WO2022253260A1 - Trousse de détection pour neutralisation d'anticorps contre un nouveau coronavirus et sa souche mutante - Google Patents

Trousse de détection pour neutralisation d'anticorps contre un nouveau coronavirus et sa souche mutante Download PDF

Info

Publication number
WO2022253260A1
WO2022253260A1 PCT/CN2022/096541 CN2022096541W WO2022253260A1 WO 2022253260 A1 WO2022253260 A1 WO 2022253260A1 CN 2022096541 W CN2022096541 W CN 2022096541W WO 2022253260 A1 WO2022253260 A1 WO 2022253260A1
Authority
WO
WIPO (PCT)
Prior art keywords
ace2
protein
fragment
cov
sars
Prior art date
Application number
PCT/CN2022/096541
Other languages
English (en)
Chinese (zh)
Inventor
覃喜建
谢琳琳
汤双双
Original Assignee
南京金斯瑞生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京金斯瑞生物科技有限公司 filed Critical 南京金斯瑞生物科技有限公司
Publication of WO2022253260A1 publication Critical patent/WO2022253260A1/fr

Links

Images

Classifications

    • 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
    • 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
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • G01N33/573—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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)

Definitions

  • the application relates to a kit for detecting neutralizing antibodies to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and/or its mutant strains, and using the kit to detect the presence of SARS-CoV-2 in samples CoV-2 and/or mutant strain neutralizing antibodies, and/or methods for evaluating the neutralizing activity of antibodies against SARS-CoV-2 and mutant strains thereof.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus type 2
  • the novel coronavirus pneumonia (COVID-19) has spread widely around the world and poses a serious threat to human health and public safety. It is caused by the novel coronavirus (SARS-CoV-2), a positive-sense single-stranded RNA virus.
  • SARS-CoV-2 novel coronavirus
  • the spike protein (S protein) of 2019-nCoV is the main molecule of the virus infecting host cells. It is composed of two parts: the S1 subunit responsible for binding to cell surface receptors and the S2 subunit that mediates membrane fusion.
  • Human angiotensin-converting enzyme 2 (ACE2) is the main binding site of S protein, and S protein binds to ACE2 protein through its receptor binding domain (RBD).
  • the novel coronavirus neutralizing antibody refers to a class of antibodies that bind to the S protein RBD to resist host cells infected by the novel coronavirus. It can inhibit the binding of host cell ACE2 receptor and SARS-CoV-2 virus S protein RBD, and the infectivity of SARS-CoV-2 is closely related to the affinity of S protein binding to human host cell surface ACE2. Mutations in the S protein may affect its infectivity by changing the infection mechanism of the virus. For example, the transmissibility of novel coronavirus mutants with S protein RBD domain K417N, E484 and N501 mutations was significantly enhanced.
  • COVID-19 vaccines plasma from convalescents and recombinant antibody drugs are effective means to prevent and treat COVID-19.
  • the new crown vaccine protects the human body by inducing the human immune system to produce neutralizing antibodies targeting the SARS-CoV-2 spike protein.
  • the active ingredient in convalescent plasma is a neutralizing antibody against the SARS-CoV-2 spike protein.
  • Recombinant antibody drugs are neutralizing antibodies developed against the SARS-CoV-2 spike protein target. Mutations in the spike protein of the new strain will affect the effectiveness of vaccine recipients, recovered patients, and new crown antibody drug injectors against new coronavirus mutants.
  • Germanians such as Markus Hoffmann used pseudoviruses with wild-type S protein, B.1.1.7 mutant S protein, B.1.351 mutant S protein, and P.1 mutant S protein to study wild-type new coronavirus recovery patients Neutralizing activity of plasma and Pfizer's BNT162b2 vaccine recipient sera against various novel coronavirus mutant strains. The results of the study showed that the plasma of wild-type 2019-nCoV patients may have a partial protective effect on the B.1.351 mutant strain and the P.1 mutant strain. The sera of BNT162b2 vaccine recipients had a strong neutralizing effect on the B.1.1.7 mutant strain, and the protection against the B.1.351 mutant strain and the P.1 mutant strain might be weakened.
  • plaque reduction neutralization test PRNT
  • pseudovirus neutralization test pVNT
  • PRNT plaque reduction neutralization test
  • pVNT pseudovirus neutralization test
  • Plaque reduction neutralization test is used to infect cells after different strains of the new coronavirus and samples with neutralizing antibodies to the new coronavirus are used to infect the cells.
  • CCID 50 half the infectious dose
  • TCID 50 tissue cell infectious dose
  • the pseudovirus neutralization test uses pseudovirus instead of real virus, which reduces the risk of virus infection.
  • it is necessary to construct a variety of new coronavirus mutant pseudoviruses, which is a heavy workload and cannot meet the needs of laboratory testing.
  • the sVNT method is a cell-free and live virus detection method for neutralizing antibody activity. This method has a short detection cycle and can obtain detection results in less than two hours in a diagnostic laboratory.
  • the inventors of the present application found that in the surrogate virus neutralization test, antibodies produced by immunization with a new coronavirus S1 protein or RBD have either good or good blocking effect on the combination of S1 protein or RBD used as an immune agent and ACE2 Poor and unpredictable.
  • SARS-CoV-2 virus strains antibodies can be used to treat infections caused by.
  • SARS-CoV-2 strains of SARS-CoV-2 virus strains generated by immunization have a preventive effect.
  • virus strain or strains these people may be infected with.
  • the application provides a combined detection kit for neutralizing antibodies to novel coronavirus and mutant strains, and a method for using the kit to detect neutralizing antibodies to novel coronavirus and mutant strains thereof, and to use the kit to evaluate vaccines or A method for neutralizing activity of antibodies against novel coronavirus and its mutants.
  • the kit of the present application can detect the level of neutralizing antibodies against various new coronavirus strains in the same sample, and by comparing the difference in the detection values of different new coronavirus neutralizing antibodies, the effect of vaccines or antibody drugs on different virus strains can be analyzed. The effectiveness of treatment and prevention can also assist in the analysis of the source of infection in infected populations.
  • the detection principle of the kit and method of the present application is that the neutralizing antibody binds to the Spike protein or its ACE2-binding fragment, thereby blocking the binding of the Spike protein or its ACE2-binding fragment to ACE2 or its fragment to a certain extent.
  • the kit and method of the present application have the advantages of simple operation, high throughput, short detection period, and low risk of infection.
  • the application provides a kit comprising:
  • Spike proteins encoded by SARS-CoV-2 or ACE2-binding fragments thereof selected from wild-type SARS-CoV-2-encoded Spike proteins or ACE2-binding fragments thereof, and SARS-CoV-2 mutations Spike protein or its ACE2 binding fragment encoded by the strain,
  • a detection signal molecule for detecting the interaction between the spike protein of i) or its ACE2-binding fragment and ii) the ACE2 protein or its fragment
  • the spike protein or its ACE2-binding fragment or ii) the ACE2 protein or its fragment is linked or coupled with a tag molecule, and iii) the signal detection molecule specifically binds to the tag molecule.
  • Two or more Spike proteins encoded by SARS-CoV-2 or ACE2 binding fragments thereof may comprise Spike proteins encoded by wild-type SARS-CoV-2 or ACE2 binding fragments thereof, and Spike proteins encoded by SARS-CoV-2 mutant strains. Spike protein or its ACE2-binding fragment. The two or more Spike proteins encoded by SARS-CoV-2 or ACE2-binding fragments thereof may only comprise Spike proteins or ACE2-binding fragments thereof encoded by SARS-CoV-2 mutant strains.
  • the Spike protein or ACE2-binding fragment thereof of i) and the ACE2 protein or fragment thereof of ii) may exist in any suitable molar ratio, such as a molar ratio of 5:1-11:1.
  • Each Spike protein or its ACE2-binding fragment can be packaged separately, and the ACE2 protein or its fragment can be packaged separately with each Spike protein or its ACE2-binding fragment at a molar ratio of 1:11-1:5.
  • the ACE2 protein or fragment thereof is packaged separately with each spike protein or ACE2-binding fragment thereof in a molar ratio of about 1:11.
  • the ACE2 protein or fragment thereof and each spike protein or ACE2-binding fragment thereof are packaged separately in an amount of about 1:10 molar ratio.
  • the ACE2 protein or fragment thereof is packaged separately with each spike protein or ACE2-binding fragment thereof in an amount of about 1:5 molar ratio.
  • the Spike protein or ACE2-binding fragment thereof or ii) the ACE2 protein or a fragment thereof may be linked with a tag molecule.
  • the Spike protein or its ACE2-binding fragment is linked with a tag molecule, and ii) the ACE2 protein or its fragment is immobilized on a solid support.
  • the Spike protein or its ACE2-binding fragment is immobilized on a solid support, and ii) the ACE2 protein or its fragment is linked with a tag molecule.
  • the spike protein encoded by SARS-CoV-2 or its ACE2 binding fragment can be selected from the spike protein encoded by wild type SARS-CoV-2 or its ACE2 binding fragment and the spike protein encoded by SARS-CoV-2 mutant strain or an ACE2-binding fragment thereof.
  • SARS-CoV-2 mutant strain can be any mutant strain, for example B.1.1.7 (GISAID Accession ID: EPI_ISL_1234251), B.1.351 (GISAID Accession ID: EPI_ISL_1191083), B.1.1.29 (GISAID Accession ID: EPI_ISL_729975) , A.23.1 (GISAID login ID: EPI_ISL_955136), B.1.427 (GISAID login ID: EPI_ISL_1291745), B.1.429 (GISAID login ID: EPI_ISL_1291171), P.1 (GISAID login ID: EPI_ISL_1289959), B.1.36.27 (GISAID login ID: EPI_ISL_1191108), B.1.1.284 (GISAID login ID: EPI_ISL_1257806), B.1.1.28 (GISAID login ID: EPI_ISL_1213565), B.1.1.106 (GISAID login ID: EPI_ISL_125
  • the Spike protein encoded by SARS-CoV-2 or its ACE2-binding fragment may comprise or consist of an S1 subunit.
  • the Spike protein encoded by SARS-CoV-2 or an ACE2-binding fragment thereof may comprise at least 70%, 75%, 80%, 85%, 90%, 91% of SEQ ID NO: 1 or 4. %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity amino acid sequence, or with SEQ ID NO: 1 or 4 has at least 70%, 75%, Amino acid sequence constituents with 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the spike protein encoded by SARS-CoV-2 or its ACE2 binding fragment comprises the amino acid sequence shown in SEQ ID NO: 1 or 4. In some specific embodiments, the amino acid sequence of the Spike protein encoded by SARS-CoV-2 or its ACE2 binding fragment is as shown in SEQ ID NO: 1 or 4.
  • the Spike protein encoded by SARS-CoV-2 or its ACE2-binding fragment may comprise, or consist of, the receptor-binding domain (RBD) of the Spike protein.
  • the Spike protein encoded by SARS-CoV-2, or an ACE2-binding fragment thereof may comprise any of SEQ ID NOs: 2, 5-11, 17 and 18 having at least 70%, 75%, An amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or consisting of an amino acid sequence with SEQ ID NO:2 , any of 5-11, 17 and 18 have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% sequence identity.
  • the spike protein encoded by SARS-CoV-2 or its ACE2 binding fragment comprises the amino acid sequence shown in any one of SEQ ID NO:2, 5-11, 17 and 18. In other embodiments, the amino acid sequence of the Spike protein encoded by SARS-CoV-2 or its ACE2 binding fragment is as shown in any one of SEQ ID NO:2, 5-11, 17 and 18.
  • the Spike protein encoded by SARS-CoV-2 or an ACE2-binding fragment thereof may comprise, or consist of, the receptor binding motif (RBM) of the Spike protein.
  • the Spike protein or ACE2-binding fragment thereof may comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% of SEQ ID NO:3 , 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 85%, 90%, 91%, Amino acid sequence constituents with 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • the spike protein or ACE2-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO:3.
  • the amino acid sequence of the Spike protein or its ACE2-binding fragment is shown in SEQ ID NO:3.
  • the ACE2 protein or fragment thereof may comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of SEQ ID NO: 12, 14 or 15 %, 97%, 98% or 99% sequence identity of the amino acid sequence, or by at least 70%, 75%, 80%, 85%, 90%, 91%, 92% with SEQ ID NO: 12, 14 or 15 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of amino acid sequences.
  • the ACE2 protein or fragment thereof may comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% of SEQ ID NO: 15 , 96%, 97%, 98% or 99% amino acid sequence identity.
  • the ACE2 protein or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 12, 14 or 15.
  • the amino acid sequence of the ACE2 protein or a fragment thereof is shown in SEQ ID NO: 12, 14 or 15.
  • the ACE2 protein or fragment thereof may comprise, or consist of, an extracellular domain.
  • the ACE2 protein or fragment thereof may comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, Amino acid sequence having 95%, 96%, 97%, 98% or 99% sequence identity, or consisting of at least 70%, 75%, 80%, 85%, 90%, 91% to SEQ ID NO: 13 or 16 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity.
  • the ACE2 protein or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 13 or 16. In other embodiments, the amino acid sequence of the ACE2 protein or a fragment thereof is shown in SEQ ID NO: 13 or 16.
  • the tag molecule can be a protein tag, such as His tag, Flag tag, StrepII tag, Fc tag, Avi tag, c-Myc tag, GST tag, and HA tag, or a small molecule tag, such as biotin.
  • the tag molecule can be attached to the N-terminus or C-terminus of i) the Spike protein or its ACE2-binding fragment or ii) the ACE2 protein or its fragment through eg recombinant expression.
  • the label molecule can be coupled to one or some sites of the spike protein or its ACE2 binding fragment of i) or ii) of the ACE2 protein or its fragment through an enzymatic reaction, etc., and the coupling site can be Randomly, it can also be deterministic, such as by making additional modifications on some amino acid residues of i) the Spike protein or its ACE2-binding fragment or ii) the ACE2 protein or its fragment.
  • the signal detection molecule can be a molecule that specifically binds to the tag molecule and generates a signal. "Specific binding” here means that, compared with other components in the kit, the signal detection molecule binds to the tag molecule with higher affinity and/or longer duration. In particular, the signal detection molecule has no binding force to the components of the kit other than the label molecule, or the binding force is very weak, or the binding time is very short.
  • Signal detection molecules can be anti-His, anti-Flag, anti-StrepII, anti-Fc, anti-Avi, anti-c-Myc labeled with horseradish peroxidase, alkaline phosphatase or acridinium ester , anti-GST or anti-HA antibody, or streptavidin labeled with horseradish peroxidase, alkaline phosphatase, etc.
  • kits of the present application may also comprise a solid support.
  • the solid support can be a microtiter plate, magnetic beads, etc.
  • the kit of the present application may be a chemiluminescence kit, an immunoturbidimetric kit, a colloidal gold kit, a time-resolved immunofluorescence detection kit, or an enzyme-linked immunosorbent assay (ELISA) kit.
  • chemiluminescence kit an immunoturbidimetric kit, a colloidal gold kit, a time-resolved immunofluorescence detection kit, or an enzyme-linked immunosorbent assay (ELISA) kit.
  • the present application also provides a kit set, comprising more than two sub-kits, each sub-kit comprising:
  • a Spike protein encoded by SARS-CoV-2 or an ACE2 binding fragment thereof selected from the Spike protein encoded by wild-type SARS-CoV-2 or an ACE2 binding fragment thereof, and a mutant strain of SARS-CoV-2 Encoded spike protein or ACE2-binding fragment thereof
  • the spike protein or its ACE2-binding fragment or ii) the ACE2 protein or its fragment is linked or coupled with a tag molecule, and iii) the signal detection molecule specifically binds to the tag molecule.
  • the Spike protein encoded by SARS-CoV-2 or its ACE2-binding fragment can be the Spike protein or its ACE2-binding fragment encoded by wild-type SARS-CoV-2, or the Spike encoded by a mutant strain of SARS-CoV-2 protein or its ACE2-binding fragment.
  • the mutant strain of SARS-CoV-2 can be any mutant strain, such as B.1.1.7, B.1.351, B.1.1.29, A.23.1, B.1.427, B.1.429, P.1, B.1.36. 27. B.1.1.284, B.1.1.28, B.1.1.106, B.1, B.1.1.317, B.1.617, B.1.617.1, B.1.617.2, B.1.617. 3. Or any newly emerging mutant strains.
  • one sub-kit may comprise the Spike protein encoded by wild-type SARS-CoV-2 or its ACE2-binding fragment
  • the other sub-kits may Each contains a spike protein encoded by a SARS-CoV-2 mutant or an ACE2-binding fragment thereof.
  • each kit may include a Spike protein encoded by a SARS-CoV-2 mutant strain or an ACE2-binding fragment thereof.
  • the Spike protein or its ACE2-binding fragment contained in each sub-kit can be made from different SARS-CoV-2 mutant strain encoding.
  • kits set can be a chemiluminescence kit, an immunoturbidimetric kit, a colloidal gold kit, a time-resolved immunofluorescence detection kit, or an enzyme-linked immunosorbent assay (ELISA) kit.
  • chemiluminescence kit an immunoturbidimetric kit
  • colloidal gold kit a colloidal gold kit
  • time-resolved immunofluorescence detection kit a time-resolved immunofluorescence detection kit
  • ELISA enzyme-linked immunosorbent assay
  • the present application provides a method of using the kit of the present application to detect whether there is an antibody selected from a wild-type SARS-CoV-2 antibody and a SARS-CoV-2 mutant antibody in a sample, including:
  • the mutant strain of SARS-CoV-2 can be any mutant strain, such as B.1.1.7, B.1.351, B.1.1.29, A.23.1, B.1.427, B.1.429, P.1, B.1.36. 27. B.1.1.284, B.1.1.28, B.1.1.106, B.1, B.1.1.317, B.1.617, B.1.617.1, B.1.617.2, B.1.617. 3. Or any newly emerging mutant strains.
  • the method may also include the step of aliquoting the sample prior to contacting each of the Spike protein or ACE2-binding fragment thereof, and the ACE2 protein or fragment thereof.
  • the aliquoted sample can be contacted with each spike protein or ACE2-binding fragment thereof separately.
  • the sample can be divided into equal portions.
  • the method may further comprise, after determining the level of interaction between each spike protein or ACE2 binding fragment thereof and ACE2 protein or fragment thereof, comparing the interaction between each spike protein or ACE2 binding fragment thereof and ACE2 protein or fragment thereof Steps for high and low levels of action.
  • more antibodies were present in the samples against SARS-CoV-2 strains containing low-level interacting spike proteins or ACE2-binding fragments thereof.
  • the amount of each Spike protein or ACE2-binding fragment thereof may be: (a) in molar ratio, lower than or equal to the amount of the Spike protein or ACE2-binding fragment thereof that can block the Spike protein or ACE2-binding fragment thereof in the sample contacted with it.
  • the amount of antibody that binds to the ACE2 protein or fragment thereof and (b) is sufficient to generate the Spike protein reflecting i) in the presence of a sample that does not contain antibodies to wild-type SARS-CoV-2 or antibodies to a mutant strain of SARS-CoV-2
  • the amount of each spike protein or ACE2-binding fragment thereof may be such that a response is generated in the presence of a sample that does not contain wild-type SARS-CoV-2 antibodies or SARS-CoV-2 mutant antibodies i) The minimum amount required for the detection signal of the interaction between the spike protein or ACE2-binding fragment thereof of ii) and the ACE2 protein or fragment thereof.
  • the sample can be a blood sample, a lymph sample, a saliva sample, or a joint obtained from a person who has been infected with wild-type SARS-CoV-2 and/or a mutant strain of SARS-CoV-2, or who has been vaccinated against SARS-CoV-2. synovial fluid.
  • the sample can be a solution containing artificially prepared antibodies, such as hybridoma supernatant, a solution containing purified antibodies, or immunized non-human mammalian blood.
  • the present application also provides a method of using the kit set of the present application to detect whether there is an antibody selected from the wild-type SARS-CoV-2 antibody and the SARS-CoV-2 mutant antibody in the sample, including:
  • a reduction in the level of interaction between the Spike protein or its ACE2-binding fragment and the ACE2 protein or its fragment in one or more sub-kits compared to the negative control indicates that there is an antibody against the corresponding Spike protein or its ACE2-binding fragment in the sample.
  • Antibodies to the SARS-CoV-2 strain are particularly useful for antigen binding to the SARS-CoV-2 strain.
  • the mutant strain of SARS-CoV-2 can be any mutant strain, such as B.1.1.7, B.1.351, B.1.1.29, A.23.1, B.1.427, B.1.429, P.1, B.1.36. 27. B.1.1.284, B.1.1.28, B.1.1.106, B.1, B.1.1.317, B.1.617, B.1.617.1, B.1.617.2, B.1.617. 3. Or any newly emerging mutant strains.
  • the method may also include, in each sub-kit, i) the spike protein encoded by SARS-CoV-2 or its ACE2 binding fragment, and ii) the spike protein or its ACE2 binding fragment of i) specifically binding
  • i) the spike protein encoded by SARS-CoV-2 or its ACE2 binding fragment and ii) the spike protein or its ACE2 binding fragment of i) specifically binding
  • Aliquoted samples can be contacted with the spike protein or ACE2-binding fragment thereof in each sub-kit. In one embodiment, the sample can be divided into equal portions.
  • the method may also include, after determining the level of interaction between the spike protein or its ACE2-binding fragment of i) in each sub-kit and (ii) the ACE2 protein or its fragment, comparing the spike protein in each sub-kit The step of increasing the level of interaction between its ACE2-binding fragment and the ACE2 protein or its fragment. Among them, more antibodies were present in the samples against SARS-CoV-2 strains containing low-level interacting spike proteins or ACE2-binding fragments thereof.
  • the amount of spike protein or ACE2-binding fragment thereof in each sub-kit can be: (a) in molar ratio, lower than or equal to the amount of the spike protein or ACE2-binding fragment in the sample contacted with it.
  • the amount of the spike protein or its ACE2-binding fragment in each sub-kit may be: in the presence of a sample that does not contain wild-type SARS-CoV-2 antibodies or SARS-CoV-2 mutant antibodies The minimum amount required to generate a detection signal reflecting the interaction between i) the Spike protein or ACE2-binding fragment thereof and ii) the ACE2 protein or a fragment thereof.
  • the sample can be a blood sample, a lymph sample, a saliva sample, or a joint obtained from a person who has been infected with wild-type SARS-CoV-2 and/or a mutant strain of SARS-CoV-2, or who has been vaccinated against SARS-CoV-2. synovial fluid.
  • the sample can be a solution containing artificially prepared antibodies, such as hybridoma supernatant, a solution containing purified antibodies, or immunized non-human mammalian blood.
  • the application provides a method for using the kit of the application to evaluate the neutralizing activity of a sample to a virus strain selected from wild-type SARS-CoV-2 and SARS-CoV-2 mutant strains, comprising:
  • the reduction of the level of interaction between one or more spike proteins or their ACE2 binding fragments and ACE2 protein or its fragments indicates that there is a reaction to SARS containing the corresponding spike protein or its ACE2 binding fragments in the sample.
  • - Neutralizing activity of CoV-2 strains - Neutralizing activity of CoV-2 strains.
  • the mutant strain of SARS-CoV-2 can be any mutant strain, such as B.1.1.7, B.1.351, B.1.1.29, A.23.1, B.1.427, B.1.429, P.1, B.1.36. 27. B.1.1.284, B.1.1.28, B.1.1.106, B.1, B.1.1.317, B.1.617, B.1.617.1, B.1.617.2, B.1.617. 3. Or any newly emerging mutant strains.
  • the method may also include the step of aliquoting the sample prior to contacting each of the Spike protein or ACE2-binding fragment thereof, and the ACE2 protein or fragment thereof.
  • the aliquoted sample can be contacted with each spike protein or ACE2-binding fragment thereof separately.
  • the sample can be divided into equal portions.
  • the method may further comprise, after determining the level of interaction between each spike protein or ACE2 binding fragment thereof and ACE2 protein or fragment thereof, comparing the interaction between each spike protein or ACE2 binding fragment thereof and ACE2 protein or fragment thereof Steps for high and low levels of action.
  • the samples had stronger neutralizing activity against SARS-CoV-2 virus strains containing the spike protein or its ACE2-binding fragment at a lower level of interaction.
  • the amount of each Spike protein or ACE2-binding fragment thereof may be: (a) in molar ratio, lower than or equal to the amount of the Spike protein or ACE2-binding fragment thereof that can block the Spike protein or ACE2-binding fragment thereof in the sample contacted with it.
  • the amount of antibody that binds to the ACE2 protein or fragment thereof and (b) is sufficient to generate the Spike protein reflecting i) in the presence of a sample that does not contain antibodies to wild-type SARS-CoV-2 or antibodies to a mutant strain of SARS-CoV-2
  • the amount of each spike protein or ACE2-binding fragment thereof may be such that a response is generated in the presence of a sample that does not contain wild-type SARS-CoV-2 antibodies or SARS-CoV-2 mutant antibodies i) The minimum amount required for the detection signal of the interaction between the spike protein or ACE2-binding fragment thereof of ii) and the ACE2 protein or fragment thereof.
  • the sample can be a blood sample, a lymph sample, a saliva sample, or a joint obtained from a person who has been infected with wild-type SARS-CoV-2 and/or a mutant strain of SARS-CoV-2, or who has been vaccinated against SARS-CoV-2. synovial fluid.
  • the sample can be a solution containing artificially prepared antibodies, such as hybridoma supernatant, a crude sample containing vector-expressed antibodies, a solution containing purified antibodies, or immunized non-human mammalian blood.
  • the present application also provides a method of using the kit set of the present application to evaluate the neutralizing activity of a sample against wild-type SARS-CoV-2 and/or SARS-CoV-2 mutant strains, including:
  • the mutant strain of SARS-CoV-2 can be any mutant strain, such as B.1.1.7, B.1.351, B.1.1.29, A.23.1, B.1.427, B.1.429, P.1, B.1.36. 27. B.1.1.284, B.1.1.28, B.1.1.106, B.1, B.1.1.317, B.1.617, B.1.617.1, B.1.617.2, B.1.617. 3. Or any newly emerging mutant strains.
  • the method may also include, in each sub-kit, i) the spike protein encoded by SARS-CoV-2 or its ACE2 binding fragment, and ii) the spike protein or its ACE2 binding fragment of i) specifically binding
  • i) the spike protein encoded by SARS-CoV-2 or its ACE2 binding fragment and ii) the spike protein or its ACE2 binding fragment of i) specifically binding
  • Aliquoted samples can be contacted with the spike protein or ACE2-binding fragment thereof in each sub-kit. In one embodiment, the sample can be divided into equal portions.
  • the method may also include, after determining the level of interaction between the spike protein or its ACE2-binding fragment of i) in each sub-kit and (ii) the ACE2 protein or its fragment, comparing the spike protein in each sub-kit The step of increasing the level of interaction between its ACE2-binding fragment and the ACE2 protein or its fragment.
  • the samples had stronger neutralizing activity against SARS-CoV-2 virus strains containing the spike protein or its ACE2-binding fragment at a lower level of interaction.
  • the amount of spike protein or ACE2-binding fragment thereof in each sub-kit can be: (a) in molar ratio, lower than or equal to the amount of the spike protein or ACE2-binding fragment in the sample contacted with it.
  • the amount of each spike protein or ACE2-binding fragment thereof in each sub-kit may be: in the presence of samples that do not contain wild-type SARS-CoV-2 antibodies or SARS-CoV-2 mutant antibodies The minimum amount required to generate a detection signal reflecting the interaction between the Spike protein or ACE2-binding fragment thereof of i) and the ACE2 protein or fragment thereof of ii).
  • the sample can be a blood sample, a lymph sample, a saliva sample, or a joint obtained from a person who has been infected with wild-type SARS-CoV-2 and/or a mutant strain of SARS-CoV-2, or who has been vaccinated against SARS-CoV-2. synovial fluid.
  • the sample can be a solution containing artificially prepared antibodies, such as hybridoma supernatant, a crude sample containing vector-expressed antibodies, a solution containing purified antibodies, or immunized non-human mammalian blood.
  • the kit or kit set of the present application can detect the level of neutralizing antibodies against various new coronaviruses (including wild-type and mutant strains) in the same sample, and the same sample can detect the level of neutralizing antibodies against various new coronaviruses Neutralizing activity of coronaviruses, including wild-type and mutant strains.
  • SARS-CoV-2 mutant strains include any mutant strains, such as B.1.1.7, P.1, B.1.351, B.1.427 and B.1.429, etc.
  • Figure 1 is a calibration curve of SARS-CoV-2 neutralizing antibodies.
  • Fig. 2 shows the binding force of wild-type SARS-CoV-2 RBD and each RBD variant to ACE2 in the kit of Example 2.
  • Figure 3 shows the blocking effect of neutralizing antibodies obtained by immunization with wild-type RBD on the binding of wild-type RBD and various RBD variants to ACE2.
  • Figure 4 shows the blocking effect of the culture supernatant of rabbit or mouse hybridoma cells immunized with RBD-B.1.351 on the binding of wild-type RBD and various RBD variants to ACE2.
  • Fig. 5 shows the binding force of wild-type SARS-CoV-2 RBD and each RBD variant to ACE2 in the kit of Example 4.
  • Fig. 6 shows the binding force of wild-type SARS-CoV-2 RBD and each RBD variant to ACE2 in the kit of Example 5.
  • the kit and method of the present application by testing the blocking activity of the antibody in the sample to two or more SARS-CoV-2 RBDs, including wild-type RBD and its RBD variants, combined with ACE2, can easily determine whether the antibody drug or Therapeutic and protective effects of vaccines against SARS-CoV-2 wild-type and/or mutant strain infection.
  • the pathogenic agent can be roughly determined with the aid of the kit and method of the present application.
  • Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is the causative agent of novel coronavirus pneumonia (COVID-19).
  • wild-type "SARS-CoV-2” refers to a virus strain whose nucleotide sequence is GenBank accession number: MN996527.1 ("Severe acute respiratory syndrome coronavirus 2 isolate WIV02, complete genome”) , or virus strains with a certain degree of genomic variation that are not defined as mutant strains in the field.
  • Some 2019-nCoV mutants have enhanced transmissibility and lead to increased hospitalization or mortality, severely reduced antibody neutralization from previous infection or immunization, and reduced therapeutic or vaccine efficacy.
  • the U.S. Centers for Disease Control and Prevention (CDC) and the SARS-CoV-2 Interdepartmental Group (SIG) classified these novel coronavirus mutants as variants of concern (VOC), including B.1.1.7, P.1, and B. 1.351 etc., see Table 1 below.
  • VOC Variable coronavirus mutants of concern
  • B.1.617 that emerged in India has two mutation sites E484Q and L452R in the coding sequence of the SARS-CoV-2 spike protein.
  • This mutant virus has a stronger ability to spread, and the US Centers for Disease Control and Prevention has classified the B.1.617 mutant as a variant to be observed (VOI), see Table 2 below. Mutations such as N501Y, E484K and K417N occur in the receptor binding motif.
  • SARS-CoV-2 mutant strains in this application include but are not limited to B.1.1.7, B.1.351, B.1.1.29, A.23.1, B.1.427, B.1.429, P.1, B.1.36 .27, B.1.1.284, B.1.1.28, B.1.1.106, B.1, B.1.1.317, B.1.617, B.1.617.1, B.1.617.2, and B. 1.617.3.
  • the spike protein of SARS-CoV-2 is the main molecule for the virus to infect host cells, including the S1 subunit responsible for binding cell surface receptors and the S2 subunit mediating membrane fusion.
  • the S1 subunit contains the receptor binding domain (RBD), through which SARS-CoV-2 binds to ACE2 expressed by host cells.
  • RBD receptor binding domain
  • virus neutralization can be accomplished by other types of antibodies, antibodies that block receptor/virus entry into cells represent the vast majority of neutralizing antibodies.
  • RBD was shown in in vitro experiments to be sufficient to bind ACE2. Most SARS-CoV-2 neutralizing antibodies block RBD-ACE2 binding by binding to RBD.
  • the RBD in turn contains the receptor binding motif (RBM), which is the region of the RBD that contacts ACE2.
  • RBM receptor binding motif
  • the kit of the present application comprises the spike protein of wild type SARS-CoV-2 or its ACE2 binding fragment, and the spike protein of SARS-CoV-2 mutant strain or its ACE2 binding fragment.
  • the ACE2-binding fragment can be any ACE2-binding fragment in the Spike protein, including but not limited to RBD and RBM.
  • B.1.1.29, B.1.36.27, B.1.1.284, B.1.1.106 and B.1 have the same or very similar RBD as wild-type SARS-CoV-2, such as SEQ ID NO: 2 shown.
  • B.1.427, B.1.429 and P.1 have the same RBD, as shown in SEQ ID NO:9.
  • B.1.617, B.1.617.1 and B.1.617.3 have the same RBD as shown in SEQ ID NO:17.
  • the Spike protein encoded by SARS-CoV-2 or its ACE2-binding fragment may contain or consist of S1 subunits.
  • the Spike protein encoded by SARS-CoV-2 or an ACE2-binding fragment thereof may comprise at least 70%, 75%, 80%, 85%, 90%, 91% of SEQ ID NO: 1 or 4. %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity amino acid sequence, or with SEQ ID NO: 1 or 4 has at least 70%, 75%, Amino acid sequence constituents with 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the Spike protein encoded by SARS-CoV-2 or its ACE2-binding fragment may comprise, or consist of, the receptor-binding domain (RBD) of the Spike protein.
  • the Spike protein encoded by SARS-CoV-2, or an ACE2-binding fragment thereof may comprise any of SEQ ID NOs: 2, 5-11, 17 and 18 having at least 70%, 75%, An amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, or consisting of an amino acid sequence with SEQ ID NO:2 , any of 5-11, 17 and 18 have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% sequence identity.
  • the Spike protein encoded by SARS-CoV-2 or an ACE2-binding fragment thereof may comprise, or consist of, the receptor binding motif (RBM) of the Spike protein.
  • the Spike protein or ACE2-binding fragment thereof may comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% of SEQ ID NO:3 , 95%, 96%, 97%, 98% or 99% sequence identity amino acid sequence, or having at least 70%, 75%, 80%, 85%, 90%, 91%, 85%, 90%, 91%, Amino acid sequence constituents with 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • sequence identity refers to the percentage of nucleotides/amino acid residues in a sequence that are identical to those in a reference sequence after alignment of the sequences, with spaces introduced, if necessary, to achieve both. The highest percentage of sequence identity between two sequences.
  • Specific binding refers to selective interaction with related molecules, as distinguished from non-specific binding.
  • a polypeptide that specifically binds a particular molecule preferably binds that particular molecule with a higher affinity, and/or for a longer duration than it binds other molecules to which it does not specifically bind.
  • polypeptide e.g., protein
  • SPR surface plasmon resonance
  • biofilm interference see e.g. Lad et al., (2015) J Biomol Screen 20(4):498-507
  • flow cytometry or radiolabeled antigen binding detection (RIA)
  • enzyme-linked immunosorbent assay Through these assays, binding to specific molecules can be identified and quantified. In some embodiments, binding can be a reaction detected in a particular assay.
  • the polypeptide binds to a non-targeting molecule about 10% less than a molecule to which the polypeptide specifically binds, as measured by ELISA, SPR, biomembrane interference, or RIA.
  • specific binding may be reflected in a binding affinity in which the polypeptide binds a non-targeted molecule with a dissociation constant (K D ) that is at least 0.1 order of magnitude higher (i.e., 0.1 x 10 n , where n is an integer representing the order of magnitude ) of K D , binding to the molecule specifically binding to the polypeptide.
  • K D dissociation constant
  • the order of magnitude may optionally be at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5 or 2.0.
  • the ACE2 protein or its fragment "specifically binds" to the Spike protein or its ACE2-binding fragment, which means that the ACE2 protein or its fragment has a higher affinity than other components in the kit or kit set, and and/or longer duration binding to the Spike protein or its ACE2-binding fragment.
  • ACE2 Angiotensin Converting Enzyme 2
  • ACE2 Angiotensin Converting Enzyme 2
  • the structure and function of ACE2 are described in Hamming et al., J Pathol (2004) 203(2):631-637, which is incorporated herein by reference in its entirety.
  • ACE2 has been identified as the key point for SARS-CoV-2 to enter cells through the spike protein. That is, ACE2 is the key receptor for SARS-CoV-2 to enter cells, and the SARS-CoV-2 spike protein binds to the extracellular domain of ACE2, and then enters the host cell.
  • ACE2 may refer to ACE2 of any species, and includes ACE2 subtypes, fragments, variants (including mutants), or homologues obtained from any species.
  • ACE2 is a mammalian species (e.g., Theria, Eutheria, Eutheria, Prototheria, Prototheria, Primates (monkeys, non-human primates, or humans) ) of ACE2.
  • the ACE2 is human, bat, pangolin, civet, or pig ACE2.
  • Isoforms, fragments, variants or homologues of ACE2 may optionally have at least 70% sequence identity, preferably 80%, 85%, 90%, 91%, with immature or mature ACE2 isoforms of a particular species, e.g. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity is characterized.
  • the ACE2 protein or a fragment thereof may comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% of SEQ ID NO: 12, 14 or 15 , 95%, 96%, 97%, 98% or 99% sequence identity amino acid sequence, or by at least 70%, 75%, 80%, 85%, 90% with SEQ ID NO: 12, 14 or 15 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences.
  • the ACE2 protein or fragment thereof may comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% of SEQ ID NO: 15 , 96%, 97%, 98% or 99% amino acid sequence identity.
  • the ACE2 protein or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 12, 14 or 15.
  • the amino acid sequence of the ACE2 protein or a fragment thereof is shown in SEQ ID NO: 12, 14 or 15.
  • the ACE2 protein or fragment thereof may comprise, or consist of, an extracellular domain.
  • the ACE2 protein or fragment thereof may comprise at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, Amino acid sequence having 95%, 96%, 97%, 98% or 99% sequence identity, or consisting of at least 70%, 75%, 80%, 85%, 90%, 91% to SEQ ID NO: 13 or 16 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity.
  • the ACE2 protein or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 13 or 16. In other embodiments, the amino acid sequence of the ACE2 protein or a fragment thereof is shown in SEQ ID NO: 13 or 16.
  • the ACE2 protein or a fragment thereof comprises an extracellular domain comprising the sequence shown in SEQ ID NO.15.
  • the detection principle of the kit and method of the present application is to neutralize the spike protein or its ACE2-binding fragment in the neutralizing antibody binding kit, thereby blocking the binding of the spike protein or its ACE2-binding fragment to ACE2 or its fragment to a certain extent.
  • the degree of binding of the spike protein or its ACE2-binding fragment to ACE2 or its fragment can be done by attaching or conjugating a tag molecule to the spike protein or its ACE2-binding fragment, or ACE2 or a fragment thereof, and by measuring the signal detection molecule specifically bound to the tag molecule.
  • the tag molecule can be a protein tag, such as His tag, Flag tag, StrepII tag, Fc tag, Avi tag, B tag, CBP tag, E tag, c-Myc tag, GST tag, HA tag, HSV tag, MBP tag, HPC4 tags, RFP tags, SNAP tags, SUMO tags, T7 tags, V5 tags, VSV-G tags, and GFP tags, and small molecule tags, such as biotin, etc.
  • the protein tag has a small molecular weight and has little impact on the tertiary structure and biological activity of the spike protein or its ACE2-binding fragment, or ACE2 or its fragment protein.
  • the tag molecule can be attached to the N-terminus or C-terminus of the Spike protein or its ACE2-binding fragment, or the ACE2 protein or its fragment, or any other suitable position via, for example, recombinant expression. Since the position of the tag molecule is fixed at a certain position according to the prior design, the spike protein or its ACE2-binding fragment or ACE2 protein or its fragment with the tag molecule obtained through recombinant expression has the same structure and characteristics. the same, or extremely similar. In one embodiment, the tag molecule is recombinantly expressed and linked to the N-terminus or C-terminus of the spike protein or its ACE2-binding fragment, or the ACE2 protein or its fragment.
  • the tag molecule can also be coupled to one or some sites of the spike protein or its ACE2-binding fragment, ACE2 protein or its fragment through enzymatic reaction or the like. Due to technical limitations in the field, most of the tag molecules coupled by this method are randomly distributed on the spike protein or its ACE2 binding fragment, or the ACE2 protein or its fragment, and the number of coupled tag molecules is also random. Therefore, the tagged spike protein or its ACE2-binding fragment, or the ACE2 protein or its fragment obtained by this method may have large differences in structure and properties. In particular, such randomly coupled tag molecules may affect the binding properties of Spike protein or its ACE2-binding fragment to ACE2 protein or its fragment.
  • the inventors compared the effects of tag molecules randomly coupled to RBD or ACE2 ectodomain, or recombined at the C-terminus of RBD or ACE2 ectodomain, on the binding of ACE2-spike protein.
  • the binding force of ACE2-spike protein was basically consistent with the binding force results obtained by thin-film interferometry; while when the tag molecules were randomly coupled to the RBD or ACE2 cells ACE2-Spike protein binding was significantly altered when the ectodomain was removed.
  • the tag molecule can be recombinantly expressed at the N-terminal or C-terminal of the extracellular domain of RBD or ACE2, preferably the C-terminal. Attaching tags such as His to the C-terminus can make the expression and purification of the molecule more complete, while linking to the N-terminus, the signal peptide may exist when the molecule is cut, making the molecule incomplete.
  • the Spike protein or its ACE2-binding fragment, or the ACE2 protein or its fragment is linked with a tag molecule, eg, via a recombinant method.
  • the tag molecule is recombined at the C-terminus of the RBD or ACE2 extracellular domain.
  • the signal detection molecule is a molecule that specifically binds to the label molecule and generates a detection signal. "Specific binding” here means that, compared with other components in the kit, the signal detection molecule binds to the tag molecule with higher affinity and/or longer duration. In particular, the signal detection molecule has no binding force to the components of the kit other than the label molecule, or the binding force is very weak, or the binding time is very short.
  • Signal detection molecules may bear, for example, fluorescent labels, luminescent labels, immunodetectable labels, radioactive labels, chemical labels, nucleic acid labels, or polypeptide labels. Detection of chemiluminescent labels is described in, for example, Kricka et al., Analyticazia acta, (2003), 500(1):279-286 and Chen et al., Chinese Journal of Analytical Chemistry (2012) 40(1):3-10 described in , both of which are incorporated by reference in their entirety.
  • a chemical substance such as an acridine compound (eg, acridinium ester or acridinium sulfonamide ester)
  • alkaline hydrogen peroxide can be used for detection.
  • Signal detection molecules can be labeled with, for example, enzymes that catalyze the emission of electromagnetic radiation from luminescent compounds, such as horseradish peroxidase (HRP), which can be used to catalyze the degradation of luminescent ammonia in the presence of hydrogen peroxide, such as alkaline phosphatase, which can Used to catalyze the degradation of AMPPD.
  • HRP horseradish peroxidase
  • the signal detection molecule can be labeled with horseradish peroxidase, luciferase, or alkaline phosphatase.
  • the signal detection molecule of the present application can be anti-His, anti-Flag, anti-StrepII, anti-Avi, anti-Fc, anti-B labeled with horseradish peroxidase, alkaline phosphatase or acridinium ester , anti-CBP, anti-E, anti-c-Myc, anti-GST, anti-HA, anti-HSV, anti-MBP, anti-HPC4, anti-RFP, anti-SNAP, anti-SUMO, anti-T7 , anti-V5, anti-VSV-G and anti-GFP antibodies, or streptavidin labeled with horseradish peroxidase, alkaline phosphatase, etc., these can be prepared by those skilled in the art according to the knowledge in the field prepared.
  • test kit of the present application may comprise:
  • Spike proteins encoded by SARS-CoV-2 or ACE2-binding fragments thereof selected from wild-type SARS-CoV-2-encoded Spike proteins or ACE2-binding fragments thereof, and SARS-CoV-2 mutations Spike protein or its ACE2 binding fragment encoded by the strain,
  • a signal detection molecule for detecting the interaction between the spike protein of i) or its ACE2-binding fragment and ii) the ACE2 protein or its fragment
  • the spike protein or its ACE2-binding fragment or ii) the ACE2 protein or its fragment is linked or coupled with a tag molecule, and iii) the signal detection molecule specifically binds to the tag molecule.
  • the spike protein or its ACE2-binding fragment and ii) the ACE2 protein or its fragment can exist in any suitable molar ratio, for example, in a molar ratio of 5:1-11:1 exist.
  • Each Spike protein or its ACE2-binding fragment can be packaged separately, and the ACE2 protein or its fragment can be packaged separately at a molar ratio of 1:11-1:5 to each Spike protein or its ACE2-binding fragment.
  • the spike protein of i) or its ACE2-binding fragment such as RBD is present in a much higher amount than the ACE2 protein or its fragment of ii) due to instability.
  • the molar ratio of ACE2 protein or its fragment to Spike protein or its ACE2 binding fragment can be 1:15-1:3.
  • the molar ratio of ACE2 protein or fragment thereof to Spike protein or ACE2-binding fragment thereof is 1:11-1:5.
  • the molar ratio of ACE2 protein or its fragment to Spike protein or its ACE2-binding fragment is 1:10-1:5.
  • the amount of ACE2 protein or its fragments in the kit is 7.86 ⁇ 10 -11 -1.57 ⁇ 10 -10 moles
  • the amount of RBD is 8.33 ⁇ 10 -10 moles.
  • the Spike protein or its ACE2-binding fragment was present in excess in the kit, when tested, the ACE2 protein or its fragment was in far excess with a molar ratio of ACE2 protein or its fragment to RBD of about 11.8:1.
  • the molar ratio of ACE2 protein or fragment thereof to RBD is about 15:1-5:1 in a single test well plate.
  • the molar ratio of the ACE2 protein or fragment thereof to RBD is about 12:1-5:1 in a single test well plate.
  • the molar ratio of ACE2 protein or fragment thereof to RBD is about 11.8:1.
  • 7.86 x 10 -13 moles of ACE2 and 6.67 x 10 -14 moles of RBD or a variant thereof are tested.
  • the Spike protein encoded by SARS-CoV-2 or its ACE2-binding fragment can be the Spike protein or its ACE2-binding fragment encoded by wild-type SARS-CoV-2, or the Spike encoded by a mutant strain of SARS-CoV-2 protein or its ACE2-binding fragment.
  • the mutant strain of SARS-CoV-2 can be any mutant strain, such as B.1.1.7, B.1.351, B.1.1.29, A.23.1, B.1.427, B.1.429, P.1, B.1.36. 27. B.1.1.284, B.1.1.28, B.1.1.106, B.1, B.1.1.317, B.1.617, B.1.617.1, B.1.617.2, B.1.617. 3. Or any newly emerging mutant strains.
  • the kit can contain the Spike protein or ACE2-binding fragment thereof of any SARS-CoV-2 virus strain, including the Spike protein or ACE2-binding fragment thereof of emerging SARS-CoV-2 mutant strains, as required.
  • Two or more Spike proteins encoded by SARS-CoV-2 or ACE2 binding fragments thereof may comprise Spike proteins encoded by wild-type SARS-CoV-2 or ACE2 binding fragments thereof, and Spike proteins encoded by SARS-CoV-2 mutant strains. Spike protein or its ACE2-binding fragment. The two or more Spike proteins encoded by SARS-CoV-2 or ACE2-binding fragments thereof may only comprise Spike proteins or ACE2-binding fragments thereof encoded by SARS-CoV-2 mutant strains.
  • kits of the present application may also comprise a solid support.
  • the solid support can be the Spike protein or an ACE2-binding fragment thereof, or the ACE2 protein or a fragment thereof can be conveniently immobilized (e.g., by adsorption or conjugation) and suitable for analysis of antibody-containing samples (e.g., obtained from any solid support for a sample of blood, such as a serum sample).
  • suitable solid supports for use in the kits of the present application are known in the art.
  • a solid support can include polystyrene, polypropylene, polycarbonate, cycloolefin, glass, or quartz.
  • a solid support can be a microtiter (or "well") plate or a microarray plate.
  • the solid support can be beads, such as magnetic beads.
  • the Spike protein or its ACE2-binding fragment according to the present application, or the ACE2 protein or its fragment can be immobilized (or "coated") on the solid support of the present application by methods known in the art.
  • the Spike protein or ACE2-binding fragment thereof, or the ACE2 protein or fragment thereof can be covalently or non-covalently immobilized to a solid support.
  • the Spike protein or its ACE2-binding fragment, or ACE2 protein or its fragment solution can be applied in a buffer, and the Spike protein or its ACE2-binding fragment, or the ACE2 protein or its fragment is adsorbed on the surface of a solid support.
  • the spike protein or its ACE2-binding fragment, or the ACE2 protein or its fragment is immobilized on the solid support.
  • the Spike protein or ACE2-binding fragment thereof, or the ACE2 protein or fragment thereof can be coupled to a solid support, eg, by a covalent bond.
  • test kit of the present application may comprise more than two sub-kits, and each sub-kit comprises:
  • a Spike protein encoded by SARS-CoV-2 or an ACE2 binding fragment thereof selected from the Spike protein encoded by wild-type SARS-CoV-2 or an ACE2 binding fragment thereof, and a mutant strain of SARS-CoV-2 Encoded spike protein or ACE2-binding fragment thereof
  • the spike protein or its ACE2-binding fragment or ii) the ACE2 protein or its fragment is linked or coupled with a tag molecule, and iii) the signal detection molecule specifically binds to the tag molecule.
  • the Spike protein encoded by SARS-CoV-2 or its ACE2-binding fragment can be the Spike protein or its ACE2-binding fragment encoded by wild-type SARS-CoV-2, or the Spike encoded by a mutant strain of SARS-CoV-2 protein or its ACE2-binding fragment.
  • the mutant strain of SARS-CoV-2 can be any mutant strain, such as B.1.1.7, B.1.351, B.1.1.29, A.23.1, B.1.427, B.1.429, P.1, B.1.36. 27. B.1.1.284, B.1.1.28, B.1.1.106, B.1, B.1.1.317, B.1.617, B.1.617.1, B.1.617.2, B.1.617. 3. Or any newly emerging mutant strains.
  • Each sub-kit can contain the Spike protein of any SARS-CoV-2 virus strain or its ACE2 binding fragment as required, including the Spike protein or its ACE2 binding fragment of the emerging SARS-CoV-2 mutant strain.
  • one sub-kit may comprise the Spike protein encoded by wild-type SARS-CoV-2 or its ACE2-binding fragment
  • the other sub-kits may Each contains a spike protein encoded by a SARS-CoV-2 mutant or an ACE2-binding fragment thereof.
  • each kit may include a Spike protein encoded by a SARS-CoV-2 mutant strain or an ACE2-binding fragment thereof.
  • the Spike protein or its ACE2-binding fragment contained in each sub-kit can be made from different SARS-CoV-2 mutant strain encoding.
  • Each sub-kit may also comprise a solid support.
  • the application detects the influence of samples on the ACE2 binding ability of various spike proteins or ACE2 binding fragments thereof to determine whether there are neutralizing antibodies against various SARS-CoV-2 virus strains in the samples.
  • the present application provides a method of using the kit of the present application to detect whether there is an antibody selected from the wild-type SARS-CoV-2 antibody and the SARS-CoV-2 mutant antibody in the sample, including:
  • the present application also provides a method of using the kit set of the present application to detect whether there is an antibody selected from the wild-type SARS-CoV-2 antibody and the SARS-CoV-2 mutant antibody in the sample, including:
  • a decrease in the level of interaction between the ACE2-binding fragment thereof and the ACE2 protein or a fragment thereof indicates the presence of antibodies against SARS-CoV-2 virus strains containing the corresponding spike protein or ACE2-binding fragment thereof in the sample.
  • the method can also compare the level of interaction between each spike protein or its ACE2-binding fragment and the ACE2 protein or its fragment, wherein, there are more spike proteins or its ACE2-binding fragment with a low interaction level in the sample.
  • the antibody detection method of the present application can simultaneously detect neutralizing antibodies against different SARS-CoV-2 virus strains in samples.
  • any spike protein or ACE2-binding fragment thereof in any kit, kit set, sub-kit, and/or (sub)kit can be selected.
  • the spike protein of the specific virus strain or its ACE2 binding fragment and wild-type spike protein as a control can be used to detect The neutralizing antibodies against the mutant virus strain in the suspected population in the area, so as to study the transmission chain of the mutant strain and predict the transmission area of the mutant strain.
  • the antibody in the suspected personnel sample is found to have an ACE2 binding blocking effect on the spike protein or its ACE2 binding fragment of the specific virus strain through qualitative or quantitative (detection value) comparison, especially when the blocking effect is significantly stronger than, for example, When there is wild-type SARS-CoV-2 or other SARS-CoV-2 strains, it can be roughly judged that the person has been infected by that specific virus strain.
  • the sample Before contacting each spike protein or its ACE2 binding fragment, and ACE2 protein or its fragment in the kit, or before contacting with the spike protein or its ACE2 binding fragment, and ACE2 protein or its fragment in each sub-kit , the sample can be divided into several parts, which are contacted with each spike protein or its ACE2 binding fragment respectively. In one embodiment, the sample can be divided into equal portions. When the sample is divided into equal aliquots, the effect of the samples on ACE2 binding by different Spike proteins or ACE2-binding fragments thereof can be compared more precisely. In the application of infection traceability, the most likely infectious virus strain can be found by dividing the sample into several equal parts.
  • the amount of each spike protein or its ACE2-binding fragment in the kit, or the spike protein or its ACE2-binding fragment in each sub-kit can be: (a) in molar ratio, is less than or equal to the amount of antibody that blocks the binding of the Spike protein or ACE2-binding fragment thereof to the ACE2 protein or fragment thereof in the contacted sample, and (b) is sufficient in the presence of no wild-type SARS-CoV-2 antibody In the case of samples of SARS-CoV-2 mutant antibodies, a detection signal reflecting the interaction between the spike protein or ACE2 binding fragment thereof of i) and the ACE2 protein or fragment thereof of ii) is generated.
  • the amount of each Spike protein or ACE2-binding fragment thereof may be such that a Spike protein reflecting i) is produced in the presence of a sample that does not contain antibodies to wild-type SARS-CoV-2 or antibodies to a mutant strain of SARS-CoV-2 The minimum amount required for the detection signal of the interaction between the ACE2 binding fragment thereof and the ACE2 protein or fragment thereof of ii).
  • the sample can be a blood sample, a lymph sample, a saliva sample, or a synovial fluid obtained from a person infected with wild-type SARS-CoV-2 and/or a mutant strain of SARS-CoV-2.
  • This application judges the neutralizing activity of antibodies against various SARS-CoV-2 virus strains by detecting the influence of antibodies in samples on the ACE2 binding ability of various spike proteins or ACE2 binding fragments thereof.
  • the application provides a method of using the kit of the application to evaluate the neutralizing activity of a sample to a virus strain selected from wild-type SARS-CoV-2 and SARS-CoV-2 mutant strains, including:
  • the present application also provides a method of using the kit set of the present application to evaluate the neutralizing activity of a sample against wild-type SARS-CoV-2 and/or SARS-CoV-2 mutant strains, including:
  • the reduction of the interaction level between one or more spike proteins or their ACE2 binding fragments in the kit and the ACE2 protein or its fragments, or the spike protein or its ACE2 binding in one or more sub-kits A decrease in the level of interaction between the fragment and the ACE2 protein or its fragment indicates that there is neutralizing activity in the sample against SARS-CoV-2 strains containing the corresponding spike protein or its ACE2-binding fragment.
  • This method can also compare the level of interaction between each spike protein or its ACE2 binding fragment and the ACE2 protein or its fragment, wherein, the samples are compared with SARS-CoV-2 containing spike protein or its ACE2 binding fragment with low interaction level. Virus strains have higher neutralizing activity.
  • the detection method of the present application can simultaneously detect the neutralizing activity of antibodies in samples against different SARS-CoV-2 strains.
  • any spike protein or ACE2-binding fragment thereof in any kit, kit set, sub-kit, and/or (sub)kit can be selected.
  • various SARS-CoV-2 mutant strains can be used to stimulate Spike protein or fragments thereof, and wild-type Spike protein or fragments thereof used as controls.
  • the serum of the recovered person has an ACE2 binding blocking effect on the Spike protein or its fragments of one or several mutant strains, and the effect is different from that on the wild-type Spike protein or its fragments. If the fragments are comparable, it indicates that the antibody has a strong neutralizing effect on the specific mutant strain.
  • the spike proteins of various SARS-CoV-2 mutant strains can be used or a fragment thereof, and wild-type Spike protein or a fragment thereof as a control.
  • the antibody drug has an ACE2 binding blocking effect on the spike protein or its fragments of one or several mutant strains, and the effect is different from that on the wild-type spike protein or its fragments. , it indicates that the antibody drug has a strong neutralizing effect on the specific mutant strain.
  • the detection object is the serum of convalescents infected with mutant strains
  • the spike protein or its fragments of infected virus strains are used as a control to analyze the neutralizing activity of convalescent serum against wild-type virus or other mutant strains.
  • the drug antibody is produced by immunization with the mutant spike protein or fragment, the spike protein or its fragment used as an immunizing agent is used as a control to analyze the neutralizing activity of the drug antibody against wild-type virus or other mutant strains .
  • the sample Before contacting each spike protein or its ACE2 binding fragment, and ACE2 protein or its fragment in the kit, or before contacting with the spike protein or its ACE2 binding fragment, and ACE2 protein or its fragment in each sub-kit , the sample can be divided into several parts, which are contacted with each spike protein or its ACE2 binding fragment respectively. In one embodiment, the sample can be divided into equal portions. In this way, the effects of samples on the binding of different spike proteins or their ACE2-binding fragments to ACE2 can be compared more accurately and conveniently.
  • the amount of each spike protein or its ACE2-binding fragment in the kit, or the spike protein or its ACE2-binding fragment in each sub-kit can be: (a) in molar ratio, is less than or equal to the amount of antibody that blocks the binding of the Spike protein or ACE2-binding fragment thereof to the ACE2 protein or fragment thereof in the contacted sample, and (b) is sufficient in the presence of no wild-type SARS-CoV-2 antibody In the case of samples of SARS-CoV-2 mutant antibodies, a detection signal reflecting the interaction between the spike protein or ACE2 binding fragment thereof of i) and the ACE2 protein or fragment thereof of ii) is generated.
  • the amount of each Spike protein or ACE2-binding fragment thereof may be such that a Spike protein reflecting i) is produced in the presence of a sample that does not contain antibodies to wild-type SARS-CoV-2 or antibodies to a mutant strain of SARS-CoV-2 The minimum amount required for the detection signal of the interaction between the ACE2 binding fragment thereof and the ACE2 protein or fragment thereof of ii).
  • the sample can be a blood sample, a lymph sample, a saliva sample, or a joint obtained from a person who has been infected with wild-type SARS-CoV-2 and/or a mutant strain of SARS-CoV-2, or who has been vaccinated against SARS-CoV-2. synovial fluid.
  • the sample can be a solution containing artificially prepared antibodies, such as hybridoma supernatant, a crude sample containing vector-expressed antibodies, a solution containing purified antibodies, or immunized non-human mammalian blood.
  • Affinity detection was performed with the help of Gator TM Label-Free Bioanalysis System (Probe LIFE), in which the ProA probe captured ACE2 and combined with different concentrations of SARS-CoV-2 RBD and its variants to detect its affinity .
  • Gator TM Label-Free Bioanalysis System Probe LIFE
  • ACE2 protein GenScript, Cat no. Z03484
  • ACE2 protein GenScript, Cat no. Z03484
  • wild-type SARS-CoV-2 RBD also known as RBD-WT, GenScript, Cat No.Z03483
  • RBD-B.1.1.7 GentScript, Cat No.Z03533
  • RBD-B wild-type SARS-CoV-2 RBD
  • the binding affinity of ACE2 to several exemplary RBD variants is higher than its binding affinity to wild-type RBD, and the ranking from high to low is RBD-B.1.1.7, RBD-B. 1.351, RBD-P.1 and RBD-B.1.427.
  • the binding affinities of ACE2 to RBD-B.1.1.7, RBD-P.1, RBD-B.1.351, and RBD-B.1.427 were 5.33 times, 3.22 times, and 3.11 times that of wild-type RBD, respectively. times and 1.84 times.
  • the exemplary kit of the present application mainly comprises the following 8 components:
  • Wild-type SARS-CoV-2 RBD with His tag also known as RBD-WT, GenScript, Cat No.Z03483), RBD-B.1.1.7 (GentScript, Cat No.Z03533), RBD-B.1.351(GingScript, Cat No.Z03537), RBD-P.1(GingScript, Cat No.Z03600), and RBD-B.1.427(GingScript, Cat No.Z03603);
  • ACE2 protein (GenScript, Cat No.Z03484) was coated with carbonate buffer (8.5g sodium chloride, 1.4g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate, dissolved in 1000ml ddH 2 O, pH 7.4) Prepared at 2 ⁇ g/ml.
  • carbonate buffer 8.5g sodium chloride, 1.4g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate, dissolved in 1000ml ddH 2 O, pH 7.4
  • carbonate buffer 8g sodium chloride, 1.4g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate, dissolved in 1000ml ddH 2 O, pH 7.4
  • the His-tagged RBD-WT, RBD-B.1.1.7, RBD-B.1.351, RBD-P.1, and RBD-B.1.427 were prepared into concentrates of the same concentration with the test buffer.
  • the concentration is 19200U/ml, 4800U/ml, 2400U/ml, 1200U/ml, 600U/ml, 300U/ml, 150U/ml, 75U/ml, 37.5U/ml, 18.75U/ml and 0U/ml.
  • 19200U/ml ⁇ 300U/ml and 0U/ml are used for the calibration curve preparation of RBD-B.1.351 neutralizing antibody; 1200U/ml ⁇ 37.5U/ml and 0U/ml are used for RBD-B.1.1.7 neutralization Calibration curve preparation of antibody; 600U/ml ⁇ 18.75U/ml and 0U/ml are used for calibration curve preparation of RBD-WT neutralizing antibody.
  • the sample signal value can be substituted into the corresponding calibration curve to calculate the concentration, which is the sample concentration.
  • concentration is the concentration after dilution
  • sample stock solution concentration test concentration ⁇ 10 (sample dilution factor).
  • the preparation concentration is 10000ng/ml, 3333ng/ml, 1111ng/ml, 370ng/ml ml, 123ng/ml, 41ng/ml, 13.72ng/ml, 4.57ng/ml, 1.52ng/ml, 0.51ng/ml, 0.17ng/ml and 0ng/ml of each RBD working solution.
  • RBD working solutions were added to the luminescence plate coated with ACE2, 100 ⁇ L/well, and reacted at 37°C for 40 minutes.
  • the EC 50 concentrations of the five groups of RBD are: RBD-WT (232ng/ml), RBD-B.1.1.7 (58ng/ml), RBD-B.1.351 (199ng/ml), RBD - P.1 (130 ng/ml), B.1.427 (261 ng/ml).
  • the binding force between RBD and ACE2 is ranked as RBD-B.1.1.7, RBD-P.1, RBD-B.1.351, RBD-WT, RBD-B.1.427 from high to low.
  • linear r should not be less than 0.99.
  • the samples with low, middle and high concentrations are used for detection, each sample is repeated 3 times, and the average concentration is calculated, and the recovery rate of each concentration should be within the range of (80%, 120%).
  • Example 2 Using the chemiluminescence detection kit in Example 2, detect the blocking effect of neutralizing antibodies prepared by immunizing SARS-CoV-2 RBD-WT on RBD-WT and RBD variants binding to ACE2.
  • COV2051 can recognize E484 of wild-type RBD, but cannot recognize RBD variants mutated at this site, such as RBD-B.1.351 mutated to E484K; COV2085 can recognize N501 of wild-type RBD, but cannot recognize the occurrence of this site Mutated RBD variants such as RBD-B.1.1.7 and RBD-B.1.351 mutated to N501Y.
  • the results are shown in Figure 3.
  • the neutralizing antibodies mAb10933 and mAb10987 had the best inhibitory effects on RBD-WT, while the inhibitory effects on RBD-B.1.427 and RBD-B.1.1.7 decreased, and on RBD-B.1.351 and RBD
  • the inhibitory effect of -P.1 decreased significantly; the neutralizing antibody COV2051 had a better inhibitory effect on RBD-WT, RBD-B.1.1.7, and RBD-B.1.427, but had a better inhibitory effect on RBD-B.1.351 and RBD-P.
  • the antibodies prepared by immunizing mice with wild-type RBD have good inhibitory effects on wild-type RBD-ACE2 binding, but have different effects on RBD variants.
  • the antibody R2B2HC has a good inhibitory effect on wild-type RBD and RBD variants.
  • the inhibitory effect of COV2051 and COV2085 on the binding of each RBD-ACE2 basically conforms to the characteristics of binding E484 but not E484K, or binding N501 but not N501Y.
  • RBD-B.1.1.7, RBD-P.1 and RBD-B.1.351 had E484 and N501 mutations, while RBD-B.1.427 did not have E484 and N501 mutations.
  • COV2051 cannot recognize RBD-B.1.351 with E484K mutation
  • COV2085 cannot recognize RBD-B.1.1.7 and RBD-B.1.351 with N501Y mutation.
  • COV2051 had no inhibitory effect on the binding of RBD-P.1 and RBD-B.1.351 to ACE2, and COV2085 had no inhibitory effect on the binding of RBD-B.1.1.7, RBD-P.1 and RBD -
  • the binding of B.1.351 to ACE2 has no inhibitory effect.
  • COV2051 and COV2085 can better inhibit its binding to ACE2.
  • Example 2 The combined chemiluminescence detection kit in Example 2 was used to detect the blocking effect of these hybridoma cell culture supernatants on RBD-WT and RBD variants binding to ACE2.
  • the antibodies prepared by immunization with RBD-B.1.351 all have good inhibitory effects on RBD-B.1.351-ACE2 binding, but have different effects on other RBDs. This result is consistent with the result obtained in 3.1.
  • Example 4 Use HRP directly labeled wild-type RBD and its variants to detect neutralizing antibodies against RBD-ACE2 binding inhibition
  • Example 2 Based on the principle of detecting neutralizing antibodies in Example 2, another kit was prepared. The main difference from the kit in Example 2 is that RBD-WT and RBD variants are directly coupled with horseradish peroxidase (HRP), and HRP is developed with TMB without the alkaline phosphate in Example 2 Enzyme (AP) conjugated anti-His and AP substrate.
  • HRP horseradish peroxidase
  • AP Enzyme
  • the kit of the present embodiment mainly comprises the following 6 components:
  • a) capture plate a 96-well microtiter plate coated with ACE2 protein
  • ACE2 protein (Gensray, Cat No.Z03484) was coated with carbonate buffer (8.5g sodium chloride, 1.4g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate, dissolved in 1000ml pure water, pH 7.4 ) was prepared at 2 ⁇ g/ml.
  • carbonate buffer 8.5g sodium chloride, 1.4g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate, dissolved in 1000ml pure water, pH 7.4
  • carbonate buffer 8.5g sodium chloride, 1.4g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate, dissolved in 1000ml pure water, pH 7.4
  • carbonate buffer 8g sodium chloride, 1.4g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate, dissolved in 1000ml pure water, pH 7.4
  • carbonate buffer 8.5g sodium chloride, 1.4g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate, dissolved in 1000ml pure
  • RBD-WT Gentry, Cat No.Z03483
  • RBD-B1.1.7 Gentry, Cat No.Z03533
  • RBD-B1.351 Gentry, Cat No.Z03537
  • RBD-P .1 GenScript, Cat No.Z03600
  • RBD-WT, RBD-B1.1.7, RBD-B.1.351, and RBD-P.1 labeled with detection diluent and horseradish peroxidase were prepared at concentrations of 1000ng/ml, 500ng/ml, and 250ng/ml , 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.6ng/ml, 7.8ng/ml, 3.9ng/ml, 1.95ng/ml, 0.98ng/ml and 0ng/ml of each RBD working solution.
  • These 4 groups of RBD working solutions were added to the plate coated with ACE2, 100 ⁇ L/well, and reacted at 37°C for 15 minutes.
  • the results are shown in Figure 5.
  • the EC 50 results of the four groups of RBD were: RBD-WT (19ng/ml), RBD-B.1.1.7 (21ng/ml), RBD-B.1.351 (48ng/ml), RBD-P.1 (20ng/ml).
  • the binding force between each RBD and ACE2 is ranked from high to low as RBD-WT, RBD-P.1, RBD-B.1.1.7, RBD-B.1.351.
  • the binding forces of ACE2 to RBD-P.1, RBD-B.1.1.7, and RBD-B.1.351 were 95/100, 90/100, and 40/100 of their binding forces to RBD-WT, respectively. This is quite different from the results of Example 1.
  • the gap in 1.1.7 has
  • Embodiment 5 uses HRP directly labeled ACE2 to detect the inhibitory effect of neutralizing antibody on RBD-ACE2 binding use
  • Example 2 Based on the principle of detecting neutralizing antibodies in Example 2, another kit was prepared. The main difference between it and the kit in Example 2 is that ACE2 is directly coupled with horseradish peroxidase (HRP). TMB was developed without the alkaline phosphatase (AP)-conjugated anti-His and AP substrates in Example 2.
  • HRP horseradish peroxidase
  • the kit of the present embodiment mainly comprises the following 6 components:
  • RBD-WT (GingScript, Cat No.Z03483), RBD-B.1.1.7 (GingScript, Cat No.Z03533), RBD-B.1.351 (GingScript, Cat No.Z03537), RBD -P.1 (Gentry, Cat No.Z03600), RBD-B.1.427 (Gentry, Cat No.Z03603) were coated with carbonate buffer (8.5g sodium chloride, 1.4g hydrogen phosphate disodium, 0.2g sodium dihydrogen phosphate, dissolved in 1000ml pure water, pH 7.4) to prepare 2 ⁇ g/ml. Add 110 ⁇ l RBD working solution to each well of the 96-well plate, and coat at 4°C for 18 hours.
  • carbonate buffer 8.5g sodium chloride, 1.4g hydrogen phosphate disodium, 0.2g sodium dihydrogen phosphate, dissolved in 1000ml pure water, pH 7.4
  • ACE2 protein (GenScript, Cat No. Z03484) was coupled with horseradish peroxidase by sodium iodate method.
  • ACE2 working solution Prepare concentrations of 1000ng/ml, 500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.6ng/ml and 0ng/ml ACE2 working solution.
  • the EC 50 of ACE2 in each group are: RBD-WT (14.5ng/ml), RBD-B.1.1.7 (10.6ng/ml), RBD-B.1.351 (22.7ng/ml) , RBD-P.1 (25.5 ng/ml), RBD-B.1.427 (27.1 ng/ml).
  • RBDs combined with ACE were ranked from high to low as RBD-B.1.1.7, RBD-WT, RBD-B.1.351, RBD-P.1, RBD-B.1.427.
  • the binding forces of RBD-B.1.1.7, RBD-B.1.351, RBD-P.1, and RBD-B.1.427 to ACE2 were 1.37 times and 64/100 of the binding force of RBD-WT to ACE2, respectively. , 57/100, 54/100. This is quite different from the results of Example 1.
  • the gap between ACE2 binding force of 1.1.7 and RBD-WT becomes smaller.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Communicable Diseases (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une trousse pour la détection d'un anticorps pour le coronavirus 2 du syndrome respiratoire aigu sévère (SARS-CoV-2) et/ou sa souche mutante, et l'utilisation de la trousse dans la détection et l'évaluation d'échantillons. Plus précisément, la trousse de détection conjointe d'un anticorps neutralisant pour le nouveau coronavirus et sa souche mutante peut détecter les taux d'anticorps neutralisants pour différentes souches du nouveau coronavirus dans le même échantillon, l'efficacité d'un vaccin ou d'un médicament anticorps pour le traitement et la prévention de différentes souches de virus peut être analysée sur la base des différences obtenues en comparant les valeurs détectées des différents anticorps neutralisants pour le nouveau coronavirus, et une source d'infection de la population infectée peut également être analysée de manière assistée.
PCT/CN2022/096541 2021-06-01 2022-06-01 Trousse de détection pour neutralisation d'anticorps contre un nouveau coronavirus et sa souche mutante WO2022253260A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110608355 2021-06-01
CN202110608355.9 2021-06-01

Publications (1)

Publication Number Publication Date
WO2022253260A1 true WO2022253260A1 (fr) 2022-12-08

Family

ID=84240664

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/096541 WO2022253260A1 (fr) 2021-06-01 2022-06-01 Trousse de détection pour neutralisation d'anticorps contre un nouveau coronavirus et sa souche mutante

Country Status (2)

Country Link
CN (1) CN115436628A (fr)
WO (1) WO2022253260A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111983226A (zh) * 2020-03-25 2020-11-24 新加坡国立大学 SARSr-CoV抗体的检测
EP3809137A1 (fr) * 2020-02-19 2021-04-21 Euroimmun Medizinische Labordiagnostika AG Procédés et réactifs pour le diagnostic d'une infection par le sars-cov-2
CN112794918A (zh) * 2021-02-22 2021-05-14 中国科学院微生物研究所 针对新型冠状病毒的人ACE2改造蛋白、ACE2-hFc类抗体蛋白

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100582781C (zh) * 2006-06-27 2010-01-20 厦门大学 联合检测hbv前s1抗原和核心抗原的方法及诊断试剂盒

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3809137A1 (fr) * 2020-02-19 2021-04-21 Euroimmun Medizinische Labordiagnostika AG Procédés et réactifs pour le diagnostic d'une infection par le sars-cov-2
CN111983226A (zh) * 2020-03-25 2020-11-24 新加坡国立大学 SARSr-CoV抗体的检测
CN112794918A (zh) * 2021-02-22 2021-05-14 中国科学院微生物研究所 针对新型冠状病毒的人ACE2改造蛋白、ACE2-hFc类抗体蛋白

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
MEYER, B. ET AL.: "Validation and clinical evaluation of a SARS-CoV-2 surrogate virus neutralisation test (sVNT)", EMERGING MICROBES & INFECTIONS, vol. 9, 31 December 2020 (2020-12-31), pages 2394 - 2403, XP055780426, DOI: 10.1080/22221751.2020.1835448 *
ROWNTREE LOUISE C, CHUA BRENDON Y, NICHOLSON SUELLEN, KOUTSAKOS MARIOS, HENSEN LUCA, DOUROS CELIA, SELVA KEVIN, MORDANT FRANCESCA : "Robust correlations across six SARS‐CoV‐2 serology assays detecting distinct antibody features", CLINICAL & TRANSLATIONAL IMMUNOLOGY, vol. 10, no. 3, 1 January 2021 (2021-01-01), GB , XP093010067, ISSN: 2050-0068, DOI: 10.1002/cti2.1258 *
TAN, C. W. ET AL.: "A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2–spike protein–protein interaction", NATURE BIOTECHNOLOGY, vol. 38, 30 September 2020 (2020-09-30), pages 1073 - 1078, XP037237853, DOI: 10.1038/s41587-020-0631-z *
TAYLOR, S. C. ET AL.: "A New SARS-CoV-2 Dual-Purpose Serology Test: Highly Accurate Infection Tracing and Neutralizing Antibody Response Detection", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 59, no. 4, 30 April 2021 (2021-04-30), XP093010034 *
WANG, JIN ET AL.: "Performance Evaluation of 3 Assays in Detecting Serum IgG and IgM Antibody Titre in Recovered COVID-19 Patients", CHINESE JOURNAL OF BLOOD TRANSFUSION, vol. 33, no. 8, 31 August 2020 (2020-08-31), pages 750 - 752, XP093010093 *

Also Published As

Publication number Publication date
CN115436628A (zh) 2022-12-06

Similar Documents

Publication Publication Date Title
US20210190797A1 (en) Methods and reagents for diagnosis of SARS-CoV-2 infection
EP3855186B1 (fr) Procédé permettant de déterminer l'efficacité d'un vaccin
US7700727B2 (en) Compositions and kits for detecting pathogen infection
WO2021254476A1 (fr) Kit de réactif de chimioluminescence de microparticules magnétiques pour la détection d'anticorps neutralisants contre le virus sras-cov-2 et application associé
US11255855B1 (en) COVID-19 spike-ACE2 binding assay for drug and antibody screening
EP2405268A1 (fr) Procédé de détection d'une substance dans un échantillon biologique
CN113215106B (zh) 杂交瘤细胞、抗SARS-CoV-2的单克隆抗体及其应用
US20100062454A1 (en) Method for predicting risk of acquiring influenza
WO2022253260A1 (fr) Trousse de détection pour neutralisation d'anticorps contre un nouveau coronavirus et sa souche mutante
US20220196658A1 (en) ANTI-VIRUS IgG3 BINDING ASSAY TO ASSESS VIRUS NEUTRALIZATION AND RELATED METHODS
EP3922262A1 (fr) Dosage pour la détection de la protéase de type cys (mpro) du sars-cov-2
WO2021217140A2 (fr) Réactifs d'amélioration de la spécificité pour un test d'anticorps du covid-19
KR20220029898A (ko) 한탄바이러스 항원 단백질을 이용한 혈청학적 진단 방법
KR102694894B1 (ko) 제2형 중증급성호흡기증후군 코로나바이러스 변이주의 중화항체 검출용 조성물 및 이를 포함하는 중화항체 검출 방법 및 키트
TWI838885B (zh) 蛋白質微陣列及其用途與檢測方法
EP3761029B1 (fr) Nouveau dosage pour le diagnostic d'infections par des nématodes
EP4039818A1 (fr) Procédé de pronostic de l'évolution d'une infection au sars-cov-2 et de la réponse à une immunisation contre le sars-cov-2 ou à une infection naturelle en mesurant la quantité d`ace2 soluble
EP3925971A1 (fr) Procédé d'identification de composés pouvant influencer la liaison au récepteur de virus
US20230266309A1 (en) Protein microarray, use and detection method thereof
Shoaib et al. The long-term characteristics of immunity conferred by COVID-19 using antibody tests
Roe et al. Performance of an envelope glycoprotein-based multiplex immunoassay for Ebola virus antibody detection in a cohort of Ebola virus disease survivors
US20130137083A1 (en) Detection of H5N1 Influenza Infection
WO2021240425A1 (fr) Procédé de détection rapide d'anticorps contre sras-cov-2 à l'aide d'une protéine de fusion nucléospicule recombinante
Davaakhuu et al. Development of serological assay for detection of antibodies in the N protein of SARS-CoV-2 in human sera in Mongolia
IL295754A (en) Methods and reagents for the diagnosis of sars-cov-2 infection

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22815302

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22815302

Country of ref document: EP

Kind code of ref document: A1