.
  • the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 41 or a variant thereof.
  • the reference antibody or antigen-binding fragment thereof may comprise antibody 12-5 or an antibody having the same HCDR1-3 and LCDR1-3.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 24 or a variant thereof; and the VL may comprise the amino acid sequence shown in SEQ ID NO: 25 or a variant thereof.
  • the nucleic acid sequence encoding VH may comprise the sequence shown in SEQ ID NO: 15; and the nucleic acid sequence encoding VL may comprise the sequence shown in SEQ ID NO: 16.
  • the reference antibody or antigen-binding fragment thereof may include antibody 12-5 or an antibody having the same light and heavy chain variable regions.
  • a reference antibody or antigen-binding fragment thereof described herein may comprise a heavy chain constant region CH and a light chain constant region CL.
  • the CH may comprise the amino acid sequence shown in SEQ ID NO: 29 or a variant thereof; and the CL may comprise the amino acid sequence shown in SEQ ID NO: 26 or a variant thereof.
  • the reference antibody or antigen-binding fragment thereof may include antibody 12-5 or an antibody having the same heavy and light chain constant regions.
  • the antibody described herein may be 12-5.
  • HCDR1, HCDR2 and HCDR3 of antibody 12-5 may comprise the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively;
  • VH may comprise the amino acid sequence shown in SEQ ID NO: 24;
  • LCDR1, LCDR2 and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41, respectively;
  • VL may comprise the amino acid sequence shown in SEQ ID NO:25.
  • a reference antibody or antigen-binding fragment thereof described herein may comprise a heavy chain variable region VH comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region VL which may comprise LCDR1, LCDR2 and LCDR3.
  • the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 47 or a variant thereof.
  • the reference antibody or antigen-binding fragment thereof may comprise antibody 12-8 or an antibody having the same HCDR1-3 and LCDR1-3.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 27 or a variant thereof; and the VL may comprise the amino acid sequence shown in SEQ ID NO: 28 or a variant thereof.
  • the nucleic acid sequence encoding VH may comprise the sequence shown in SEQ ID NO: 18; and the nucleic acid sequence encoding VL may comprise the sequence shown in SEQ ID NO: 19.
  • the reference antibody or antigen-binding fragment thereof may include antibody 12-8 or an antibody having the same light chain variable region and heavy chain variable region.
  • a reference antibody or antigen-binding fragment thereof described herein may comprise a heavy chain constant region CH and a light chain constant region CL.
  • the CH may comprise the amino acid sequence shown in SEQ ID NO: 29 or a variant thereof; and the CL may comprise the amino acid sequence shown in SEQ ID NO: 23 or a variant thereof.
  • the reference antibody or antigen-binding fragment thereof may include antibody 12-8 or an antibody having the same heavy and light chain constant regions.
  • the antibody described herein may be 12-8.
  • HCDR1, HCDR2 and HCDR3 of antibodies 12-8 may comprise the amino acid sequence shown in SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44, respectively;
  • VH may comprise the amino acid sequence shown in SEQ ID NO:27;
  • LCDR1, LCDR2 and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47, respectively;
  • VL may comprise the amino acid sequence shown in SEQ ID NO:28.
  • the concentration of the reference antibody may be 50ng/mL-1000ng/mL.
  • the concentration of the reference antibody can be 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL, 500ng/mL , 600ng/mL, 700ng/mL, 800ng/mL, 900ng/mL and 1000ng/mL.
  • the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, which may include: using the kit.
  • the present application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, which may include:
  • step 2) adding the sample to be tested in step 1) to react with the SARS-CoV-2 spike protein or its functionally active fragment;
  • the competitive binding agent may comprise a substance capable of competing with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or its functionally active fragment; the SARS-CoV-2 spike The protein or a functionally active fragment thereof may comprise a receptor binding region RBD; the competitive binding agent may comprise an ACE2 receptor protein or a functionally active fragment thereof.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof may comprise the full-length SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or functionally active fragment thereof may comprise a trimeric full-length SARS-CoV-2 spike protein.
  • the RBD can be linked directly or indirectly to the Fc region. In some embodiments, the RBD can be fused in-frame to the Fc.
  • the Fc region may be selected from the group consisting of rFc, mFc, and hFc.
  • the Fc region can be rFc.
  • the Fc region can be mFc.
  • the Fc region can be hFc.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:8.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:9.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO: 10.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO: 11.
  • the concentration of the SARS-CoV-2 spike protein or functionally active fragment thereof may be 50ng/mL-4000ng/mL.
  • it can be 50ng/mL-3500ng/mL, 50ng/mL-3000ng/mL, 50ng/mL-2000ng/mL, 50ng/mL-1500ng/mL, 50ng/mL-1000ng/mL, 50ng/mL-900ng/mL or 50ng/mL-800ng/mL.
  • the concentration of the SARS-CoV-2 spike protein or its functionally active fragment may be 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL, 800ng/mL, 900ng/mL, 1000ng /mL, 1500ng/mL, 2000ng/mL, 2500ng/mL, 3000ng/mL, 3500ng/mL or 4000ng/mL.
  • the ACE2 receptor protein or functionally active fragment thereof may comprise a human ACE2 receptor protein or functionally active fragment thereof.
  • the ACE2 receptor protein or functionally active fragment thereof may comprise an extracellular domain ECD.
  • the ACE2 receptor protein or functionally active fragment thereof may be linked directly or indirectly to the Fc region.
  • the ACE2 receptor protein or functionally active fragment thereof can be fused in-frame to the Fc region.
  • the Fc region is selected from the group consisting of rFc, mFc, and hFc.
  • rFc for example, mFc.
  • hFc for example, rFc.
  • rFc for example, mFc.
  • hFc for example, rFc.
  • the ACE2 receptor protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the ACE2 receptor protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the ACE2 receptor protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:4.
  • the ACE2 receptor protein or functionally active fragment thereof may comprise one or more markers capable of producing, directly or indirectly, the presence of the ACE2 receptor protein or functionally active fragment thereof and/or content signals.
  • the marker can include horseradish peroxidase (HRP) or an HRP-like enzyme.
  • HRP horseradish peroxidase
  • HRP-like enzyme an HRP-like enzyme
  • the reaction concentration of the ACE2 receptor protein or its functionally active fragment may be 100 ng/mL-2000 ng/mL.
  • it can be 100ng/mL-1500ng/mL, 100ng/mL-1000ng/mL, 100ng/mL-800ng/mL, 500ng/mL-2000ng/mL, 500ng/mL-1500ng/mL, 500ng/mL-1000ng/mL or 500ng/mL-800ng/mL.
  • it can be 200ng/mL, 300ng/mL, 400ng/mL, 500ng/mL, 600ng/mL, 700ng/mL, 800ng/mL, 900ng/mL, 1000ng/mL, 1500ng/mL or 2000ng/mL.
  • the RBD can be coated at a concentration of 50 ng/mL to 4000 ng/mL.
  • it can be 50ng/mL-3500ng/mL, 50ng/mL-3000ng/mL, 50ng/mL-2000ng/mL, 50ng/mL-1500ng/mL, 50ng/mL-1000ng/mL, 50ng/mL-900ng/mL mL or 50ng/mL-800ng/mL.
  • it can be 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL, 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL, 500ng/mL, 600ng/mL, 700ng/mL , 800ng/mL, 900ng/mL or 1000ng/mL.
  • the coating concentration of the RBD may be 50ng/mL-4000ng/mL
  • the reaction concentration of the ACE2 receptor protein or its functionally active fragment may be 500ng/mL-1500ng/mL.
  • it may include a sample diluent, which may have a pH of 6.0-9.6.
  • a calibrator may include one or more reference antibody molecules or antigens thereof that specifically recognize the SARS-CoV-2 spike protein or functionally active fragment thereof Combine fragments.
  • the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof .
  • the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof .
  • the reference antibodies can include antibodies 32-1, 12-5, and 12-8.
  • the concentration of the reference antibody may be from 50 ng/mL to 1000 ng/mL.
  • the application provides a method for detecting SARS-CoV-2 neutralizing antibodies in a sample, which can include:
  • step 3 adding the incubated product in step 2) to step 1);
  • SARS-CoV-2 spike protein or functionally active fragment thereof may comprise a receptor binding region RBD, and the RBD may comprise one or more markers capable of producing, directly or indirectly, the Signals for the presence and/or content of RBD.
  • the marker can include horseradish peroxidase (HRP) or an HRP-like enzyme.
  • HRP horseradish peroxidase
  • HRP-like enzyme an HRP-like enzyme
  • the RBD can be linked directly or indirectly to the Fc region.
  • the RBD can be fused in-frame to the Fc.
  • the Fc region may be selected from the group consisting of rFc, mFc, and hFc.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:7.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:8.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:9.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO: 10.
  • the SARS-CoV-2 spike protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO: 11.
  • the concentration of the SARS-CoV-2 spike protein or functionally active fragment thereof may be 50ng/mL-4000ng/mL.
  • the ACE2 receptor protein or functionally active fragment thereof may be linked directly or indirectly to the Fc region.
  • the ACE2 receptor protein or functionally active fragment thereof can be fused in-frame to the Fc region.
  • the Fc region may be selected from the group consisting of rFc, mFc, and hFc.
  • the ACE2 receptor protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:2.
  • the ACE2 receptor protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the ACE2 receptor protein or functionally active fragment thereof may comprise the amino acid sequence shown in SEQ ID NO:4.
  • the coating concentration of the ACE2 receptor protein or functionally active fragment thereof may be 1 ⁇ g/mL-20 ⁇ g/mL.
  • it can be 1 ⁇ g/mL-15 ⁇ g/mL, 1 ⁇ g/mL-10 ⁇ g/mL, 1 ⁇ g/mL-8 ⁇ g/mL, 1 ⁇ g/mL-7 ⁇ g/mL, 1 ⁇ g/mL-6 ⁇ g/mL or 1 ⁇ g/mL-5 ⁇ g/mL .
  • it can be 2 ⁇ g/mL-10 ⁇ g/mL, 2 ⁇ g/mL-8 ⁇ g/mL, 2 ⁇ g/mL-6 ⁇ g/mL, 3 ⁇ g/mL-10 ⁇ g/mL, 3 ⁇ g/mL-8 ⁇ g/mL or 3 ⁇ g/mL-6 ⁇ g/mL .
  • it can be 1 ⁇ g/mL, 2 ⁇ g/mL, 3 ⁇ g/mL, 4 ⁇ g/mL, 5 ⁇ g/mL, 6 ⁇ g/mL, 7 ⁇ g/mL, 8 ⁇ g/mL, 9 ⁇ g/mL, 10 ⁇ g/mL, 15 ⁇ g/mL or 20 ⁇ g/mL .
  • the reaction concentration of the RBD can be 10 ng/mL-200 ng/mL.
  • it can be 10ng/mL-150ng/mL, 10ng/mL-100ng/mL, 10ng/mL-80ng/mL, 10ng/mL-50ng/mL, 10ng/mL-40ng/mL, 10ng/mL-30ng/mL or 10ng/mL-20ng/mL.
  • it can be 20ng/mL-150ng/mL, 20ng/mL-100ng/mL, 20ng/mL-80ng/mL, 20ng/mL-50ng/mL, 20ng/mL-40ng/mL or 20ng/mL-30ng/mL .
  • it can be 10 ng/mL, 20 ng/mL, 30 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL or 100 ng/mL.
  • the coating concentration of the ACE2 receptor protein or its functionally active fragment may be 1 ⁇ g/mL-10 ⁇ g/mL
  • the reaction concentration of the RBD may be 20 ng/mL-150 ng/mL.
  • the coating concentration of the ACE2 receptor protein or its functionally active fragment may be 3 ⁇ g/mL-6 ⁇ g/mL
  • the reaction concentration of the RBD may be 20 ng/mL-40 ng/mL.
  • it may include a sample diluent, which may have a pH of 6.0-9.6.
  • a calibrator may include one or more reference antibody molecules or antigens thereof that specifically recognize the SARS-CoV-2 spike protein or functionally active fragment thereof Combine fragments.
  • the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 2 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof .
  • the one or more reference antibody molecules or antigen-binding fragments thereof can specifically recognize at least 3 different epitopes on the SARS-CoV-2 spike protein or functionally active fragment thereof .
  • the reference antibodies can include antibodies 32-1, 12-5, and 12-8.
  • the reference antibody may be at a concentration of 50 ng/mL to 1000 ng/mL.
  • the sample may be of natural or synthetic origin, and may be obtained by any means known to those skilled in the art.
  • the sample can be a tissue or fluid sample isolated from a subject, including but not limited to urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirate, and bronchial lavage fluid.
  • the samples can be research samples and clinical samples.
  • the sample can also be a blood sample for blood transfusion or therapy.
  • the sample can also be synthetic and include, but is not limited to, in vitro cell culture components, including but not limited to conditioned media, recombinant cells, and cell components.
  • sample and “sample” are used interchangeably.
  • the sample is derived from a subject.
  • the subject generally refers to any organism, including but not limited to mammals such as mice, rats, dogs, guinea pigs, ferrets, rabbits, and primates.
  • a subject may be a human, pet or livestock.
  • the subject can be a recovered patient from COVID-19, a healthy subject and/or a subject who has been treated with a COVID-19 vaccine.
  • the method may include the steps of:
  • Step 1 coating antigen: Dilute the SARS-CoV-2 spike protein or its functionally active fragment with the coating solution, and add the SARS-CoV-2 spike protein or its functionally active fragment dilution into the enzyme-labeled well of the reaction plate .
  • Step 2 add the sample to be tested and incubate for 30-60 minutes (eg, incubate for 30 minutes, incubate for 35 minutes, incubate for 40 minutes, incubate for 45 minutes, incubate for 50 minutes, incubate for 55 minutes, or incubate for 60 minutes).
  • Step 3 add a competitive binding agent and incubate for 30-60 minutes (eg, incubate for 30 minutes, incubate for 35 minutes, incubate for 40 minutes, incubate for 45 minutes, incubate for 50 minutes, incubate for 55 minutes, or incubate for 60 minutes).
  • a competitive binding agent eg, incubate for 30 minutes, incubate for 35 minutes, incubate for 40 minutes, incubate for 45 minutes, incubate for 50 minutes, incubate for 55 minutes, or incubate for 60 minutes).
  • Step 4 color development, stop reading.
  • the sample to be tested can be incubated in a 35°C-40°C environment (eg, the incubation temperature can be 35°C, 35.5°C, 36°C, 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37°C, 37.1°C, 37.2°C, 37.3°C, 37.4°C, 37.5°C, 37.6°C, 37.7°C, 37.8°C, 37.9°C, 38°C, 38.5°C, 39°C, 39.5°C or 40°C).
  • the incubation temperature can be 35°C, 35.5°C, 36°C, 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37°C, 37.1°C, 37.2°C, 37.3°C, 37.4°C, 37.5°C, 37.6°C, 37.7°C, 37.8°C, 37.9°C, 38°C,
  • the competing binding agent can be incubated in a 35°C-40°C environment (eg, the incubation temperature can be 35°C, 35.5°C, 36°C, 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37°C, 37.1°C, 37.2°C, 37.3°C, 37.4°C, 37.5°C, 37.6°C, 37.7°C, 37.8°C, 37.9°C, 38°C, 38.5°C, 39°C, 39.5°C or 40°C).
  • the incubation temperature can be 35°C, 35.5°C, 36°C, 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37°C, 37.1°C, 37.2°C, 37.3°C, 37.4°C, 37.5°C, 37.6°C, 37.7°C, 37.8°C, 37.9°C, 38°C, 3
  • the color development process can be performed in a 35°C-40°C environment (eg, can be 35°C, 35.5°C, 36°C, 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37°C , 37.1°C, 37.2°C, 37.3°C, 37.4°C, 37.5°C, 37.6°C, 37.7°C, 37.8°C, 37.9°C, 38°C, 38.5°C, 39°C, 39.5°C or 40°C).
  • a detection kit for SARS-CoV-2 neutralizing antibody comprising:
  • SARS-CoV-2 spike protein or a functionally active fragment thereof
  • a competitive binding agent comprising a substance capable of competing with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or a functionally active fragment thereof.
  • kits of embodiment 1, wherein the SARS-CoV-2 spike protein or functionally active fragment thereof comprises a receptor binding region RBD.
  • kits according to embodiment 7, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:7.
  • kits according to embodiment 7, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:8.
  • kits according to embodiment 7, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:9.
  • kits according to embodiment 3, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 10.
  • kits according to embodiment 4, wherein the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 11.
  • kits of embodiment 13, wherein the ACE2 receptor protein or a functionally active fragment thereof comprises a human ACE2 receptor protein or a functionally active fragment thereof.
  • kit of embodiment 16 wherein the ACE2 receptor protein or functionally active fragment thereof is fused in-frame to the Fc region.
  • kit of embodiment 18, wherein the competitive binding agent comprises the amino acid sequence set forth in SEQ ID NO:4.
  • kit of embodiment 13, wherein the competing binding agent comprises one or more markers capable of directly or indirectly generating a signal showing the presence and/or content of the competing binding agent .
  • kits of embodiment 22, wherein the marker comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
  • HRP horseradish peroxidase
  • kit of any one of embodiments 1-23, comprising a sample diluent, the sample diluent having a pH of 6.0-9.6.
  • the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 33-35;
  • the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 36-38
  • the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 39-41; or
  • the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 42-44
  • the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 45-47.
  • the VH comprises the amino acid sequence shown in SEQ ID NO: 21, and the VL comprises the amino acid sequence shown in SEQ ID NO: 22;
  • the VH comprises the amino acid sequence shown in SEQ ID NO:24, and the VL comprises the amino acid sequence shown in SEQ ID NO:25; or
  • the VH comprises the amino acid sequence shown in SEQ ID NO:27
  • the VL comprises the amino acid sequence shown in SEQ ID NO:28.
  • kits of embodiment 32, wherein the marker comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
  • HRP horseradish peroxidase
  • a method for detecting SARS-CoV-2 neutralizing antibodies in a sample comprising:
  • a method for detecting SARS-CoV-2 neutralizing antibodies in a sample comprising:
  • step 2) adding the sample to be tested in step 1) to react with the SARS-CoV-2 spike protein or its functionally active fragment;
  • the competitive binding agent comprises a substance capable of competing with the SARS-CoV-2 neutralizing antibody for binding to the SARS-CoV-2 spike protein or its functionally active fragment;
  • the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a receptor binding region RBD; the competitive binding agent comprises an ACE2 receptor protein or a functionally active fragment thereof.
  • SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:10.
  • SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:11.
  • ACE2 receptor protein or functionally active fragment thereof comprises a human ACE2 receptor protein or functionally active fragment thereof.
  • ACE2 receptor protein or functionally active fragment thereof comprises one or more markers capable of producing, directly or indirectly, a marker showing the Signals for the presence and/or amount of ACE2 receptor protein or a functionally active fragment thereof.
  • the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 33-35;
  • the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 36-38
  • the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 39-41; or
  • the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 42-44
  • the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 45-47.
  • the VH comprises the amino acid sequence shown in SEQ ID NO: 21, and the VL comprises the amino acid sequence shown in SEQ ID NO: 22;
  • the VH comprises the amino acid sequence shown in SEQ ID NO:24, and the VL comprises the amino acid sequence shown in SEQ ID NO:25; or
  • the VH comprises the amino acid sequence shown in SEQ ID NO:27
  • the VL comprises the amino acid sequence shown in SEQ ID NO:28.
  • sample is selected from the group consisting of urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirate, and bronchial lavage fluid.
  • a method for detecting neutralizing antibodies to SARS-CoV-2 in a sample comprising:
  • step 3 adding the incubated product in step 2) to step 1);
  • the SARS-CoV-2 spike protein or a functionally active fragment thereof comprises a receptor binding region RBD
  • the RBD comprises one or more markers capable of directly or indirectly producing a protein indicative of the RBD presence and/or content signals.
  • the marker comprises horseradish peroxidase (HRP) or an HRP-like enzyme.
  • SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:7.
  • SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:8.
  • SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:9.
  • SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:10.
  • SARS-CoV-2 spike protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 11.
  • ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:2.
  • ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:3.
  • ACE2 receptor protein or functionally active fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 30-32, and the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 33-35;
  • the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 36-38
  • the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 39-41; or
  • the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID NOs: 42-44
  • the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID NOs: 45-47.
  • the VH comprises the amino acid sequence shown in SEQ ID NO: 21, and the VL comprises the amino acid sequence shown in SEQ ID NO: 22;
  • the VH comprises the amino acid sequence shown in SEQ ID NO:24, and the VL comprises the amino acid sequence shown in SEQ ID NO:25; or
  • the VH comprises the amino acid sequence shown in SEQ ID NO:27
  • the VL comprises the amino acid sequence shown in SEQ ID NO:28.
  • test sample is selected from the group consisting of urine, saliva, serum, plasma, whole blood, nasopharyngeal aspirate, and bronchial lavage fluid.
  • test sample is derived from a subject.
  • HRP horseradish peroxidase
  • HRP horseradish peroxidase labeled human ACE2 receptor protein: add human ACE2 receptor protein and activated horseradish peroxidase (HRP) in a mass ratio of 1:4, add human ACE2 receptor protein and Sodium carbonate buffer (pH 9.6) with 1/5 of the total volume of horseradish peroxidase (HRP) was placed at room temperature for 1 hour in the dark; 1/10 of the volume of horseradish peroxidase (HRP) was added as a reducing agent, Place in the dark at room temperature for 1 hour; add 1/10 volume of horseradish peroxidase (HRP) terminator, place in the dark at room temperature for 30 minutes, and then use or store at 4°C.
  • HRP horseradish peroxidase
  • Coating antigen use 1x coating solution (10x coating solution is 15.9g/L Na 2 CO 3 +29.3g/L NaHCO 3 , diluted with ddH 2 O to 1x use) to dilute RBD antigen to 0.3 ⁇ g/mL Mix well, add the RBD antigen dilution solution to the enzyme-labeled wells of the reaction plate, 100 ⁇ L per well, cover the reaction plate and place it at 4°C overnight to coat, the upper layer is liquid, and the coated RBD antigen is adsorbed on the reaction plate. superior.
  • Color development Combine color development solution A (2.1g/L citric acid monohydrate+8.448g/L sodium acetate+0.5g/L carbamide peroxide+0.08g/L EDTA+2mL/L P300) and B solution (10mL /L 100x basic buffer (105.6g/L citric acid monohydrate+10.3g/L sodium acetate+2mL/L P300)+100mL/L glycerin+0.5g/L TMB hydrochloride+1.67mL/L Na 2 S 2 O 3 (20mg/mL)+2mL/L P300) was mixed at a volume ratio of 1:1 to form a working solution for color development, and 100 ⁇ L of working solution for color development was added to each well. 10 minutes.
  • stop solution 252.5g/L citric acid monohydrate + 26.6mL/L concentrated sulfuric acid
  • HRP horseradish peroxidase
  • HRP horseradish peroxidase labeled RBD protein: add RBD protein and activated horseradish peroxidase (HRP) in a mass ratio of 1:4, add RBD protein and horseradish peroxidase (HRP) 1/5 of the total volume of sodium carbonate buffer (pH 9.6), placed in the dark at room temperature for 1 hour; add 1/10 volume of horseradish peroxidase (HRP) reducing agent, placed in the dark at room temperature for 1 hour; add 1 Terminator/10 volume of horseradish peroxidase (HRP), placed at room temperature for 30 minutes in the dark, ready to use or stored at 4°C.
  • HRP horseradish peroxidase
  • Coating protein Use 1x coating solution to dilute the ACE2 protein to 0.3 ⁇ g/mL and mix well. Add the ACE2 protein dilution to the enzyme-labeled wells of the reaction plate, 100 ⁇ L per well, cover the reaction plate and place it in 4. °C overnight coating, the upper layer is liquid, and the coated ACE2 protein is adsorbed on the reaction plate.
  • Color development Mix the color development solution A and B solution at a volume ratio of 1:1 to form a color development working solution, add 100 ⁇ L of the color development working solution to each well, replace the plate and stick it well, and place it at 37 °C for reaction 10 in the dark. minute.
  • titer value using the above method to detect the sample, when the inhibition rate of the sample under a certain dilution factor is >15%, and the inhibition rate of the sample under the next dilution gradient of the dilution factor is ⁇ 15%, then the dilution rate titer for this sample.
  • the titer is the largest dilution that still yields a positive result and reflects the titer of the antibody.
  • Example 2 Using three different ACE2-mFc concentration reaction plates, use five dilution ratios of 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL and 30ng/mL to dilute RBD-mFc-HRP, according to the operation of Example 2 Steps: Detect the antibody at gradient dilution concentrations, and compare the detection limit differences under different ACE2-mFc coating concentrations and RBD-mFc-HRP dilution ratios.
  • the cut-off value is the magnitude of the sample being examined and is used to determine whether the result is above or below the clinical or analytical cut-off point.
  • the cut-off value is set to give a brief combination of sensitivity and specificity.
  • the Cut-off value was determined based on the evaluation results of 27 recovered patient sera, 1452 non-COVID-19 infected human serum samples, and 72 vaccine sera (one third of placebo), and was analyzed by SPSS software. off value, according to the operation steps of Example 1, the sensitivity of our kit is >90% and the specificity is >99%. The vaccine effect evaluation is 95% consistent with the real virus test results.
  • Sensitivity Sensitivity, which indicates the detection rate of positive samples.
  • 0.997 means that 99.7% of positive samples can be correctly judged as positive results.
  • Specificity Indicates the detection rate of negative samples.
  • 0.991 means that 99.1% of negative samples can be correctly judged as negative results.
  • the test variable results are shown in Table 7.
  • the receiver operating characteristic curve (or ROC curve) is shown in Figure 2.
  • the curve coordinates and test results As shown in Table 8.

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Abstract

La présente demande concerne un kit et une méthode de détection d'un anticorps neutralisant le SARS-CoV-2. La présente demande se rapporte au domaine de la médecine biologique, et concerne un kit et une méthode de détection d'un anticorps neutralisant le SARS-CoV-2. Une étiquette protéique est utilisée pour modifier RBD et ACE2 de manière à les rendre plus stables et plus réactives, et la sensibilité de détection est ainsi améliorée.
PCT/CN2021/120953 2020-11-11 2021-09-27 Kit et méthode de détection d'anticorps neutralisant le sars-cov-2 WO2022100303A1 (fr)

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US20180043014A1 (en) * 2008-08-20 2018-02-15 Celldex Therapeutics, Inc. Compositions Using Antibodies Directed to GPNMB and Uses Thereof
CN108690134A (zh) * 2017-04-07 2018-10-23 厦门大学 用于治疗乙肝感染及相关疾病的抗体
CN111562369A (zh) * 2020-06-18 2020-08-21 威海威高生物科技有限公司 SARS-CoV-2中和抗体检测试剂盒
CN111562368A (zh) * 2020-06-18 2020-08-21 威海威高生物科技有限公司 SARS-CoV-2中和抗体检测试剂盒
CN111592594A (zh) * 2020-03-13 2020-08-28 北京大学 一种抗新型冠状病毒的单克隆抗体及其应用

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US20180043014A1 (en) * 2008-08-20 2018-02-15 Celldex Therapeutics, Inc. Compositions Using Antibodies Directed to GPNMB and Uses Thereof
CN108690134A (zh) * 2017-04-07 2018-10-23 厦门大学 用于治疗乙肝感染及相关疾病的抗体
CN111592594A (zh) * 2020-03-13 2020-08-28 北京大学 一种抗新型冠状病毒的单克隆抗体及其应用
CN111562369A (zh) * 2020-06-18 2020-08-21 威海威高生物科技有限公司 SARS-CoV-2中和抗体检测试剂盒
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