WO2021170090A1 - SARS-CoV-2病毒的检测方法及检测试剂盒 - Google Patents

SARS-CoV-2病毒的检测方法及检测试剂盒 Download PDF

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WO2021170090A1
WO2021170090A1 PCT/CN2021/078179 CN2021078179W WO2021170090A1 WO 2021170090 A1 WO2021170090 A1 WO 2021170090A1 CN 2021078179 W CN2021078179 W CN 2021078179W WO 2021170090 A1 WO2021170090 A1 WO 2021170090A1
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antibody
cov
sars
virus
antigen
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PCT/CN2021/078179
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English (en)
French (fr)
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覃喜建
吴东明
汤双双
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南京金斯瑞生物科技有限公司
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Priority to CN202180016690.8A priority Critical patent/CN115151824A/zh
Publication of WO2021170090A1 publication Critical patent/WO2021170090A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • 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

Definitions

  • the invention belongs to the field of viral molecular biology, and relates to an immunological detection method and a detection kit for the SARS-CoV-2 virus, in particular to a method for detecting the presence of the SARS-CoV-2 virus or its antigen or antibody in a sample and its detection Reagent test kit.
  • SARS-CoV-2 virus (Severe Acute Respiratory Syndrome Coronavirus Type 2), also known as 2019 new coronavirus (2019-nCoV), is a new coronavirus that has not been found in humans before and can cause pneumonia.
  • the SARS-CoV-2 virus was found in patients with pneumonia and caused a rapid outbreak of pneumonia. So far, the epidemic has spread to more than 20 countries in Asia, Europe, North America and Oceania. More than 60,000 people were infected, causing more than 1,500 deaths. Since humans have never been exposed to this virus before, they cannot be protected by existing vaccines or natural immunity, and there is no specific medicine for the prevention or treatment of SARS-CoV-2 virus. Therefore, the virus may cause a large-scale outbreak of the disease and eventually evolve into a pandemic.
  • SARS-CoV-2 virus is the same as Middle East Respiratory Syndrome-related Coronavirus (MERSr-CoV) and Severe Acute Respiratory Syndrome-related Coronavirus (SARSr-CoV) Belongs to the beta coronavirus genus.
  • MERSr-CoV Middle East Respiratory Syndrome-related Coronavirus
  • SARSr-CoV Severe Acute Respiratory Syndrome-related Coronavirus
  • N nucleocapsid protein
  • the S protein is the largest structural protein of SARS-CoV-2, which mediates the virus binding to the host cell receptor to enter the cell for replication.
  • the S protein can be cleaved into S1 and S2 subunits under the action of host enzymes.
  • the S1 subunit contains the receptor binding domain (RBD), which is the main target antigen that causes the host immune response and produces neutralizing antibodies.
  • RBD receptor binding domain
  • Spike protein S is a type I transmembrane glycoprotein, composed of an N-terminal S1 domain and a C-terminal S2 domain.
  • the main function of S1 is to bind to host cell surface receptors, and the S2 subunit mediates virus-cell and cell-cell membrane fusion.
  • the S1 protein receptor binding region RBD binds to the receptor ACE2 to complete the virus adsorption process, and then the S2 protein executes the fusion of the virus envelope and cell membrane to complete the virus infection process.
  • S protein plays an important role in inducing the body's immune protection response.
  • Spike protein S antibodies are widespread in the sera of patients recovered from SARS.
  • the spike protein S of SARS-CoV can effectively induce the production of neutralizing antibodies.
  • Spike protein S has the main antigenic determinants of the virus, and it is one of the potential diagnostic targets for virus detection.
  • the existing SARS-CoV-2 virus clinical detection reagents are RT-PCR nucleic acid molecular detection methods for the virus genetics.
  • the SARS-CoV-2 nucleic acid molecular diagnostic test method has shortcomings. It has high technical requirements, long operation time, special processing of specimens and corresponding equipment, which must be concentrated in a central laboratory for testing. Moreover, there is a certain false positive rate in nucleic acid diagnosis. In addition, with the mutation of the virus, the false negative rate of nucleic acid diagnosis will also increase.
  • the serum antibody detection method for SARS-CoV-2 virus includes enzyme-linked immunosorbent method.
  • the production of antibodies after virus infection requires a window period of 1-3 weeks, and the incubation period from infection to onset is very short during SARS-CoV-2 infection (usually within a week).
  • antibody detection is of no significance for the early diagnosis of SARS-CoV-2 virus, it can be used as a basis for the diagnosis of SARS-CoV-2 virus.
  • the ELISA test kit requires the patient's positive serum as a positive control.
  • the number of positive sera from patients is limited and there is a risk of infection. Therefore, preparing an equivalent that can replace the patient's positive serum as a positive control is a big problem in the development of a SARS-CoV-2 virus antibody detection kit.
  • SARS-CoV-2 virus antigen diagnosis method has irreplaceable significance in the diagnosis of SARS-CoV-2.
  • SARS-CoV-2 virus antigen diagnostic reagents there is no report about SARS-CoV-2 virus antigen diagnostic reagents in the world. There is no report of using spike protein RBD fragment as the detection antigen.
  • the SARS-CoV human monoclonal antibody CR3022 is an antibody constructed from the variable regions of the light and heavy chains obtained from the CR3022 single-chain variable fragment (scFv) and the human IgG1 type constant region.
  • CR3022scFv was screened from the immune scFv phage library constructed from lymphocytes of convalescent SARS patients in Singapore. It can be combined with SARS-CoV inactivated by ultraviolet light. There is no report on the use of CR3022 antibody for SARS-CoV-2 virus or antibody detection.
  • One aspect of the present invention provides a method for detecting the presence of SARS-CoV-2 virus or its antigen in a sample and a detection kit thereof. Another aspect provides a method for detecting the presence of antibodies against SARS-CoV-2 virus in a sample and a detection kit thereof. The present invention further provides an application of an antibody that specifically binds to SARS-CoV-2 virus in a detection kit.
  • the heavy chain sequence of the antibody is shown in SEQ ID NO: 6, and the light chain sequence of the antibody As shown in SEQ ID NO: 7.
  • One aspect of the present invention provides a method for detecting the presence of SARS-CoV-2 virus or its antigen in a sample, the method comprising:
  • the antibody that specifically binds to the SARS-CoV-2 virus is an antibody against the SARS-CoV-2 S protein.
  • the antibody is an antibody against the SARS-CoV-2 S RBD protein.
  • the heavy chain variable region of the antibody comprises an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 4, and the light chain variable region of the antibody comprises the same amino acid sequence as SEQ ID NO: 5.
  • the sequence shown is an amino acid sequence with at least 80% identity.
  • the heavy chain variable region of the antibody comprises the sequence shown in SEQ ID NO: 4, and the light chain variable region of the antibody comprises the sequence shown in SEQ ID NO: 5.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO: 4, and the light chain variable region sequence of the antibody is shown in SEQ ID NO: 5.
  • the heavy chain sequence of the antibody contains an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 6, and the light chain sequence of the antibody contains at least the same sequence as that shown in SEQ ID NO: 7. 80% identical amino acid sequence.
  • the heavy chain sequence of the antibody includes the sequence shown in SEQ ID NO: 6, and the light chain sequence of the antibody includes the sequence shown in SEQ ID NO: 7.
  • the heavy chain sequence of the antibody is shown in SEQ ID NO: 6, and the light chain sequence of the antibody is shown in SEQ ID NO: 7.
  • the step of contacting the antibody with the sample includes first coating the antibody on a solid support, and then adding the sample to the solid support coated with the antibody for incubation.
  • the suitable condition refers to incubating the antibody and the sample at 25-37°C for 15 minutes to 1.5 hours.
  • the step of detecting the immune complex includes adding a second antibody, and the second antibody is an antibody against the SARS-CoV-2 virus.
  • the second antibody is an anti-SARS-CoV-2 S protein antibody.
  • the antibody is an anti-SARS-CoV-2 S RBD protein antibody.
  • the second antibody is suitable for detecting immune complexes by an ELISA method, particularly a sandwich ELISA method for detecting immune complexes.
  • the second antibody is an antibody against SARS-CoV-2 S RBD protein, which is used in the sandwich ELISA method to detect immune complexes.
  • the second antibody is labeled with biotin, radioisotope, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase.
  • kits for detecting SARS-CoV-2 virus or its antigen in a sample comprising:
  • the antibody that specifically binds to the SARS-CoV-2 virus is an anti-SARS-CoV-2 S protein antibody, preferably, the antibody is an anti-SARS-CoV-2 S RBD protein antibody.
  • the heavy chain variable region of the antibody comprises an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 4, and the light chain variable region of the antibody comprises the same amino acid sequence as SEQ ID NO: The sequence shown in 5 is an amino acid sequence with at least 80% identity.
  • the heavy chain variable region of the antibody comprises the sequence shown in SEQ ID NO: 4, and the light chain variable region of the antibody comprises the sequence shown in SEQ ID NO: 5.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO: 4, and the light chain variable region sequence of the antibody is shown in SEQ ID NO: 5.
  • the heavy chain sequence of the antibody comprises an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 6, and the light chain sequence of the antibody comprises the sequence shown in SEQ ID NO: 7 Amino acid sequence with at least 80% identity.
  • the heavy chain sequence of the antibody comprises the sequence shown in SEQ ID NO: 6, and the light chain sequence of the antibody comprises the sequence shown in SEQ ID NO: 7.
  • the heavy chain sequence of the antibody is shown in SEQ ID NO: 6, and the light chain sequence of the antibody is shown in SEQ ID NO: 7.
  • the antibody is coated on a solid support.
  • the solid support includes inorganic and organic polymers.
  • the solid support is an inner wall of a container, such as an inner wall of an orifice plate.
  • the tool for detecting immune complexes includes a second antibody.
  • the second antibody is an IgG, IgM and/or IgA antibody.
  • the second antibody is an antibody against SARS-CoV-2 virus.
  • the second antibody is suitable for detecting immune complexes by an ELISA method, particularly a sandwich ELISA method for detecting immune complexes.
  • the second antibody is an antibody against SARS-CoV-2 S RBD protein, which is used in the sandwich ELISA method to detect immune complexes.
  • the second antibody is labeled with a biotin, a radioisotope, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase.
  • the kit for detecting SARS-CoV-2 virus or its antigen in a sample of the present invention is selected from an ELISA detection kit, a chemiluminescence detection kit, a colloidal gold detection kit or a time-resolved immunochromatography detection kit, preferably The ELISA detection kit is more preferably a sandwich ELISA detection kit.
  • the present invention specifically provides an ELISA kit for detecting SARS-CoV-2 virus or its antigen in a sample, the kit comprising:
  • a solid support coated with a first antibody which is an antibody that specifically binds to SARS-CoV-2 virus antigen
  • a second antibody labeled with biotin or horseradish peroxidase which is an antibody that specifically binds to SARS-CoV-2 virus antigen
  • the first antibody and the second antibody are respectively selected from the following antibody sequences: the heavy chain variable region of the first/second antibody comprises the amino acid sequence shown in SEQ ID NO: 64, the The light chain variable region of the first/second antibody comprises the amino acid sequence shown in SEQ ID NO:5.
  • the positive control includes SARS-CoV-2 S RBD protein or SARS-CoV-2 S pseudovirus.
  • the positive control may be a negative control including SARS-CoV-2 S RBD protein or SARS-CoV-2 S pseudovirus.
  • the negative control comprises healthy human serum.
  • the negative control comprises a PBS buffer of BSA.
  • Another aspect of the present invention provides a method for detecting the presence of antibodies against SARS-CoV-2 virus in a sample, the method comprising:
  • the SARS-CoV-2 virus antigen comprises an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 2 or 3. In other embodiments, the SARS-CoV-2 virus antigen comprises the amino acid sequence shown in SEQ ID NO: 2 or 3. In a specific embodiment, the SARS-CoV-2 virus antigen sequence is shown in SEQ ID NO: 2 or 3.
  • the step of contacting the viral antigen with the sample includes first coating the viral antigen on a solid support, and then adding the sample to the solid support coated with the antigen and incubating.
  • the suitable condition is that the viral antigen and the sample are incubated at 25-37°C for 15 minutes to 1.5 hours.
  • the detecting step includes adding a second antibody, and the second antibody is an anti-human antibody.
  • the second antibody may be an anti-human IgG, anti-human IgM and/or anti-human IgA antibody.
  • the second antibody is labeled with biotin, radioisotope, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase.
  • kits for detecting antibodies against SARS-CoV-2 virus in a sample comprising:
  • SARS-CoV-2 virus antigen SARS-CoV-2 virus antigen
  • a tool for detecting the formation of immune complexes between the virus antigen and the antibody that may be present in the sample is a tool for detecting the formation of immune complexes between the virus antigen and the antibody that may be present in the sample.
  • the SARS-CoV-2 virus antigen is SARS-CoV-2 S RBD protein.
  • the SARS-CoV-2 virus antigen comprises an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 2 or 3.
  • the SARS-CoV-2 virus antigen comprises the amino acid sequence shown in SEQ ID NO: 2 or 3.
  • the SARS-CoV-2 virus antigen sequence is shown in SEQ ID NO: 2 or 3.
  • the viral antigen is coated on a solid support.
  • the solid support includes inorganic and organic polymers.
  • the solid support is an inner wall of a container, such as an inner wall of an orifice plate.
  • the tool for detecting immune complexes includes a detection antibody, the antibody being biotin, radioisotope, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase or acetylcholine Esterase labeled.
  • the detection antibody is an anti-human antibody labeled with horseradish peroxidase.
  • the detection antibody is horseradish peroxidase-labeled anti-human IgG, anti-human IgM and/or anti-human IgA antibody.
  • the kit of the present invention for detecting antibodies against SARS-CoV-2 virus in a sample is selected from an ELISA detection kit, a chemiluminescence detection kit, a colloidal gold detection kit or a time-resolved immunochromatography detection kit, preferably an ELISA
  • the detection kit is more preferably an indirect ELISA detection kit.
  • the present invention specifically provides an ELISA kit for detecting antibodies against SARS-CoV-2 virus in a sample, the kit comprising:
  • the SARS-CoV-2 virus antigen is SARS-CoV-2 S RBD protein.
  • the SARS-CoV-2 virus antigen comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2 or 3.
  • the SARS-CoV-2 virus antigen comprises the amino acid sequence shown in SEQ ID NO: 2 or 3.
  • the SARS-CoV-2 virus antigen sequence is shown in SEQ ID NO: 2 or 3.
  • the positive control includes an antibody that specifically binds to the SARS-CoV-2 virus.
  • the heavy chain variable region of the antibody included in the positive control includes the sequence shown in SEQ ID NO: 4, and the light chain variable region of the antibody includes the sequence shown in SEQ ID NO: 5.
  • the heavy chain variable region sequence of the antibody in the positive control is shown in SEQ ID NO: 4, and the light chain variable region sequence of the antibody is shown in SEQ ID NO: 5.
  • the heavy chain sequence of the antibody included in the positive control is shown in SEQ ID NO: 6, and the light chain sequence of the antibody is shown in SEQ ID NO: 7.
  • the negative control comprises healthy human serum.
  • the negative control comprises BSA in PBS buffer.
  • the present invention also provides an application of an antibody that specifically binds to SARS-CoV-2 virus in a detection kit.
  • an application of an anti-SARS-CoV-2 virus antibody in a kit for detecting SARS-CoV-2 virus antibody is provided.
  • the heavy chain sequence of the antibody is shown in SEQ ID NO: 6, and the light chain sequence of the antibody is shown in SEQ ID NO: 7.
  • the antibody is added to a negative control as a positive control.
  • the negative control can be healthy human serum or PBS buffer containing BSA.
  • the method of the present invention for detecting whether there is an antibody against SARS-CoV-2 virus in a sample or a kit for detecting an antibody against SARS-CoV-2 virus in a sample wherein the sample is derived from an infection or a suspected SARS infection -CoV-2 virus plasma, serum, whole blood, pleural effusion, cerebrospinal fluid or tissue specimens.
  • SARS-CoV-2 also known as 2019-nCoV
  • 2019-nCoV belongs to the ⁇ -coronavirus, has an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 60- 140nm. Its genetic characteristics are significantly different from SARSr-Cov and MERSr-CoV. Studies have shown that it has more than 85% homology with bat SARS-like coronavirus (bat-SL-CoVZC45).
  • bat SARS-like coronavirus bat SARS-like coronavirus
  • 2019-nCov can be found in human respiratory epithelial cells in about 96 hours, while isolation and culture in Vero E6 and Huh-7 cell lines takes about 6 days.
  • SARS-CoV-2 virus antigen refers to the SARS-CoV-2 whole virus or the antigen of its lysate or the recombinantly expressed SARS-CoV-2 antigen.
  • SARS-CoV-2 virus includes spike protein (S), envelope protein (E), matrix protein (M) and nucleocapsid protein (N) antigens, among which S protein is the largest structural protein of SARS-CoV-2.
  • S protein can be cleaved into S1 and S2 subunits under the action of host enzymes.
  • the S1 subunit contains the receptor binding region RBD and is the main target antigen.
  • the SARS-CoV-2 virus antigen is selected from spike protein (S), envelope protein (E), matrix protein (M) and/or nucleocapsid protein (N) antigen.
  • the SARS-CoV-2 virus antigen is SARS-CoV-2 S RBD antigen.
  • the SARS-CoV-2 S RBD antigen in the present invention can be produced by conventional recombinant expression methods, by constructing a plasmid expressing SARS-CoV-2 S RBD, such as pFastBac1, pTT5, and the expression vector containing the target gene is transfected into the expression cell , Such as CHO cells and SF9 cells, express and purify SARS-CoV-2 S RBD recombinant protein.
  • antibody is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains (two heavy chains (H) and two light chains (L) are connected to each other by disulfide bonds (ie, "complete antibody molecules")) , And its multimers (for example, IgM) or antigen-binding fragments thereof.
  • Each heavy chain is composed of a heavy chain variable region ("HCVR” or "VH”) and a heavy chain constant region (composed of domains CH1, CH2, and CH3).
  • Each light chain is composed of a light chain variable region ("LCVR or "VL”) and a light chain constant region (CL).
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDR), There is a more conserved region inserted in between called the framework region (FR).
  • CDR complementarity determining regions
  • FR framework region
  • Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the hydroxyl terminus: FR1, CDR1, FR2, CDR2, FR3 , CDR3, FR4.
  • the FR of the antibody may be the same as the human germline sequence or may be naturally or artificially modified.
  • the "percent (%) amino acid sequence identity" of a peptide or polypeptide sequence is defined as comparing the sequences and introducing gaps when necessary to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Candidates The percentage of amino acid residues in the sequence that are identical to the amino acid residues in the specific peptide or polypeptide sequence. Sequence comparisons can be performed in a variety of ways within the skill of the art to determine percent amino acid sequence identity, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required to obtain the maximum comparison over the entire length of the sequence being compared.
  • the “solid support” includes organic and inorganic polymers well known in the art, such as including but not limited to: dextran, natural or modified cellulose, polyethylene, polystyrene, polyacrylamide , Agarose, latex, the inner wall of the container such as test tube, titration plate, glass cup, etc.
  • organic and inorganic polymers well known in the art, such as including but not limited to: dextran, natural or modified cellulose, polyethylene, polystyrene, polyacrylamide , Agarose, latex, the inner wall of the container such as test tube, titration plate, glass cup, etc.
  • Various methods known in the art can be used to coat the solid support. For example: using bifunctional reagents for activation, see US Patent No. 5,399,501.
  • the solid support is the wall of a 96-well plate.
  • subject refers to an animal in need of alleviation, prevention and/or treatment of a disease or condition such as a viral infection, preferably a mammal, more preferably a human.
  • a disease or condition such as a viral infection
  • the term includes human subjects who have a coronavirus such as SARS-CoV-2 infection or are at risk of having a coronavirus such as SARS-CoV-2 infection.
  • a healthy subject refers to a healthy animal that has not been infected with the SARS-CoV-2 virus, preferably a mammal, and more preferably a human.
  • administering and “treatment” are used to refer to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, it means to combine exogenous drugs, therapeutic agents, diagnostic agents or compositions with animals, humans, and recipients. Contact with the person being treated, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, treatment methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting the reagent with the cell and contacting the reagent with a fluid, where the fluid is in contact with the cell.
  • administering and “treatment” also mean the treatment of cells in vitro and ex vivo, for example, by reagents, diagnostic agents, binding compositions, or by other cells.
  • One aspect of the present invention provides a method for detecting the presence of SARS-CoV-2 virus or its antigen in a sample, the method comprising:
  • the antibody that specifically binds to the SARS-CoV-2 virus antigen is contacted with the sample, and under suitable conditions, the antibody forms an immune complex with the virus or its antigen in the sample;
  • the antibody that specifically binds to the SARS-CoV-2 virus antigen may be selected from polyclonal antibodies or monoclonal antibodies.
  • the antibody that specifically binds to SARS-CoV-2 virus antigen is selected from antibodies that specifically bind to SARS-CoV-2 S protein (anti-SARS-CoV-2 S antibody).
  • the antibody that specifically binds to the SARS-CoV-2 S protein is an antibody against the SARS-CoV-2 S RBD protein.
  • variable region of the heavy chain of the antibody that specifically binds to the SARS-CoV-2 virus antigen comprises at least 80%, at least 85%, at least 90%, or at least 95% of the sequence shown in SEQ ID NO: 4 , At least 97% or at least 99% identical to the amino acid sequence, the light chain variable region of the antibody contains at least 80%, at least 85%, at least 90%, at least 95%, at least 97% of the sequence shown in SEQ ID NO: 5 % Or at least 99% identical amino acid sequence.
  • the variable region of the antibody heavy chain is shown in SEQ ID NO: 4, and the variable region of the antibody light chain is shown in SEQ ID NO: 5.
  • the antibody heavy chain sequence that specifically binds to the SARS-CoV-2 virus antigen comprises at least 80%, at least 85%, at least 90%, at least 95%, at least the sequence shown in SEQ ID NO: 6 97% or at least 99% identical amino acid sequence
  • the light chain sequence of the antibody contains at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least the sequence shown in SEQ ID NO: 7 Amino acid sequence with 99% identity.
  • the heavy chain sequence of the antibody is shown in SEQ ID NO: 6, and the light chain sequence of the antibody is shown in SEQ ID NO: 7.
  • the antibody that specifically binds to SARS-CoV-2 virus antigen is CR3022 antibody.
  • the antibody specifically binding to SARS-CoV-2 virus antigen can be constructed, expressed and produced by conventional molecular biology methods.
  • the SARS-CoV-2 virus antigen antibody such as the CR3022 antibody
  • the SARS-CoV-2 virus antigen antibody can be obtained by constructing plasmids expressing the heavy and light chains of the antibody, co-transfecting the expressing cells, and culturing and purifying the antibody.
  • the activity of the antibody can be detected by immunoassay methods, such as the use of indirect ELISA to determine the titer of the antibody.
  • the step of contacting the antibody that specifically binds to the SARS-CoV-2 virus antigen and the viral antigen that may be present in the sample includes first coating the antibody on a solid support, and then adding the sample to the sample. Incubate on a solid support coated with antibody.
  • the coating can use a variety of methods known in the art to coat the solid support. For example: using bifunctional reagents for activation, see US Patent No. 5,399,501.
  • suitable conditions of the antigen-antibody immune complex are well-known in the art and refer to the incubation of antibodies that specifically bind to SARS-CoV-2 virus antigens with SARS-CoV-2 virus antigens under appropriate temperature and time conditions.
  • the sample mixture for example, is incubated at about 20°C to 39°C, preferably about 25°C to 37°C, for about 20 minutes to about 4 hours, preferably for about 50 minutes to about 1.5 hours.
  • the suitable conditions refer to the incubation of the antibody and the sample at 25-37°C for 15 minutes to 1.5 hours, preferably, the incubation of the antibody and the sample at 37°C for 1 hour.
  • the solid phase support is washed with an appropriate washing solution commonly used in the art, such as washing the test plate several times, to remove unbound labeled antigen.
  • the washing liquid used is generally a phosphate buffer solution with a pH of about 5-8, preferably a pH of about 7.4.
  • the step of detecting the immune complex includes adding a second antibody, and the second antibody is an antibody against the SARS-CoV-2 virus antigen.
  • the second antibody may be the same as the aforementioned antibody that specifically binds to the SARS-CoV-2 virus antigen, or may be different from the aforementioned antibody that specifically binds to the SARS-CoV-2 virus antigen. According to the method used to label the second antibody, select an appropriate quantitative detection program.
  • the second antibody is labeled with biotin, radioisotope, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase or acetylcholinesterase.
  • biotin is used when labeling the second antibody, and then streptavidin (SA)-horseradish peroxidase is used to interact with the antigen-antibody immune complex.
  • SA streptavidin
  • the enzyme is at a wavelength of 450nm
  • the indicator reads the absorbance value. Compare the obtained reading with the Cutoff value to judge the result of the measured sample.
  • kits for detecting SARS-CoV-2 virus or its antigen in a sample comprising:
  • an antibody that specifically binds to the SARS-CoV-2 virus antigen is an anti-SARS-CoV-2 S protein antibody, preferably, the antibody is an anti-SARS-CoV-2 S RBD protein antibody;
  • a tool for detecting the virus or its antigen that may be present in the sample to form an immune complex with the antibody is provided.
  • variable region of the heavy chain of the antibody that specifically binds to the SARS-CoV-2 virus antigen comprises at least 80%, at least 85%, at least 90%, or at least 95% of the sequence shown in SEQ ID NO: 4 , At least 97% or at least 99% identical to the amino acid sequence, the light chain variable region of the antibody contains at least 80%, at least 85%, at least 90%, at least 95%, at least 97% of the sequence shown in SEQ ID NO: 5 % Or at least 99% identical amino acid sequence.
  • the variable region of the antibody heavy chain is shown in SEQ ID NO: 4, and the variable region of the antibody light chain is shown in SEQ ID NO: 5.
  • the heavy chain sequence of the antibody that specifically binds to the SARS-CoV-2 virus antigen comprises at least 80%, at least 85%, at least 90%, at least 95% of the sequence shown in SEQ ID NO: 6, At least 97% or at least 99% identical amino acid sequence, and the light chain sequence of the antibody contains at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least the sequence shown in SEQ ID NO: 7 Amino acid sequence with 99% identity.
  • the heavy chain sequence of the antibody is shown in SEQ ID NO: 6, and the light chain sequence of the antibody is shown in SEQ ID NO: 7.
  • the antibody that specifically binds to SARS-CoV-2 virus antigen is CR3022 antibody.
  • the antibody that specifically binds to SARS-CoV-2 virus antigen is coated on a solid support.
  • the solid support includes inorganic and organic polymers.
  • the solid support is an inner wall of a container, such as an inner wall of an orifice plate.
  • the solid support is the inner wall of a 96-well plate.
  • the solid support is the coating.
  • the solid support can be coated by various methods known in the art. For example: using bifunctional reagents for activation, see US Patent No. 5,399,501.
  • the anti-SARS-CoV-2 S RBD antibody at a suitable concentration is coated at 4°C-37°C for 1-30 hours, then the plate is washed, blocked with a blocking solution, and patted dry to obtain an antibody-coated plate.
  • the concentration of the anti-SARS-CoV-2 S RBD antibody, such as CR3022 antibody is coated at 4°C for 18 hours at a concentration of 1-10 ⁇ g/ml, then the plate is washed, blocked with blocking solution, and patted dry to obtain the antibody Coated plate, that is, SARS-CoV-2 virus antigen capture plate.
  • the means for detecting the immune complex includes a second antibody.
  • the second antibody is an anti-IgG, anti-IgM, and/or anti-IgA antibody.
  • the second antibody is an anti-human IgG antibody or an anti-human IgM antibody.
  • the second antibody is an anti-human IgG antibody.
  • the second antibody is an antibody against SARS-CoV-2 virus antigen.
  • the second antibody may be the same as the aforementioned antibody that specifically binds to the SARS-CoV-2 virus antigen, or may be different from the aforementioned antibody that specifically binds to the SARS-CoV-2 virus antigen.
  • the anti-SARS-CoV-2 virus antibody is CR3022 antibody.
  • the second antibody is labeled with biotin, radioisotope, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase.
  • biotin is used when labeling the second antibody, and then streptavidin-horseradish peroxidase is used to interact with the antigen-antibody immune complex. Take the absorbance value. The obtained readings are compared with the readings of the negative control to judge the result of the tested sample.
  • the kit for detecting SARS-CoV-2 virus or its antigen in the sample is selected from an ELISA detection kit, a chemiluminescence detection kit, a colloidal gold detection kit or a time-resolved immunochromatography Detection kit.
  • the ELISA test kit is designed according to the principle of antigen-antibody specific immune reaction, and the SARS-CoV-2 virus antigen is detected by the double-antibody sandwich method.
  • the method is to coat specific antibodies on the surface of a solid-phase carrier to form a solid-phase antibody, add the sample to be tested, make the corresponding antigen in it bind to the antibody on the solid-phase carrier, and then add an enzyme-labeled second antibody to form The second antibody complex is labeled with antigen-antibody-enzyme, and finally the substrate solution is added for color development. After the enzyme reacts with the substrate, the substrate develops color, and the antigen in the sample to be tested is detected qualitatively or quantitatively according to the color of the substrate ⁇ The amount.
  • the present invention specifically provides an ELISA kit for detecting SARS-CoV-2 virus or its antigen in a sample, the kit comprising:
  • a solid support coated with a first antibody which is an antibody that specifically binds to SARS-CoV-2 virus antigen
  • a second antibody labeled with biotin or horseradish peroxidase which is an antibody that specifically binds to SARS-CoV-2 virus antigen
  • the first antibody and the second antibody may be monoclonal antibodies or polyclonal antibodies.
  • the second antibody and the first antibody are the same antibody or different antibodies.
  • the second antibody and the first antibody are the same antibody.
  • the second antibody and the first antibody are different antibodies.
  • the first antibody and the second antibody are respectively selected from the following sequences: the heavy chain sequence of the first/second antibody is shown in SEQ ID NO: 6, the first/second antibody The light chain sequence is shown in SEQ ID NO: 7.
  • the heavy chain of the first antibody is shown in SEQ ID NO: 6, and the light chain is shown in SEQ ID NO: 7.
  • the heavy chain of the second antibody is shown in SEQ ID NO: 6, and the light chain is shown in SEQ ID NO: 7.
  • the first antibody is CR3022 antibody
  • the second antibody is an antibody against SARS-CoV-2 S RBD protein.
  • the second antibody is CR3022 antibody
  • the first antibody is an antibody against SARS-CoV-2 S RBD protein.
  • the solid phase support coated with the first antibody is a microtiter plate coated with an anti-SARS-CoV-2 virus antibody.
  • the solid support coated with the first antibody is a 96-well microtiter plate coated with CR3022 antibody.
  • the preparation method of the first antibody-coated solid support is a well-known method in the art, including coating the anti-SARS-CoV-2 S RBD antibody at a suitable concentration at 4°C-37°C for 1-30 hours, and then Wash the plate, block with blocking solution, and pat dry to obtain an antibody-coated plate.
  • the concentration of the anti-SARS-CoV-2 S RBD antibody such as CR3022 antibody, is coated at 4°C for 18 hours at a concentration of 2 ⁇ g/ml, then the plate is washed, blocked with blocking solution, and patted dry to obtain antibody coating Plate, that is, SARS-CoV-2 virus antigen capture plate.
  • the positive control includes SARS-CoV-2 S RBD protein or SARS-CoV-2 S pseudovirus.
  • the negative control comprises a PBS buffer of BSA.
  • the positive control is a phosphate buffer solution added with SARS-CoV-2 S RBD protein or SARS-CoV-2 S pseudovirus.
  • the positive control is healthy human plasma, serum, whole blood, pleural effusion or cerebrospinal fluid added with SARS-CoV-2 S RBD protein or SARS-CoV-2 S pseudovirus.
  • the positive control is a negative sample added with SARS-CoV-2 S RBD protein or SARS-CoV-2 S pseudovirus.
  • the positive control is healthy human serum added with SARS-CoV-2 S RBD protein or SARS-CoV-2 S pseudovirus.
  • the biotin or horseradish peroxidase-labeled second antibody is a biotin or horseradish peroxidase-labeled CR3022 antibody, preferably, a biotin-labeled CR3022 antibody.
  • the kit includes 0.05-4 ⁇ g/ml biotin-labeled SARS-CoV-2 S RBD antibody, preferably 1 ⁇ g/ml biotin-labeled CR3022 antibody.
  • the kit contains 1 ⁇ g/m biotin-labeled CR3022 antibody.
  • the sample diluent, washing solution, substrate reaction solution and termination reaction solution included in the ELISA kit for detecting SARS-CoV-2 virus or its antigen in a sample of the present invention are all solutions commonly used in the art.
  • the sample diluent includes sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, Tween 20, BSA, water and adjusted to pH 7.4.
  • the washing solution includes sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, Tween 20, water and adjusted to pH 7.4.
  • the substrate reaction solution includes a TMB substrate solution.
  • the invention also provides the application of the ELISA kit for detecting SARS-CoV-2 virus antigen in the sample in detecting subjects infected or suspected of being infected with SARS-CoV-2 virus.
  • the detection operation steps of the ELISA kit of the present invention include:
  • the ELISA kit further includes instructions for detection operation steps.
  • the sample is derived from plasma, serum, whole blood, sputum, oral/nasopharyngeal secretions or lotions, urine, feces, chest and abdomen that are infected or suspected of being infected with SARS-CoV-2 virus Effusion, cerebrospinal fluid, tissue specimens or non-biological samples such as water, beverages.
  • the present invention provides a method for detecting the presence of SARS-CoV-2 virus antibodies in a sample, the method comprising:
  • the SARS-CoV-2 virus antigen is in contact with the sample, and under suitable conditions, the antigen and the SARS-CoV-2 virus antibody in the sample form an immune complex;
  • the SARS-CoV-2 virus antigen is SARS-CoV-2 S protein. In some preferred embodiments, the SARS-CoV-2 virus antigen is SARS-CoV-2 S RBD protein. In some embodiments, the SARS-CoV-2 virus antigen comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity with SEQ ID NO: 2 or 3. Amino acid sequence. In a specific embodiment, the SARS-CoV-2 virus antigen is shown in SEQ ID NO: 2 or 3.
  • the contacting step includes first coating the viral antigen on a solid support, and then adding the sample to the solid support coated with the antigen and incubating.
  • the coating can use a variety of methods known in the art to coat the solid support. For example: using bifunctional reagents for activation, see US Patent No. 5,399,501.
  • the "appropriate conditions" of the antigen-antibody immune complex are well-known in the art, and refer to the incubation of the SARS-CoV-2 virus or the mixture of its antigen and a sample containing SARS-CoV-2 virus antibodies under appropriate temperature and time conditions For example, incubate at about 20°C-39°C, preferably about 25°C-37°C, for about 3 minutes to about 4 hours, preferably for about 5 minutes to about 1.5 hours.
  • the suitable condition refers to the incubation of the antigen and the sample at 25-37°C for 15 minutes to 1.5 hours, preferably, the incubation of the antigen and the sample at 37°C for 1 hour.
  • the solid support is washed with an appropriate washing solution commonly used in the art, such as a test plate, several times to remove unbound labeled antigen.
  • the washing liquid used is generally a phosphate buffer solution with a pH of about 5-8, preferably a pH of about 7.4.
  • the step of detecting immune complexes includes adding a second antibody, and the second antibody is an anti-IgG, IgM and/or IgA antibody.
  • the second antibody is an anti-human IgG or anti-IgM antibody.
  • the second antibody is an anti-human IgG antibody. According to the method used to label the second antibody, select an appropriate quantitative detection protocol.
  • the second antibody is labeled with biotin, radioisotope, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase or acetylcholinesterase.
  • the labeled second antibody is horseradish peroxidase labeled, and then the substrate solution and the stop solution are added, and after color development, the absorbance value is read on a 450nm microplate reader. The obtained readings are compared with the readings of the negative control to judge the result of the tested sample.
  • the present invention provides a kit for detecting SARS-CoV-2 virus antibodies in a sample, the kit comprising:
  • SARS-CoV-2 virus antigen the antigen is SARS-CoV-2 S RBD protein
  • a tool for detecting the formation of immune complexes between the virus antigen and the antibody that may be present in the sample is a tool for detecting the formation of immune complexes between the virus antigen and the antibody that may be present in the sample.
  • the SARS-CoV-2 virus antigen comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 3. .
  • the SARS-CoV-2 virus antigen is shown in SEQ ID NO: 3.
  • the SARS-CoV-2 virus or its antigen is coated on a solid support.
  • the solid support includes inorganic and organic polymers.
  • the solid support is an inner wall of a container, such as an inner wall of an orifice plate.
  • the solid support is the inner wall of a 96-well plate.
  • the coating may use various methods known in the art to coat the solid support. For example: using bifunctional reagents for activation, see US Patent No. 5,399,501.
  • the SARS-CoV-2 S RBD antigen at a suitable concentration is coated at 25°C-37°C for 0.2-3 hours, and the plate is washed, sealed with a blocking solution, and patted dry to obtain an antigen-coated plate.
  • the SARS-CoV-2 S RBD recombinant antigen at a concentration of 1-10 ⁇ g/ml is coated at 37°C for 1 hour, then the plate is washed, sealed with a blocking solution, and patted dry to obtain an antigen-coated plate. That is, the SARS-CoV-2 antibody capture plate.
  • the means for detecting the immune complex includes a second antibody.
  • the second antibody is an anti-IgG, anti-IgM, and/or anti-IgA antibody.
  • the second antibody is an anti-human IgG antibody or an anti-IgM antibody.
  • the second antibody is an anti-human IgG antibody.
  • the second antibody is labeled with biotin, radioisotope, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase.
  • biotin, radioisotope horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase acetylcholinesterase
  • the labeled second antibody is horseradish peroxidase labeled
  • the substrate reaction solution and the stop solution are used for the reaction
  • the absorbance value is read on a 450nm microplate reader after color development. Compare the obtained reading with the Cutoff value to judge the result of the measured sample.
  • the kit for detecting SARS-CoV-2 virus antibodies in the sample is selected from an ELISA detection kit, a chemiluminescence detection kit, a colloidal gold detection kit or a time-resolved immunochromatographic detection reagent box.
  • kits for detecting SARS-CoV-2 virus antibodies in a sample comprising:
  • the SARS-CoV-2 virus antigen is SARS-CoV-2 S RBD protein.
  • the SARS-CoV-2 virus antigen comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 3. .
  • the SARS-CoV-2 virus antigen is shown in SEQ ID NO: 3.
  • the well plate coated with SARS-CoV-2 virus antigen is a 96-well microtiter plate coated with SARS-CoV-2 S RBD recombinant protein. The coating can use a variety of methods known in the art to coat the solid support.
  • the SARS-CoV-2 S RBD antigen at a suitable concentration is coated at 25°C-37°C for 0.2-3 hours, and the plate is washed, sealed with a blocking solution, and patted dry to obtain an antigen-coated plate.
  • the SARS-CoV-2 S RBD recombinant antigen at a concentration of 10 ⁇ g/ml is coated at 37°C for 1 hour, then the plate is washed, sealed with a blocking solution, and patted dry to obtain an antigen-coated plate, that is SARS-CoV-2 antibody capture plate.
  • the labeled antibody is horseradish peroxidase labeled anti-human IgG or anti-human IgM antibody. In a preferred embodiment, the labeled antibody is an anti-human IgG antibody labeled with horseradish peroxidase.
  • the positive control includes an antibody that specifically binds to SARS-CoV-2 virus antigen.
  • the antibody that specifically binds to SARS-CoV-2 virus antigen is an anti-SARS-CoV-2 S RBD antibody, preferably, the antibody is CR3022 antibody.
  • the positive control is a sample of a healthy subject to which an anti-SARS-CoV-2 S RBD antibody is added.
  • the positive control is healthy human serum, whole blood, pleural effusion or cerebrospinal fluid added with anti-SARS-CoV-2 S RBD antibody.
  • the positive control is healthy human serum supplemented with anti-SARS-CoV-2 S RBD antibodies.
  • the negative control may be healthy human serum or PBS buffer containing BSA.
  • the sample diluent includes 1% BSA and 2% sucrose.
  • the washing solution includes sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, Tween 20, water and adjusted to pH 7.4.
  • the substrate reaction solution includes a TMB substrate solution.
  • the present invention also provides the application of the ELISA kit containing the SARS-CoV-2 virus antibody in detecting subjects infected or suspected of being infected with the SARS-CoV-2 virus.
  • the detection operation steps of the ELISA kit of the present invention include:
  • the negative control can be healthy human serum, or PBS buffer containing BSA.
  • the ELISA kit further includes instructions for detection operation steps.
  • the invention provides an application of an antibody that specifically binds to SARS-CoV-2 virus as a positive control in a SARS-CoV-2 virus antibody detection kit.
  • the antibody that specifically binds to the SARS-CoV-2 virus is an anti-SARS-CoV-2 S RBD antibody. In some embodiments, the SARS-CoV-2 virus antibody is CR3022 antibody.
  • the positive control is a negative sample with anti-SARS-CoV-2 S RBD antibody added. In a preferred embodiment, the positive control is a negative sample with anti-SARS-CoV-2 S RBD antibody added. In a more preferred embodiment, the positive control is a negative sample with anti-SARS-CoV-2 S RBD antibody added. In a specific embodiment, the positive control is a negative sample with an anti-SARS-CoV-2 S RBD antibody added at a concentration of 0.008-25 ⁇ g/ml. In a preferred embodiment, the positive control is a negative sample with an anti-SARS-CoV-2 S RBD antibody added at a concentration of 0.2-5 ⁇ g/ml.
  • the negative control can be healthy human serum or PBS buffer containing BSA.
  • the sample is derived from plasma, serum, whole blood, thoracic and abdominal cavity infected or suspected of being infected with SARS-CoV-2 virus Effusion, cerebrospinal fluid, or tissue specimen.
  • CR3022 antibody can be used to determine the presence or content of SARS-CoV-2 virus antigen or its related antibody in a sample.
  • the invention provides an ELISA detection method for SARS-CoV-2 virus antigen. Compared with SARS-CoV-2 fluorescent PCR detection method, this method has low technical requirements, simple operation and no special equipment. Compared with the SARS-CoV-2 virus antibody detection method, the SARS-CoV-2 virus antigen ELISA detection reagent method can realize the early diagnosis of the virus.
  • the invention provides an ELISA detection method for SARS-CoV-2 virus antibody.
  • the innovation of this method is to use a negative control with CR3022 antibody as a positive control for SARS-CoV-2 antibody detection.
  • the negative control infected with SARS-CoV-2 virus is used to prepare the positive control of SARS-CoV-2 virus antibody detection kit.
  • the serum of SARS-CoV-2 infected persons is not easy to obtain and there is a risk of potential infection.
  • the CR3022 antibody used in the present invention prepares a positive control serum, which has the advantages of being capable of mass production, controllable batch-to-batch stability, and no potential infection risk.
  • the spike protein S (spike, S) encoded by the S gene of SARS-CoV-2 virus has a total length of 1273 amino acids, which is shown in SEQ ID NO:1.
  • the S1 subunit of the SARS-CoV-2 virus is located at the N-terminus of the spike protein S and consists of 650 amino acids, as shown in SEQ ID NO: 2.
  • the RBD domain of the SARS-CoV-2 virus is located in the spike protein S1 and consists of 325 amino acids, shown in SEQ ID NO: 3.
  • GenBank accession number of the CR3022 antibody heavy chain variable region is DQ168569, and the GenBank accession number of the CR3022 antibody light chain variable region is DQ168570.
  • the sequence of the CR3022 antibody heavy chain variable region is shown in SEQ ID NO: 4; the sequence of the CR3022 antibody light chain variable region is shown in SEQ ID NO: 5.
  • the CR3022 antibody heavy chain sequence is shown in SEQ ID NO: 6; the CR3022 antibody light chain sequence is shown in SEQ ID NO: 7.
  • SARS-CoV-2 virus spike protein RBD SARS-CoV-2 S RBD
  • the SARS-CoV-2 S RBD protein fragment was added with a His tag at the C-terminus and used for affinity purification on a nickel column to obtain the SARS-CoV-2 S RBD protein.
  • Resuscitate SF9 cells (Thermo Fisher, 11496015), culture the cells until the growth state is in the logarithmic phase;
  • SARS-CoV-2 S RBD recombinant protein to an equal volume of loading buffer (1.5g Tris base, 4g SDS, 20ml glycerol, 2ml ⁇ -mercaptoethanol, 0.02g bromophenol blue, 100ml ddH 2 O, pH 6.8) After mixing, heat to 100°C in a water bath for 5 minutes, and separate the samples by SDS-PAGE using BioRad electrophoresis system and 10% precast gel (GenScript, M01012C). When the color strip of bromophenol blue is 1cm near the bottom, stop the electrophoresis and turn off the power.
  • loading buffer 1.5g Tris base, 4g SDS, 20ml glycerol, 2ml ⁇ -mercaptoethanol, 0.02g bromophenol blue, 100ml ddH 2 O, pH 6.8
  • loading buffer 1.5g Tris base, 4g SDS, 20ml glycerol, 2ml ⁇ -mercaptoethanol, 0.02
  • SARS-CoV-2 S RBD recombinant protein to an equal volume of loading buffer (1.5g Tris base, 4g SDS, 20ml glycerol, 2ml ⁇ -mercaptoethanol, 0.02g bromophenol blue, 100ml ddH 2 O, pH 6.8) , After mixing, heat to 100°C in a water bath for 5 minutes. The samples were separated by SDS-PAGE using BioRad electrophoresis system and 10% precast gel (GenScript, M01012C). When the color strip of bromophenol blue is 1cm near the bottom, stop the electrophoresis and turn off the power.
  • loading buffer 1.5g Tris base, 4g SDS, 20ml glycerol, 2ml ⁇ -mercaptoethanol, 0.02g bromophenol blue, 100ml ddH 2 O, pH 6.8
  • loading buffer 1.5g Tris base, 4g SDS, 20ml glycerol, 2ml ⁇ -mercaptoethanol,
  • CR3022 heavy chain including signal peptide + heavy chain variable region + constant region, the antibody heavy chain protein sequence is as SEQ ID NO: 6
  • CR3022 light chain including signal peptide + light chain can be Variable region + constant region, the antibody light chain protein sequence is a DNA fragment of SEQ ID NO: 7).
  • the antibody was quantified by OD280nm with an extinction coefficient Ec (0.1%) of 1.43, and the purified antibody was identified by HPLC. See the HPLC chart shown in Figure 3. The purity was 98.95%. The purified antibody was stored at -70°C. spare.
  • Coating plate Reconstitute the coating antigen SARS-CoV-2 S RBD with coating buffer (8.5g sodium chloride, 1.4g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate 1000ml ddH 2 O, pH 7.4) Dilute the protein to 0.5 ⁇ g/mL coating plate, 100 ⁇ l/well, overnight coating at 4°C or coating at 37°C for 2 hours;
  • Blocking 1% BSA/PBS blocking solution, 100 ⁇ l/well, blocking for 1 hour at 37°C;
  • Secondary antibody add horseradish peroxidase-labeled goat anti-human at a dilution of 1:5000, 50 ⁇ l/well, react at 37°C for 0.5 hours;
  • Stop add stop solution, 50 ⁇ l/well;
  • composition of the SARS-CoV-2 virus antibody ELISA detection kit 3.1 The composition of the SARS-CoV-2 virus antibody ELISA detection kit
  • SARS-CoV-2 antibody capture plate 96-well ELISA plate coated with SARS-CoV-2 S RBD recombinant protein
  • Positive control a negative control supplemented with anti-SARS-CoV-2 recombinant human antibody (CR3022 antibody) at a concentration of 100 ⁇ g/ml;
  • Negative control PBS buffer (8.5g sodium chloride, 1.4g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate, 0.5ml Tween 20, 1000ml ddH2O, pH 7.4), 10g BSA;
  • HRP-labeled anti-human antibody working solution horseradish peroxidase-labeled anti-human IgG antibody (GenScript, catalog number A01854) diluted 1:1000 with sample diluent;
  • washing solution PBS buffer (8.5g sodium chloride, 1.4g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate, 0.5ml Tween20, 1000ml ddH 2 O, pH 7.4);
  • the ORF DNA sequence of SARS-CoV-2 coronavirus S protein was digested with restriction DNA endonucleases HindIII and XbaI, and the plasmid vector p3XFLAG-CMV14 (Sigma, catalog number E4901) was digested with the same restriction enzymes. After digestion, the S protein ORF with sticky ends and plasmid vector fragments were ligated with T4 ligase and transformed into E. coli competent cells to obtain plasmid p3XFLAG-CMV14-2019nCoV-S (DOI: https://doi.org /10.1371/journal.pone.0076469).
  • HEK293FT cells Inoculate 4x10 6 HEK293FT cells (Thermo Fisher Scientific, Catalog No. R70007) in a 10 cm cell culture dish, aspirate the original medium on the second day after inoculation, and add 10 ml of fresh DMEM complete medium. After 2 hours, 5 ⁇ g of the plasmid p3XFLAG-CMV14-2019nCoV-S expressing the NCP coronavirus S protein was mixed with 20 ⁇ g of HIV-Luc plasmid and added to 500 ⁇ l of OptiMEM serum-free medium, and 50 ⁇ l of PEI was added to 500 ⁇ l of OptiMEM serum-free medium. In, let stand at room temperature for 5 minutes.
  • the OptiMEM serum-free medium containing PEI was mixed with 500 ⁇ l of OptiMEM serum-free medium containing plasmids, and the mixture was allowed to stand at room temperature for 8 minutes, and 1 ml of the mixture was added to HEK293FT cells. 24 hours after transfection, the medium containing the transfection mixture was replaced with 10 ml of fresh DMEM complete medium. The culture supernatant containing the pseudovirus was harvested 48 hours after transfection, filtered with a 0.45 ⁇ m pore filter, and frozen at -80°C.
  • SARS-CoV-2 virus antigen capture plate 96-well ELISA plate coated with anti-SARS-CoV antibody (CR3022 antibody);
  • Negative control PBS buffer (8.5g sodium chloride, 1.4g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate, 0.5ml Tween 20, 1000ml ddH2O, pH 7.4), 10g BSA;
  • Positive control a negative control with SARS-CoV-2 S RBD protein or SARS-CoV-2 S pseudovirus
  • Biotin-labeled anti-SARS-CoV-2 S RBD antibody such as CR3022 antibody
  • Streptavidin-HRP working solution use sample diluent 1:50,000 Streptavidin-HRP (GenScript, catalog number M00091);
  • washing solution PBS buffer (8.5g sodium chloride, 1.4g disodium hydrogen phosphate, 0.2g sodium dihydrogen phosphate, 0.5ml Tween 20, 1000ml ddH 2 O, pH 7.4);
  • the RBD protein standard curve of SARS-CoV-2 antigen detection kit is shown in Figure 6.
  • the SARS-CoV-2 antigen detection kit pseudovirus standard curve is shown in Figure 7.
  • the SARS-CoV-2 antigen detection kit has a very good linear relationship in the detection of virus antigens and viruses, and has potential application value in the detection of SARS-CoV-2 virus.
  • SEQ ID NO: 1 SARS-CoV-2 S protein
  • SEQ ID NO: 2 SARS-CoV-2 S1 protein
  • SEQ ID NO: 3 SARS-CoV-2 S RBD protein

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Abstract

属于病毒分子领域，涉及SARS-CoV-2病毒的检测方法及检测试剂盒，具体涉及一种检测样品中是否存在SARS-CoV-2病毒或其抗原的方法及其检测试剂盒。还涉及一种检测样品中是否存在抗SARS-CoV-2病毒抗体的方法及其检测试剂盒。进一步提供了一种特异性结合SARS-CoV-2病毒的抗体在检测试剂盒中的应用，所述抗体的重链序列如SEQ ID NO:6所示，和所述抗体的轻链序列如SEQ ID NO:7所示。所述的SARS-CoV-2检测方法操作简单且不需要特殊设备，能够实现病毒的早期免疫诊断。

Description

SARS-CoV-2病毒的检测方法及检测试剂盒 技术领域
本发明属于病毒分子生物学领域，涉及SARS-CoV-2病毒的免疫检测方法及检测试剂盒，具体涉及一种检测样品中是否存在SARS-CoV-2病毒或其抗原或抗体的方法及其检测试剂盒。
背景技术
SARS-CoV-2病毒(严重急性呼吸道综合征冠状病毒2型)，又称2019新型冠状病毒(2019-nCoV)，是以前在人类中尚未发现的、能引发肺炎疾病的新冠状病毒。SARS-CoV-2病毒在肺炎患者身体内发现，并引起肺炎疫情迅速爆发。迄今为止，该疫情已经扩散到亚洲、欧洲、北美洲和大洋洲的二十多个国家。感染者超过六万，造成超过1500人死亡。由于人类以前从未暴露于这种病毒，无法通过现有疫苗或天然免疫受到保护，也没有用于预防或治疗SARS-CoV-2病毒的特效药物。所以，该病毒可能导致疾病大面积暴发并最终演变成大流行。以前SARS和MERS的暴发就是这种情况。为此，世界卫生组织在2020年1月30日宣布该疫情构成“国际突发公共卫生事件”。中国卫生健康委员会将SARS-CoV-2病毒感染引起的急性呼吸道疾病定为法定乙类传染病，按甲类管理。2020年1月10日中国***门公布了SARS-CoV-2病毒的完整基因组。目前，已有多个研究团队在努力开发SARS-CoV-2病毒的疫苗、诊断试剂和治疗方法。
SARS-CoV-2病毒与中东呼吸综合征相关冠状病毒(Middle East Respiratory Syndrome-related Coronavirus,MERSr-CoV)和严重急性呼吸综合征相关冠状病毒(Severe Acute Respiratory Syndrome-related Coronavirus,SARSr-CoV)同属于β冠状病毒属。2020年1月8日上海市公共卫生临床中心向NCBI GenBank数据库提交SARS-CoV-2病毒基因组全序列。该病毒的全基因组序列(29903nt)的GenBank登录号为MN908947。根据同源性分析，SARS-CoV-2病毒的四种主要结构蛋白是刺突蛋白(S)、包膜蛋白(E)、基质蛋白(M)和核衣壳蛋白(N)。S蛋白是SARS-CoV-2最大的结构蛋白，介导病毒结合宿主细胞受体进入细胞内复制。S蛋白在宿主酶的作用下能裂解为S1和S2亚单位，其中S1亚单位含有受体结合区(Receptor binding domain,RBD)，是引起宿主免疫反应和产生中和抗体的主要靶抗原，也是疫苗研发和病原检测的重要靶标。刺突蛋白S是Ⅰ型跨膜糖蛋白，由N端的S1结构域和C端S2结构域组成。S1主要功能是与宿主细胞表面受体结合，S2亚基介导病毒-细胞以及细胞-细胞膜融合。在病毒感染过程中，首先通过S1蛋白受体结合区RBD与受体ACE2结合，完成病毒吸附过程，然后通过S2蛋白执行病毒包膜与细胞膜的融合，完成病毒感染过程。S蛋白在诱导机体免疫保护应答中发挥重要作用。SARS康复病人的血清中普遍存在着刺突蛋白S的抗体。SARS-CoV的毒刺突蛋白S能够有效地诱导中和抗体的产生。刺突蛋白S具有病毒的主要抗原决定簇，它是病毒检测的潜在诊断靶标之一。
目前已有的SARS-CoV-2病毒临床检测试剂是针对该病毒遗传物的RT-PCR核酸分子检测法。但SARS-CoV-2核酸分子诊断检测法存在不足之处。它对技术要求高，操作时间长，标本需要特殊处理及需要相应的设备，必须集中到中心实验室才能进行检测。而且核酸诊断 存在一定的假阳性率，另外随着病毒的突变，核酸诊断假阴性率也会上升。对SARS-CoV-2病毒的血清抗体检测方法包括酶联免疫方法。病毒感染后产生抗体需要1-3周的窗口期，而从感染到发病的潜伏期在SARS-CoV-2感染时很短(一般一周之内)。虽然抗体检测对SARS-CoV-2病毒早期诊断没有意义，但可以作为SARS-CoV-2病毒确诊的依据。但ELISA检测试剂盒中需要病人的阳性血清作为阳性对照。但病人的阳性血清数量有限且存在传染的风险。因此，制备能够替代作为阳性对照的病人阳性血清的等同物是开发SARS-CoV-2病毒抗体检测试剂盒的一大难题。SARS-CoV-2病毒的检测特别需要一种能在感染早期快速且灵敏地进行诊断的方法。快速、敏感、特异的SARS-CoV-2病毒抗原诊断方法在SARS-CoV-2诊断中具有无可取代的意义。目前世界上尚没有关于SARS-CoV-2病毒抗原诊断性试剂的报道。也未见以刺突蛋白RBD片段作为检测抗原的报道。
SARS-CoV人单克隆抗体CR3022是从CR3022单链抗体(single-chain variable fragment，scFv)获得的轻链和重链的可变区和人的IgG1型恒定区构建的抗体。CR3022scFv是从新加坡的恢复期SARS患者的淋巴细胞构建了免疫scFv噬菌体文库中筛选的，它可以与紫外线灭活的SARS-CoV结合。目前没有关于CR3022抗体用于SARS-CoV-2病毒或者抗体检测的报道。
发明内容
本发明一方面提供了一种检测样品中是否存在SARS-CoV-2病毒或其抗原的方法及其检测试剂盒。另一方面提供了一种检测样品中是否存在抗SARS-CoV-2病毒的抗体的方法及其检测试剂盒。本发明进一步提供了一种特异性结合SARS-CoV-2病毒的抗体在检测试剂盒中的应用，所述抗体的重链序列如SEQ ID NO:6所示，和所述抗体的轻链序列如SEQ ID NO:7所示。
发明简述
本发明一方面提供了一种检测样品中是否存在SARS-CoV-2病毒或其抗原的方法，所述方法包括：
(1)将特异性结合SARS-CoV-2病毒的抗体与样品接触，在合适条件下，所述抗体与样品中所述病毒或其抗原形成免疫复合物；
(2)检测所述免疫复合物的存在。
在一些实施方案中，所述特异性结合SARS-CoV-2病毒的抗体为抗SARS-CoV-2 S蛋白的抗体。在一个优选实施方案中，所述抗体为抗SARS-CoV-2 S RBD蛋白的抗体。
在一些实施方案中，所述抗体的重链可变区包含与SEQ ID NO:4所示序列至少80％一致性的氨基酸序列，所述抗体的轻链可变区包含与SEQ ID NO:5所示序列至少80％一致性的氨基酸序列。在另一些实施方案中，所述抗体的重链可变区包含SEQ ID NO:4所示序列，所述抗体的轻链可变区包含SEQ ID NO:5所示序列。在一个具体实施方案中，所述抗体的重链可变区序列如SEQ ID NO:4所示，所述抗体的轻链可变区序列如SEQ ID NO:5所示。
在一些实施方案中，所述抗体的重链序列包含与SEQ ID NO:6所示序列至少80％一致性氨基酸序列，和所述抗体的轻链序列包含与SEQ ID NO:7所示序列至少80％一致性的氨基酸序列。在另一些实施方案中，所述抗体的重链序列包含SEQ ID NO:6所示的序列，所述 抗体的轻链序列包含SEQ ID NO:7所示的序列。在一个具体实施方案中，所述抗体的重链序列如SEQ ID NO:6所示，和所述抗体的轻链序列如SEQ ID NO:7所示。
在一些实施方案中，所述抗体与样品接触步骤包括先将所述抗体包被在固相支持物上，再将样品加入到包被抗体的固相支持物上孵育。
在一些实施方案中，所述合适条件是指所述抗体与所述样品在25-37℃孵育15分钟到1.5小时。
在一些实施方案中，所述检测免疫复合物步骤包括加入第二抗体，所述第二抗体为抗SARS-CoV-2病毒的抗体。在一些优选实施方案中，所述第二抗体为抗SARS-CoV-2 S蛋白抗体。在另一些优选实施例中，所述抗体为抗SARS-CoV-2 S RBD蛋白抗体。所述第二抗体适用于ELISA方法检测免疫复合物，特别地是夹心ELISA方法检测免疫复合物。在一些实施方案中，所述第二抗体为抗SARS-CoV-2 S RBD蛋白的抗体，用于夹心ELISA方法检测免疫复合物。
在一些实施方案中，所述第二抗体是生物素、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶进行标记的。
本发明另一方面提供了一种检测样品中SARS-CoV-2病毒或其抗原的试剂盒，所述试剂盒包括：
(1)特异性结合SARS-CoV-2病毒的抗体；
(2)固相支持物；
(3)检测所述样品中可能存在的病毒抗原与所述抗体形成免疫复合物的工具。
在一些实施方案中，所述特异性结合SARS-CoV-2病毒的抗体为抗SARS-CoV-2 S蛋白抗体，优选地，所述抗体为抗SARS-CoV-2 S RBD蛋白的抗体。在另一些实施方案中，所述抗体的重链可变区包含与SEQ ID NO:4所示序列至少80％一致性的氨基酸序列，所述抗体的轻链可变区包含与SEQ ID NO:5所示序列至少80％一致性的氨基酸序列。在一些实施方案中，所述抗体的重链可变区包含SEQ ID NO:4所示序列，所述抗体的轻链可变区包含SEQ ID NO:5所示序列。在一个具体实施方案中，所述抗体的重链可变区序列如SEQ ID NO:4所示，所述抗体的轻链可变区序列如SEQ ID NO:5所示。在另一些实施方案中，所述抗体的重链序列包含与SEQ ID NO:6所示序列至少80％一致性氨基酸序列，和所述抗体的轻链序列包含与SEQ ID NO:7所示序列至少80％一致性的氨基酸序列。在一些实施方案中，所述抗体的重链序列包含SEQ ID NO:6所示序列，所述抗体的轻链序列包含SEQ ID NO:7所示序列。在一个具体实施方案中，所述抗体的重链序列如SEQ ID NO:6所示，和所述抗体的轻链序列如SEQ ID NO:7所示。
在一些实施方案中，所述抗体包被在固相支持物上。在一些实施方案中，所述固相支持物包括无机和有机多聚物，优选地，所述固相支持物为容器内壁，如孔板内壁。
在一些实施方案中，所述检测免疫复合物的工具包括第二抗体。在一些实施方案中，所述第二抗体为IgG、IgM和/或IgA抗体。在另一些实施方案中，所述第二抗体为抗SARS-CoV-2病毒的抗体。所述第二抗体适用于ELISA方法检测免疫复合物，特别地是夹心ELISA方法检测免疫复合物。在一些实施方案中，所述第二抗体为抗SARS-CoV-2 S RBD蛋白的抗体，用于夹心ELISA方法检测免疫复合物。在一些实施方案中，所述第二抗体是生 物素、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶标记的。
本发明所述的检测样品中SARS-CoV-2病毒或其抗原的试剂盒选自ELISA检测试剂盒、化学发光检测试剂盒、胶体金检测试剂盒或时间分辨免疫层析检测试剂盒，优选为ELISA检测试剂盒，更优选为夹心ELISA检测试剂盒。
本发明具体地提供了一种检测样品中SARS-CoV-2病毒或其抗原的ELISA试剂盒，所述试剂盒包括：
(1)第一抗体包被的固相支持物，所述第一抗体为特异性结合SARS-CoV-2病毒抗原的抗体；
(2)生物素或辣根过氧化物酶标记的第二抗体，所述第二抗体为特异性结合SARS-CoV-2病毒抗原的抗体；
(3)阴性对照及阳性对照；
(4)样本稀释液、洗涤液、可选择的辣根过氧化物酶标记链霉亲和素以及底物反应液。
在一些实施方案中，所述第一抗体和第二抗体分别选自如下抗体序列：所述第一/第二抗体的重链可变区包含SEQ ID NO:64所示的氨基酸序列，所述第一/第二抗体的轻链可变区包含SEQ ID NO:5所示的氨基酸序列。
在一些实施方案中，所述阳性对照包括SARS-CoV-2 S RBD蛋白或SARS-CoV-2 S假病毒。所述阳性对照可以是包括SARS-CoV-2 S RBD蛋白或SARS-CoV-2 S假病毒的阴性对照。在另一些实施方案中，所述阴性对照包含健康人血清。在一些实施方案中，所述阴性对照包含BSA的PBS缓冲液。
本发明上述的检测样品中是否存在SARS-CoV-2病毒或其抗原的方法或者检测样品中SARS-CoV-2病毒或其抗原的试剂盒，其中所述样品是来源于感染或疑似感染SARS-CoV-2病毒的血浆、血清、全血、痰液、口腔/鼻咽分泌物或洗液、尿液、粪便、胸腹腔积液、脑脊液、组织标本或非生物学样品如水、饮料。
本发明又一方面提供了一种检测样品中是否存在抗SARS-CoV-2病毒的抗体的方法，所述方法包括：
(1)将SARS-CoV-2病毒抗原与样品接触，在合适条件下，所述抗原与所述样品中SARS-CoV-2病毒抗体形成免疫复合物；
(2)检测所述免疫复合物的存在。
在一些实施方案中，所述SARS-CoV-2病毒抗原包含与SEQ ID NO:2或3所示序列至少80％一致性的氨基酸序列。在另一些实施方案中，所述SARS-CoV-2病毒抗原包含SEQ ID NO:2或3所示的氨基酸序列。在一个具体实施方案中，所述SARS-CoV-2病毒抗原序列如SEQ ID NO:2或3所示。
在一些实施方案中，所述病毒抗原与所述样品接触步骤包括先将所述病毒抗原包被在固相支持物上，再将样品加入到包被抗原的固相支持物上孵育。
在一些实施方案中，所述合适条件为所述病毒抗原与所述样品在25-37℃孵育15分钟到1.5小时。
在一些实施方案中，所述检测步骤包括加入第二抗体，所述第二抗体为抗人抗体。进一步，所述第二抗体可以是抗人IgG、抗人IgM和/或抗人IgA抗体。在另一些实施方案中， 所述第二抗体是生物素、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶标记的。
本发明另一方面提供了一种检测样品中抗SARS-CoV-2病毒的抗体的试剂盒，所述试剂盒包括：
(1)SARS-CoV-2病毒抗原；
(2)固相支持物；
(3)检测所述病毒抗原与所述样品中可能存在的抗体形成免疫复合物的工具。
在一些实施方案中，所述SARS-CoV-2病毒抗原为SARS-CoV-2 S RBD蛋白。在另一些实施方案中，所述SARS-CoV-2病毒抗原包含与SEQ ID NO:2或3所示序列至少80％一致性的氨基酸序列。在另一些实施方案中，所述SARS-CoV-2病毒抗原包含SEQ ID NO:2或3所示的氨基酸序列。在一个具体实施方案中，所述SARS-CoV-2病毒抗原序列如SEQ ID NO:2或3所示。
在一些实施方案中，所述病毒抗原包被在固相支持物上。在另一些实施方案中，所述固相支持物包括无机和有机多聚物，优选地，所述固相支持物为容器内壁，如孔板内壁。
在一些实施方案中，所述检测免疫复合物的工具包括一种检测抗体，所述抗体是生物素、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶标记的。在一个具体实施方案中，所述检测抗体为辣根过氧化物酶标记的抗人抗体。在另一个具体实施方案中，所述检测抗体为辣根过氧化物酶标记的抗人IgG、抗人IgM和/或抗人IgA抗体。
本发明所述检测样品中抗SARS-CoV-2病毒的抗体的试剂盒选自ELISA检测试剂盒、化学发光检测试剂盒、胶体金检测试剂盒或时间分辨免疫层析检测试剂盒，优选为ELISA检测试剂盒，更优选为间接ELISA检测试剂盒。
本发明具体地提供了一种检测样品中抗SARS-CoV-2病毒的抗体的ELISA试剂盒，所述试剂盒包括：
(1)包被SARS-CoV-2病毒抗原的孔板；
(2)辣根过氧化物酶碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶标记的抗人抗体；
(3)阴性对照及阳性对照；
(4)样本稀释液、洗涤液、底物反应液以及终止液。
在一些实施方案中，所述SARS-CoV-2病毒抗原为SARS-CoV-2 S RBD蛋白。在另一些实施方案中，所述SARS-CoV-2病毒抗原包含与SEQ ID NO:2或3至少80％一致性的氨基酸序列。在另一些实施方案中，所述SARS-CoV-2病毒抗原包含SEQ ID NO:2或3所示的氨基酸序列。在一个具体实施方案中，所述SARS-CoV-2病毒抗原序列如SEQ ID NO:2或3所示。
在另一些实施方案中，所述阳性对照包括特异性结合SARS-CoV-2病毒的抗体。在一些实施方案中，所述阳性对照包含的抗体的重链可变区包含SEQ ID NO:4所示序列，所述抗体的轻链可变区包含SEQ ID NO:5所示的序列。在一个具体实施方案中，所述阳性对照中抗体的重链可变区序列如SEQ ID NO:4所示，所述抗体的轻链可变区序列如SEQ ID NO:5所示。在一个具体实施方案中，所述阳性对照包括的抗体的重链序列如SEQ ID NO:6所示，和所述抗体的轻链序列如SEQ ID NO:7所示。在一些实施方案中，所述阴性对照包含健康人血清。在另一些实施方案中，所述阴性对照包含BSA的PBS缓冲液。本发明还提供了一种特异性 结合SARS-CoV-2病毒的抗体在检测试剂盒中的应用。优选地，提供了一种抗SARS-CoV-2病毒的抗体在检测SARS-CoV-2病毒抗体的试剂盒中的应用。
在一些实施方案中，所述抗体的重链序列如SEQ ID NO:6所示，和所述抗体的轻链序列如SEQ ID NO:7所示。在一些实施方案中，通过将所述抗体添加至阴性对照作为阳性对照。所述阴性对照可以是健康人血清，也可以是包含BSA的PBS缓冲液。
本发明所述的检测样品中是否存在抗SARS-CoV-2病毒的抗体的方法或检测样品中抗SARS-CoV-2病毒的抗体的试剂盒，其中所述样品是来源于感染或疑似感染SARS-CoV-2病毒的血浆、血清、全血、胸腹腔积液、脑脊液或组织标本。
发明详述
除非另有说明，本发明所用的技术和科学术语具有与本发明所属领域的普通技术员通常所理解的含义。
术语“新型冠状病毒”(SARS-CoV-2)，亦称为2019-nCoV，，其属于β属冠状病毒，有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60-140nm。其基因特征与SARSr-Cov和MERSr-CoV有明显区别。研究显示，其与蝙蝠SARS样冠状病毒(bat-SL-CoVZC45)同源性达85％以上。体外分离培养时，2019-nCov 96个小时左右即可在人呼吸道上皮细胞内发现，而在Vero E6和Huh-7细胞系中分离培养需约6天。
“SARS-CoV-2病毒抗原”是指SARS-CoV-2全病毒或其裂解液的抗原或者重组表达的SARS-CoV-2抗原。SARS-CoV-2病毒包括刺突蛋白(S)、包膜蛋白(E)、基质蛋白(M)和核衣壳蛋白(N)抗原，其中S蛋白是SARS-CoV-2最大的结构蛋白。S蛋白在宿主酶的作用下能裂解为S1和S2亚单位，其中S1亚单位含有受体结合区RBD，是主要的靶抗原。本发明中，所述SARS-CoV-2病毒抗原选自刺突蛋白(S)、包膜蛋白(E)、基质蛋白(M)和/或核衣壳蛋白(N)抗原。优选地，所述SARS-CoV-2病毒抗原为SARS-CoV-2 S RBD抗原。本发明中所述SARS-CoV-2 S RBD抗原可采用常规重组表达的方法生产，通过构建表达SARS-CoV-2 S RBD的质粒，如pFastBac1、pTT5，包含目的基因的表达载体转染表达细胞，如CHO细胞、SF9细胞，表达纯化得到SARS-CoV-2 S RBD重组蛋白。
术语"抗体"意在指由四条多肽链组成的免疫球蛋白分子(其中两条重链(H)和两条轻链(L)通过二硫键相互连接(即"完整的抗体分子"))，以及其多聚体(例如IgM)或其抗原结合片段。每条重链由重链可变区(“HCVR”或“VH”)和重链恒定区(由结构域CH1、CH2和CH3组成)组成。每条轻链由轻链可变区(“LCVR或“VL”)和轻链恒定区(CL)组成。VH和VL区可进一步细分为称为互补决定区(CDR)的高变区，其间插有更保守的区称为框架区(FR)。每个VH和VL由三个CDR和四个FR组成，以下列顺序从氨基末端至羟基末端排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。在本发明的一些实施方案中，抗体(或其抗原结合片段)的FR可与人种系序列相同或可经天然或人工修饰。
关于肽或多肽序列的“百分比(％)氨基酸序列一致性”定义为对比序列并在必要时引入缺口以获取最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分，候选序列中与特定肽或多肽序列中的氨基酸残基相同的氨基酸残基的百分率。可以本领域技术范围内的多种方式进行序列对比以测定百分比氨基酸序列同一性，例如使用公众可得到的计算 机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可决定测量对比的适宜参数，包括对所比较的序列全长获得最大对比所需的任何算法。
所述的“固相支持物”包括本领域周知的有机和无机多聚物，诸如包括但不局限于：葡聚糖、天然或经修饰的纤维素、聚乙烯、聚苯乙烯、聚丙烯酰胺，琼脂糖，乳胶，容器内壁如试管、滴定板、玻璃杯等。可使用本领域周知的多种方法包被固相支持物。例如：利用双功能试剂活化，参见美国专利5399501。在一些具体实施方案中，所述固相支持物为96孔板的孔壁。
如本文使用的术语“受试者”指需要缓解、预防和/或治疗疾病或病症如病毒感染的动物，优选哺乳动物，更优选人。术语包括具有冠状病毒如SARS-CoV-2感染或处于具有冠状病毒如SARS-CoV-2感染风险的人受试者。健康受试者是指未感染SARS-CoV-2病毒的健康动物，优选哺乳动物，更优选人。
当用“给予”和“治疗”提及动物、人、实验对象、细胞、组织、器官或生物液时，是指将外源性药物、治疗剂、诊断剂或组合物与动物、人、受治疗者、细胞、组织、器官或生物液接触。“给予”和“治疗”可指例如治疗方法、药动学方法、诊断方法、研究方法和实验方法。治疗细胞包括让试剂与细胞接触以及让试剂与流液接触，其中所述流液与细胞接触。“给予”和“治疗”还意味着例如通过试剂、诊断剂、结合组合物或通过其他细胞对细胞进行体外和离体治疗。
除非另外特别说明，否则单数的使用包括复数。除非另外特别说明，否则词语“一个(a)”或“一个(an)”意指“至少一个”。除非另外说明，否则“或”的使用意指“和/或”。短语“至少一个”的含义等同于短语“一个或多个”的含义。此外，术语“包括(including)”以及其他形式诸如“包括(includes)”和“包括(included)”的使用不是限制性的。此外，除非另外特别说明，否则术语诸如“要素”或“组分”包括包含一个单元的元素或组分以及包含多于一个单元的元素和组分。
检测SARS-CoV-2病毒或其抗原的方法
本发明一方面提供一种检测样品中是否存在SARS-CoV-2病毒或其抗原的方法，所述方法包括：
(1)特异性结合SARS-CoV-2病毒抗原的抗体与样品接触，在合适条件下，所述抗体与样品中所述病毒或其抗原形成免疫复合物；
(2)检测所述免疫复合物的存在。
在一些实施方案中，特异性结合SARS-CoV-2病毒抗原的抗体可选自多克隆抗体或者单克隆抗体。所述特异性结合SARS-CoV-2病毒抗原的抗体选自特异性结合SARS-CoV-2 S蛋白的抗体(抗SARS-CoV-2 S抗体)。优选地，特异性结合SARS-CoV-2 S蛋白的抗体为抗SARS-CoV-2 S RBD蛋白的抗体。
在一些实施方案中，所述特异性结合SARS-CoV-2病毒抗原的抗体重链可变区包含与SEQ ID NO:4所示序列至少80％，至少85％，至少90％，至少95％，至少97％或至少99％一致性的氨基酸序列，所述抗体轻链可变区包含与SEQ ID NO:5所示序列至少80％，至少85％，至少90％，至少95％，至少97％或至少99％一致性的氨基酸序列。在一些实施方案中，所述抗体重链可变区如SEQ ID NO:4所示，所述抗体轻链可变区如SEQ ID NO:5所示。
在一些实施方案中，所述特异性结合SARS-CoV-2病毒抗原的抗体重链序列包含与SEQ ID NO:6所示序列至少80％，至少85％，至少90％，至少95％，至少97％或至少99％一致性的氨基酸序列，所述抗体的轻链序列包含与SEQ ID NO:7所示序列至少80％，至少85％，至少90％，至少95％，至少97％或至少99％一致性的氨基酸序列。在一些具体实施方案中，所述抗体的重链序列如SEQ ID NO:6所示，所述抗体的轻链序列如SEQ ID NO:7所示。在一些具体实施方案中，所述特异性结合SARS-CoV-2病毒抗原的抗体为CR3022抗体。
所述特异性结合SARS-CoV-2病毒抗原的抗体可通过常规的分子生物学方法构建表达和生产。本发明中，所述SARS-CoV-2病毒抗原的抗体如CR3022抗体，可通过构建分别表达抗体重链和轻链的质粒，共转染表达细胞，培养纯化后获得抗体。进一步，可通过免疫测定方法检测抗体的活性，如采用间接ELISA测定抗体的效价。
在一些实施方案中，所述特异性结合SARS-CoV-2病毒抗原的抗体与样品中可能存在的病毒抗原的接触步骤包括先将所述抗体包被在固相支持物上，再将样品加入到包被抗体的固相支持物上孵育。所述包被可使用本领域周知的多种方法包被固相支持物。例如：利用双功能试剂活化，参见美国专利5399501。
所述的抗原抗体免疫复合物“合适条件”为本领域周知，是指在适当的温度及时间条件下孵育特异性结合SARS-CoV-2病毒抗原的抗体与含SARS-CoV-2病毒抗原的样品的混合物，例如在约20℃-39℃，优选约25℃-37℃下，孵育约20分钟至约4小时，优选孵育约50分钟至约1.5小时。在一些具体实施方案中，所述合适条件是指所述抗体与所述样品在25-37℃孵育15分钟到1.5小时，优选地，所述抗体与所述样品在37℃孵育1小时。
经孵育形成抗原抗体免疫复合物后，用本领域常用的适当洗涤溶液洗涤固相支持物，如洗涤检测板数次，以清除未结合的经标记的抗原。所用洗液一般为pH约5-8，优选为pH约7.4的磷酸盐缓冲溶液。
可采用多种本领域周知的方法定量检测抗原抗体免疫复合物的形成。在一些实施方案中，所述检测免疫复合物的步骤包括加入第二抗体，所述第二抗体为抗SARS-CoV-2病毒抗原的抗体。所述第二抗体可与前述特异性结合SARS-CoV-2病毒抗原的抗体相同，也可与前述特异性结合SARS-CoV-2病毒抗原的抗体不同。根据标记第二抗体所使用的方法，选择适当的定量检测方案。本发明一些实施方案中，所述第二抗体是生物素、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶标记的。
在一个优选的实施方案中，标记第二抗体时使用生物素，再使用链霉亲和素(SA)-辣根过氧化物酶与抗原抗体免疫复合物作用，显色后在波长为450nm酶标仪读取吸光值。将所得读数与Cutoff值的读数加以比较，判断所测样本的结果。
检测SARS-CoV-2病毒或其抗原的试剂盒
本发明另一方面提供一种检测样品中SARS-CoV-2病毒或其抗原的试剂盒，所述试剂盒包括：
(1)特异性结合SARS-CoV-2病毒抗原的抗体，所述抗体为抗SARS-CoV-2 S蛋白抗体，优选地，所述抗体为抗SARS-CoV-2 S RBD蛋白的抗体；
(2)固相支持物；
(3)检测所述样品中可能存在的病毒或其抗原与所述抗体形成免疫复合物的工具。
在一些实施方案中，所述特异性结合SARS-CoV-2病毒抗原的抗体重链可变区包含与SEQ ID NO:4所示序列至少80％，至少85％，至少90％，至少95％，至少97％或至少99％一致性的氨基酸序列，所述抗体轻链可变区包含与SEQ ID NO:5所示序列至少80％，至少85％，至少90％，至少95％，至少97％或至少99％一致性的氨基酸序列。在一些实施方案中，所述抗体重链可变区如SEQ ID NO:4所示，所述抗体轻链可变区如SEQ ID NO:5所示。
在一些实施方案中，所述特异性结合SARS-CoV-2病毒抗原的抗体的重链序列包含与SEQ ID NO:6所示序列至少80％，至少85％，至少90％，至少95％，至少97％或至少99％一致性氨基酸序列，所述抗体的轻链序列包含与SEQ ID NO:7所示序列至少80％，至少85％，至少90％，至少95％，至少97％或至少99％一致性的氨基酸序列。在一些具体实施方案中，所述抗体的重链序列如SEQ ID NO:6所示，所述抗体的轻链序列如SEQ ID NO:7所示。在一些具体实施方案中，所述特异性结合SARS-CoV-2病毒抗原的抗体为CR3022抗体。
在一些实施方案中，所述特异性结合SARS-CoV-2病毒抗原的抗体包被在固相支持物上。在一些实施方案中，所述固相支持物包括无机和有机多聚物，优选地，所述固相支持物为容器内壁，如孔板内壁。在一个具体实施方案中，所述固相支持物为96孔板内壁。在另一个具体实施方案中，所述固相支持物为所述包被可使用本领域周知的多种方法包被固相支持物。例如：利用双功能试剂活化，参见美国专利5399501。在一些实施方案中，合适浓度的所述抗SARS-CoV-2 S RBD抗体在4℃-37℃包被1-30小时，再洗板，封闭液封闭，拍干，得到抗体包被板。在一个优选实施方案中，浓度为1-10μg/ml所述抗SARS-CoV-2 S RBD抗体，如CR3022抗体在4℃包被18小时，再洗板，封闭液封闭，拍干，得到抗体包被板，也即SARS-CoV-2病毒抗原捕获板。
在一些实施方案中，检测所述免疫复合物的工具包括第二抗体。在一些实施方案中，所述第二抗体为抗IgG、抗IgM和/或抗IgA抗体。在一个具体实施方案中，所述第二抗体为抗人IgG抗体或抗人IgM抗体。在一个优选实施方案中，所述第二抗体为抗人IgG抗体。在另一些实施方案中，所述第二抗体为抗SARS-CoV-2病毒抗原的抗体。所述第二抗体可与前述特异性结合SARS-CoV-2病毒抗原的抗体相同，也可与前述特异性结合SARS-CoV-2病毒抗原的抗体不同。在一个具体实施方案中，所述抗SARS-CoV-2病毒的抗体为CR3022抗体。
在一些实施方案中，所述第二抗体是生物素、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶标记的。根据标记第二抗体所使用的方法，选择适当的定量检测方案。在一个优选的实施方案中，标记第二抗体时使用生物素，再使用链霉亲和素-辣根过氧化物酶与抗原抗体免疫复合物作用，显色后在波长为450nm酶标仪读取吸光值。将所得读数与阴性对照的读数加以比较，判断所测样本的结果。根据定性或定量检测方案的不同，所述检测样品中SARS-CoV-2病毒或其抗原的试剂盒选自ELISA检测试剂盒、化学发光检测试剂盒、胶体金检测试剂盒或时间分辨免疫层析检测试剂盒。
ELISA检测试剂盒是根据抗原抗体特异性免疫反应原理设计，采用双抗体夹心法检测SARS-CoV-2病毒抗原。该方法是将特异性抗体包被在固相载体的表面，形成固相抗体，加入待检样品，使其中的相应抗原结合到固相载体的抗体上，再加入酶标记的第二抗体，形成 抗原-抗体-酶标记第二抗体复合物，最后加入底物溶液显色，酶与底物发生反应后，底物显色，根据底物显色的深浅定性或定量地检测待检样本中抗原的量。
本发明具体的提供一种检测样品中SARS-CoV-2病毒或其抗原的ELISA试剂盒，所述试剂盒包括：
(1)第一抗体包被的固相支持物，所述第一抗体为特异性结合SARS-CoV-2病毒抗原的抗体；
(2)生物素或辣根过氧化物酶标记的第二抗体，所述第二抗体为特异性结合SARS-CoV-2病毒抗原的抗体；
(3)阴性对照及阳性对照；
(4)样本稀释液、洗涤液、可选择的辣根过氧化物酶标记链霉亲和素、底物反应液及终止反应液。
在一些实施方案中，所述第一抗体和第二抗体可为单克隆抗体或者多克隆抗体。所述第二抗体与第一抗体为相同抗体或不同的抗体。在一些实施方案中，述第二抗体与第一抗体为相同抗体。在另一些实施方案中，述第二抗体与第一抗体为不同的抗体。在一些实施方案中，所述第一抗体和第二抗体分别选自如下序列：所述第一/第二抗体的重链序列如SEQ ID NO:6所示，所述第一/第二抗体的轻链序列如SEQ ID NO:7所示。在一些实施方案中，所述第一抗体的重链如SEQ ID NO:6所示，轻链如SEQ ID NO:7所示。在一些实施方案中，所述第二抗体的重链如SEQ ID NO:6所示，轻链如SEQ ID NO:7所示。在一个具体实施方案中，所述第一抗体为CR3022抗体，所述第二抗体为抗SARS-CoV-2 S RBD蛋白的抗体。在另一个具体实施方案中，所述第二抗体为CR3022抗体，所述第一抗体为抗SARS-CoV-2 S RBD蛋白的抗体。
在一些实施方案中，所述第一抗体包被的固相支持物为包被抗SARS-CoV-2病毒抗体的微量滴定板。在一些优选实施方案中，所述第一抗体包被的固相支持物为包被CR3022抗体的96孔酶标板。第一抗体包被的固相支持物的制备方法为本领域公知的方法，包括将合适浓度的所述抗SARS-CoV-2 S RBD抗体在4℃-37℃包被1-30小时，再洗板，封闭液封闭，拍干，得到抗体包被板。在一个优选实施方案中，浓度为2μg/ml所述抗SARS-CoV-2 S RBD抗体，如CR3022抗体在4℃包被18小时，再洗板，封闭液封闭，拍干，得到抗体包被板，也即SARS-CoV-2病毒抗原捕获板。
在一些实施方案中，所述阳性对照包括SARS-CoV-2 S RBD蛋白或SARS-CoV-2 S假病毒。在一些实施方案中，所述阴性对照包含BSA的PBS缓冲液。在一个具体实施方案中，所述阳性对照为添加SARS-CoV-2 S RBD蛋白或SARS-CoV-2 S假病毒的磷酸盐缓冲液。在另一些实施方案中，所述阳性对照为添加SARS-CoV-2 S RBD蛋白或SARS-CoV-2 S假病毒的健康人血浆、血清、全血、胸腹腔积液或脑脊液。在一些实施方案中，所述阳性对照为添加SARS-CoV-2 S RBD蛋白或SARS-CoV-2 S假病毒的阴性样本。在一个优选实施方案中，所述阳性对照为添加SARS-CoV-2 S RBD蛋白或SARS-CoV-2 S假病毒的健康人血清。
在一些实施方案中，所述生物素或辣根过氧化物酶标记的第二抗体为生物素或辣根过氧化物酶标记的CR3022抗体，优选为，生物素标记的CR3022抗体。在一个具体实施方案中，所述试剂盒中包括0.05-4μg/ml生物素标记的SARS-CoV-2 S RBD抗体，优选为1μg/ml生物 素标记的CR3022抗体。在一个具体实施方案中，所述试剂盒包含1μg/m生物素标记的CR3022抗体。
本发明的检测样品中SARS-CoV-2病毒或其抗原的ELISA试剂盒所包括的样本稀释液、洗涤液、底物反应液及终止反应液均为本领域常用的溶液。所述样本稀释液包括氯化钠，磷酸氢二钠，磷酸二氢钠，Tween 20，BSA，水并调节pH 7.4。所述洗涤液包括氯化钠，磷酸氢二钠，磷酸二氢钠，Tween 20，水并调节pH 7.4。所述底物反应液包含TMB底物溶液。
本发明还提供了所述检测样品中SARS-CoV-2病毒抗原的ELISA试剂盒在检测感染或疑似感染SARS-CoV-2病毒受试者中的应用。
本发明所述ELISA试剂盒检测操作步骤包括：
(1)向酶标板中加入优化稀释的待检样品，同时设立包含BSA的PBS缓冲液和的阴性对照孵育；
(2)加入洗涤液，洗板拍干；
(3)加入生物素标记的SARS-CoV-2 S RBD抗体，孵育；
(4)加入洗涤液，洗板拍干；
(5)加入Streptavidin(SA)-HRP工作液孵育；
(6)加入洗涤液，洗板拍干；
(7)加入底物显色液孵育；
(8)加入终止液反应，酶标仪测定。
在一些实施方案中，ELISA试剂盒中进一步包括检测操作步骤的说明书。
在一些实施方案中，所述样品是来源于感染或疑似感染SARS-CoV-2病毒的血浆、血清、全血、痰液、口腔/鼻咽分泌物或洗液、尿液、粪便、胸腹腔积液、脑脊液、组织标本或非生物学样品如水、饮料。
检测SARS-CoV-2病毒抗体的方法
本发明提供了一种检测样品中是否存在SARS-CoV-2病毒抗体的方法，所述方法包括：
(1)SARS-CoV-2病毒抗原与样品接触，在合适条件下，所述抗原与所述样品中SARS-CoV-2病毒的抗体形成免疫复合物；
(2)检测所述免疫复合物的存在。
在一些实施方案中，所述SARS-CoV-2病毒抗原为SARS-CoV-2 S蛋白。在一些优选实施方案中，所述SARS-CoV-2病毒抗原为SARS-CoV-2 S RBD蛋白。在一些实施方案中，所述SARS-CoV-2病毒抗原包含与SEQ ID NO:2或3至少80％、至少85％、至少90％、至少95％、至少97％或至少99％一致性的氨基酸序列。在一个具体实施方案中，所述SARS-CoV-2病毒抗原如SEQ ID NO:2或3所示。
在一些实施方案中，所述接触步骤包括先将所述病毒抗原包被在固相支持物上，再将样品加入到包被抗原的固相支持物上孵育。所述包被可使用本领域周知的多种方法包被固相支持物。例如：利用双功能试剂活化，参见美国专利5399501。
所述的抗原抗体免疫复合物“合适条件”为本领域周知，是指在适当的温度及时间条件下孵育SARS-CoV-2病毒或其抗原与含SARS-CoV-2病毒抗体的样品的混合物，例如在约20℃-39℃，优选约25℃-37℃下，孵育约3分钟至约4小时，优选孵育约5分钟至约1.5小 时。在一些具体实施方案中，所述合适条件是指所述抗原与所述样品在25-37℃孵育15分钟到1.5小时，优选地，所述抗原与所述样品在37℃孵育1小时。
经孵育形成抗原抗体免疫复合物后，用本领域常用的适当洗涤溶液洗涤固相支持物，如检测板数次，以清除未结合的经标记的抗原。所用洗液一般为pH约5-8，优选为pH约7.4的磷酸盐缓冲溶液。
可采用多种本领域周知的方法定量检测抗原抗体免疫复合物的形成。在一些实施方案中，所述检测免疫复合物的步骤包括加入第二抗体，所述第二抗体为抗IgG、IgM和/或IgA抗体。在一些优选的实施方案中，所述第二抗体为抗人IgG或抗IgM抗体。在另一些优选的实施方案中，所述第二抗体为抗人IgG抗体。根据标记第二抗体所使用的方法，选择适当的定量检测方案。本发明一些实施方案中，所述第二抗体是生物素、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶进行标记的。
在一个优选的实施方案中，标记第二抗体是辣根过氧化物酶标记，再加入底物液和终止液，显色后在波长为450nm酶标仪读取吸光值。将所得读数与阴性对照的读数加以比较，判断所测样本的结果。
检测SARS-CoV-2病毒抗体的试剂盒
本发明提供了一种检测样品中SARS-CoV-2病毒抗体的试剂盒，所述试剂盒包括：
(1)SARS-CoV-2病毒抗原，所述抗原为SARS-CoV-2 S RBD蛋白；
(2)固相支持物；
(3)检测所述病毒抗原与所述样品中可能存在的抗体形成免疫复合物的工具。
在一些实施方案中，所述SARS-CoV-2病毒抗原包含与SEQ ID NO:3至少80％、至少85％、至少90％、至少95％、至少97％或至少99％一致性的氨基酸序列。在一个具体实施方案中，所述SARS-CoV-2病毒抗原如SEQ ID NO:3所示。
在一些实施方案中，所述SARS-CoV-2病毒或其抗原包被在固相支持物上。在一些实施方案中，所述固相支持物包括无机和有机多聚物，优选地，所述固相支持物为容器内壁，如孔板内壁。在一个具体实施方案中，所述固相支持物为96孔板内壁。在另一个具体实施方案中，所述包被可使用本领域周知的多种方法包被固相支持物。例如：利用双功能试剂活化，参见美国专利5399501。在一些实施方案中，合适浓度的所述SARS-CoV-2 S RBD抗原在25℃-37℃包被0.2-3小时，再洗板，封闭液封闭，拍干，得到抗原包被板。在一个优选实施方案中，浓度为1-10μg/ml所述SARS-CoV-2 S RBD重组抗原在37℃包被1小时，再洗板，封闭液封闭，拍干，得到抗原包被板，也即SARS-CoV-2抗体捕获板。
在一些实施方案中，检测所述免疫复合物的工具包括第二抗体。在一些实施方案中，所述第二抗体为抗IgG、抗IgM和/或抗IgA抗体。在一个具体实施方案中，所述第二抗体为抗人IgG抗体或抗IgM抗体。在一个优选实施方案中，所述第二抗体为抗人IgG抗体。
在一些实施方案中，所述第二抗体是生物素、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶标记的。根据标记第二抗体所使用的方法，选择适当的检测方案。在一个优选的实施方案中，标记第二抗体是辣根过氧化物酶标记的，使用底物反应液和终止液作用，显色后在波长为450nm酶标仪读取吸光值。将所得读数与Cutoff值加以比较，判断所测样本的结果。根据定性或定量检测方案的不同，所述检测样品中SARS- CoV-2病毒抗体的试剂盒选自ELISA检测试剂盒、化学发光检测试剂盒、胶体金检测试剂盒或时间分辨免疫层析检测试剂盒。
本发明另一方面提供了一种检测样品中SARS-CoV-2病毒抗体的ELISA试剂盒，所述试剂盒包括：
(1)包被SARS-CoV-2病毒抗原的孔板；
(2)辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶标记的抗人IgG或抗人IgM抗体；
(3)阴性对照及阳性对照；
(4)样本稀释液、洗涤液、底物反应液以及终止液。
在一些实施方案中，所述SARS-CoV-2病毒抗原为SARS-CoV-2 S RBD蛋白。在一些实施方案中，所述SARS-CoV-2病毒抗原包含与SEQ ID NO:3至少80％、至少85％、至少90％、至少95％、至少97％或至少99％一致性的氨基酸序列。在一个具体实施方案中，所述SARS-CoV-2病毒抗原如SEQ ID NO:3所示。在一些实施方案中，所述包被SARS-CoV-2病毒抗原的孔板为包被有SARS-CoV-2 S RBD重组蛋白的96孔酶标板。所述包被可使用本领域周知的多种方法包被固相支持物。例如：利用双功能试剂活化，参见美国专利5399501。在一些实施方案中，合适浓度的所述SARS-CoV-2 S RBD抗原在25℃-37℃包被0.2-3小时，再洗板，封闭液封闭，拍干，得到抗原包被板。在一个优选实施方案中，浓度为10μg/ml所述SARS-CoV-2 S RBD重组抗原在37℃包被1小时，再洗板，封闭液封闭，拍干，得到抗原包被板，也即SARS-CoV-2抗体捕获板。
在一些实施方案中，所述标记抗体为辣根过氧化物酶标记的抗人IgG或抗人IgM抗体。在一个优选实施方案中，所述标记抗体为辣根过氧化物酶标记的抗人IgG抗体。
在一些实施方案中，所述阳性对照包括特异性结合SARS-CoV-2病毒抗原的抗体。所述特异性结合SARS-CoV-2病毒抗原的抗体为抗SARS-CoV-2 S RBD抗体，优选地，所述抗体为CR3022抗体。在一个具体实施方案中，所述阳性对照为添加抗SARS-CoV-2 S RBD抗体的健康受试者样本。在一个优选实施方案中，所述阳性对照为添加抗SARS-CoV-2 S RBD抗体的健康人血清、全血、胸腹腔积液或脑脊液。在一个更优选的实施方案中，所述阳性对照为添加抗SARS-CoV-2 S RBD抗体的健康人血清。在一些实施例中，所述阴性对照可以是健康人血清，也可以包含BSA的PBS缓冲液。
在一些方案中，所述样本稀释液包括1％BSA和2％蔗糖。在一些方案中，所述洗涤液包括氯化钠，磷酸氢二钠，磷酸二氢钠，Tween 20，水并调整pH 7.4。在一些方案中那个，所述底物反应液包含TMB底物溶液。
本发明还提供了包含所述SARS-CoV-2病毒抗体的ELISA试剂盒在检测感染或疑似感染SARS-CoV-2病毒受试者中的应用。
本发明所述ELISA试剂盒检测操作步骤包括：
(1)向酶标板中加入优化稀释的待测样品、包含抗SARS-CoV-2 S RBD抗体的阳性对照和阴性对照孵育；
(2)加入洗涤液，洗板拍干；
(3)加入辣根过氧化物酶标记的抗人IgG抗体工作液孵育；
(4)加入洗涤液，洗板拍干；
(5)加入底物液显色孵育；
(6)加入终止液，酶标仪检测。
其中所述阴性对照可以是健康人血清，也可以包含BSA的PBS缓冲液。
在一些实施方案中，ELISA试剂盒中进一步包括检测操作步骤的说明书。
特异性结合SARS-CoV-2病毒抗原的抗体在检测试剂盒中的应用
本发明提供了一种特异性结合SARS-CoV-2病毒的抗体作为SARS-CoV-2病毒抗体检测试剂盒中阳性对照的应用。
在一些实施方案中，所述特异性结合SARS-CoV-2病毒的抗体为抗SARS-CoV-2 S RBD抗体。在一些实施方案中，所述SARS-CoV-2病毒抗体为CR3022抗体。
在一个具体实施方案中，所述阳性对照为添加抗SARS-CoV-2 S RBD抗体的阴性样本。在一个优选实施方案中，所述阳性对照为添加抗SARS-CoV-2 S RBD抗体的阴性样本。在一个更优选的实施方案中，所述阳性对照为添加抗SARS-CoV-2 S RBD抗体的阴性样本。在一个具体实施方案中，所述阳性对照为添加浓度为0.008-25μg/ml抗SARS-CoV-2 S RBD抗体的阴性样本。在一个优选实施方案中，所述阳性对照为添加浓度为0.2-5μg/ml抗SARS-CoV-2 S RBD抗体的阴性样本。所述阴性对照可以是健康人血清，也可以包含BSA的PBS缓冲液。
本发明检测SARS-CoV-2病毒抗体方法或检测SARS-CoV-2病毒抗体试剂盒中，所述样品是来源于感染或疑似感染SARS-CoV-2病毒的血浆、血清、全血、胸腹腔积液、脑脊液或组织标本。
本发明出乎意料地发现，CR3022抗体可以用于测定样品中SARS-CoV-2病毒抗原或其相关抗体的存在情况或者含量。
本发明提供了SARS-CoV-2病毒抗原ELISA检测方法。与SARS-CoV-2荧光PCR检测法相比，该方法对技术要求低，操作简单且不需特殊设备。与SARS-CoV-2病毒抗体检测法相比，SARS-CoV-2病毒抗原ELISA检测试剂法能够实现病毒的早期诊断。
本发明提供了SARS-CoV-2病毒抗体ELISA检测方法。该方法的创新之处是使用添加CR3022抗体的阴性对照作为SARS-CoV-2抗体检测的阳性对照。通常情况下用感染SARS-CoV-2病毒的阴性对照制备SARS-CoV-2病毒抗体检测试剂盒的阳性对照。但SARS-CoV-2感染者血清不易获得且存在潜在传染的风险。本发明使用的CR3022抗体制备阳性对照血清，具有能够大量生产、批次间稳定性可控、且不存在潜在传染风险等优点。
附图说明
图1.SARS-CoV-2 S RBD蛋白SDS-PAGE鉴定图
图2.SARS-CoV-2 S RBD蛋白Western Blot鉴定图
图3.CR3022抗体HPLC鉴定图
图4.间接ELISA测定CR3022抗体效价
图5.SARS-CoV-2抗体检测试剂盒检测阳性对照样本
图6.SARS-CoV-2抗原检测试剂盒RBD蛋白标准曲线
图7.SARS-CoV-2抗原检测试剂盒假病毒标准曲线
具体实施方式
根据同源性分析，SARS-CoV-2病毒的S基因编码的刺突蛋白S(spike,S)氨基酸序列全长共有1273个氨基酸组成，示于SEQ ID NO：1中。SARS-CoV-2病毒的S1亚基位于刺突蛋白S的N末端，由650个氨基酸组成，示于SEQ ID NO：2。SARS-CoV-2病毒的RBD结构域位于刺突蛋白S1内由325个氨基酸组成，示于SEQ ID NO：3。
CR3022抗体重链可变区的GenBank登录号为DQ168569，CR3022抗体轻链可变区的GenBank登录号为DQ168570。CR3022抗体重链可变区的序列，示于SEQ ID NO：4；CR3022抗体轻链可变区的序列，示于SEQ ID NO：5。CR3022抗体重链序列，示于SEQ ID NO：6；CR3022抗体轻链序列，示于SEQ ID NO：7。
实施例1 SARS-CoV-2病毒刺突蛋白RBD(SARS-CoV-2 S RBD)片段的表达和鉴定
通过SARS-CoV-2 S RBD蛋白片段的C端加His标签，用于镍柱亲和纯化，得到SARS-CoV-2 S RBD蛋白。
1.1SARS-CoV-2 S RBD表达质粒制备
1)使用金斯瑞密码子优化软件(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)对编码带有His标签SARS-CoV-2 S RBD蛋白片段(氨基酸序列如SEQ ID NO:3所示)的核苷酸序列进行密码子优化；
2)合成密码子优化后的SARS-CoV-2 S RBD DNA片段；
3)将合成后的SARS-CoV-2 S RBD DNA片段***pFastBac1表达载体中，构建表达质粒pFastBac1-S RBD；
4)对质粒***片段进行DNASanger测序，确认***序列正确后，抽提pFastBac1-S RBD质粒。
1.2 SARS-CoV-2 S RBD重组蛋白的制备
1)复苏SF9细胞(Thermo Fisher，11496015)，培养细胞至生长状态处于对数期；
2)将pFastBac1-S RBD质粒转染SF9细胞，37℃摇瓶中培养7天；
3)收集细胞培养物上清液，使用2×PBS上述缓冲液(16g氯化钠，400mg氯化钾，2.88磷酸氢二钠，480mg磷酸二氢钾，ddH2O 1000ml,pH 7.4)1:1稀释并过滤除菌；
4)纯化之前，将管道和镍亲和层析柱用0.2M NaOH去热原；
5)将过滤的上清液和镍亲和层析柱室温孵育1小时，并用1×上述缓冲液洗涤柱后，使用咪唑洗脱SARS-CoV-2 S RBD蛋白。
6)PBS透析浓缩后，使用BCA法对蛋白进行定量，将纯化的蛋白进行SDS-PAGE和Western Blot鉴定，纯化的蛋白放-70℃保存备用。
1.3 SARS-CoV-2 S RBD重组蛋白的SDS-PAGE鉴定
将SARS-CoV-2 S RBD重组蛋白分别加入等体积的上样缓冲液(1.5g Tris碱，4g SDS，20ml甘油，2mlβ-巯基乙醇，0.02g溴酚蓝，100ml ddH 2O，pH 6.8)，混匀后加热到100℃水浴5分钟，使用BioRad电泳***和10％预制胶(金斯瑞，M01012C)通过SDS-PAGE分离样品。溴酚蓝的色条带走至近底端1cm时，停止电泳，关闭电源。然后从电泳槽内小心取出凝胶板，用薄板将凝胶板外的两块板轻轻撬开，染色脱色。SDS-PAGE鉴定的结果如图1所示，说明成功制备了SARS-CoV-2 S RBD重组蛋白，蛋白纯度大于85％。
1.4 SARS-CoV-2 S RBD重组蛋白的Western blot鉴定
将SARS-CoV-2 S RBD重组蛋白分别加入等体积的上样缓冲液(1.5g Tris碱，4g SDS，20ml甘油，2mlβ-巯基乙醇，0.02g溴酚蓝，100ml ddH 2O，pH 6.8)，混匀后加热到100℃水浴5分钟。使用BioRad电泳***和10％预制胶(金斯瑞，M01012C)通过SDS-PAGE分离样品。溴酚蓝的色条带走至近底端1cm时，停止电泳，关闭电源。然后从电泳槽内小心取出凝胶板，用薄板将凝胶板外的两块板轻轻撬开。通过BioRad转膜***将凝胶上的蛋白转移到PDFV膜上，封闭后，用抗His单克隆抗体(金斯瑞，A00186)孵育PDFV膜1小时，PBST洗膜后，用IR dye 800标记的羊抗小鼠抗体(LI-COR,INC.，926-32210)孵育PDFV膜1小时；PBS洗膜后，用
Figure PCTCN2021078179-appb-000001
Infrared Imaging System仪器(LI-COR,INC.，Odyssey CLx)中进行WB荧光检测，如图2所示的结果，说明获得的蛋白是SARS-CoV-2 S RBD重组蛋白。
实施例2抗体CR3022的表达和鉴定
2.1 CR3022重链和轻链表达质粒制备
1)使用金斯瑞密码子优化软件(https://www.genscript.com/gensmart-free-gene-codon-optimization.html)对CR3022重链可变区(GenBank，DQ168569，SEQ ID NO:4)和轻链可变区(GenBank，DQ168570，SEQ ID NO:5)的核苷酸序列进行密码子优化。
2)分别合成密码子优化后的CR3022重链(包含信号肽+重链可变区+恒定区，抗体重链蛋白序列如SEQ ID NO:6)和CR3022轻链(包含信号肽+轻链可变区+恒定区，抗体轻链蛋白序列如SEQ ID NO:7)的DNA片段。
3)将合成后的CR3022重链和轻链DNA片段分别***pTT5表达载体中，构建表达质粒pTT5-CR3022HC和pTT5-CR3022LC。
3)对质粒进行DNAsanger测序，确认***片段序列正确后，大抽pTT5-CR3022HC质粒和pTT5-CR3022LC质粒。
2.2 CR3022抗体生产
1)复苏CHO-3E7细胞(从Canadian National Research Council获得)，培养细胞至生长状态处于对数期；
2)将pTT5-CR3022HC质粒和pTT5-CR3022LC质粒共转染CHO-3E7细胞，37℃摇瓶中培养7天；
3)收集细胞培养物上清液，使用2×上述缓冲液(16g氯化钠，400mg氯化钾，2.88磷酸氢二钠，480mg磷酸二氢钾，ddH 2O 1000ml,pH 7.4)1:1稀释并过滤除菌。
4)纯化之前，将管道和蛋白A柱用0.2M NaOH去热原，将柱用含有0.05M Tris和1.5M NaCl(pH8.0)的缓冲液重新平衡；
5)将过滤的上清液和蛋白A柱室温孵育2小时，并用1×上述缓冲液(8g氯化钠，200mg氯化钾，1.44磷酸氢二钠，240mg磷酸二氢钾，ddH 2O 1000ml，pH 7.4)洗涤柱后，使用无菌0.1M柠檬酸钠(pH3.5)洗脱IgG，收集了洗脱液并用九分之一体积的无菌1M Tris-HCl(pH9)中和；
6)浓缩之后，使用1.43的消光系数Ec(0.1％)通过OD280nm对抗体进行定量，将纯化的抗体进行HPLC鉴定，见图3的HPLC图谱，纯度为98.95％，纯化的抗体放-70℃保存备用。
2.2 CR3022抗体活性测定
通过间接ELISA测定CR3022抗体的活性：
1)包板：用包被缓冲液(8.5g氯化钠，1.4g磷酸氢二钠，0.2g磷酸二氢钠1000ml ddH 2O,pH 7.4)将包被抗原SARS-CoV-2 S RBD重组蛋白稀释至0.5μg/mL包板，100μl/孔，4℃过夜包被或37℃包被2小时；
2)洗板:PBST缓冲液洗涤一次，200μl/孔；
3)封闭：1％BSA/PBS封闭液，100μl/孔，37℃封闭1小时；
4)洗板：PBST缓冲液洗涤三次，200μl/孔；
5)加样：将CR3022抗体梯度稀释，50μl/孔，37℃反应1小时；
6)洗板：PBST缓冲液洗涤三次，200μl/孔；
7)二抗：加入1：5000稀释的辣根过氧化物酶标山羊抗人，50μl/孔，37℃反应0.5小时；
8)洗板：PBST缓冲液洗涤三次，200μl/孔；
9)显色：加入TMB底物反应液，100μl/孔，37℃反应15分钟；
10)终止：加入终止液，50μl/孔；
11)读数：OD450nm读数。
间接ELISA测定人单克隆抗体CR3022效价如图4所示说明制备的CR3022抗体能够识别SARS-CoV-2 S RBD重组蛋白且灵敏度高，可用于SARS-CoV-2检测试剂盒的开发。
实施例3 SARS-CoV-2病毒IgG抗体ELISA检测方法的建立
3.1 SARS-CoV-2病毒抗体ELISA检测试剂盒组成
1)SARS-CoV-2抗体捕获板：包被有SARS-CoV-2 S RBD重组蛋白的96孔酶标板；
2)阳性对照：一份添加有抗SARS-CoV-2重组人抗体(CR3022抗体)浓度为100μg/ml的阴性对照；
3)阴性对照：PBS缓冲液(8.5g氯化钠，1.4g磷酸氢二钠，0.2g磷酸二氢钠，0.5ml Tween 20，1000ml ddH2O，pH 7.4)，10g BSA；
4)HRP标记的抗人抗体工作液：用样本稀释液1：1000稀释的辣根过氧化物酶标记的抗人IgG抗体(金斯瑞，货号A01854)；
5)样本稀释液：1*PBST中包含1％BSA+2％蔗糖；
6)洗涤液：PBS缓冲液(8.5g氯化钠，1.4g磷酸氢二钠，0.2g磷酸二氢钠，0.5ml Tween20，1000ml ddH 2O，pH 7.4)；
7)底物反应液：Thermofisher货号34028；
8)终止液：8.3ml 12mol/L盐酸，91.7ml ddH 2O；
3.2 SARS-CoV-2抗体捕获板的制备
1)用碳酸盐缓冲液将SARS-CoV-2 S RBD重组蛋白稀释成2μg/ml的缓冲液；
2)向96孔酶标板板孔加100μlSARS-CoV-2 S RBD重组蛋白缓冲液，37℃孵育1小时；
3)每孔加入200μl洗涤液，洗板3次拍干；
4)每孔加100μl封闭液(1*PBST中包含1％脱脂奶粉+2％蔗糖)，37℃孵育1小时；
5)每孔加入200μl洗涤液，洗板3次拍干，拍干用铝膜真空封闭保存、备用。
3.3 ELISA操作步骤
1)用样品稀释液1：50稀释待检测样品、阳性对照及阴性对照；
2)向SARS-CoV-2抗体捕获板分别加入100μl的待检测样品、阳性对照及阴性对照，37℃孵育1小时；
3)每孔加入200μl洗涤液，洗板3次并拍干；
4)每孔加入100μlHRP标记的抗人IgG抗体工作液，37℃孵育10-30分钟；
5)每孔加入200μl洗涤液，洗涤3次并拍干；
6)每孔加入100μl底物液，避光显色15分钟；
7)每孔加入50μl终止液；
8)酶标仪OD450nm读数。
3.4 结果判断和Cut-off值的确定
计算阴性吸光度值的均值(NC OD450)，则Cut-off值CO＝NC OD450均值+0.12。样本吸光度值(S)，计算S/CO；当S/CO＜1时，样本为阴性；当S/CO≥1.0时，样本为阳性。
3.5 标准阳性对照的制备和测定
在阴性对照中添加入CR3022抗体,浓度分别为25μg/ml,5μg/ml,1μg/ml,0.2μg/ml,0.04μg/ml和0.008μg/ml按3.3ELISA操作步骤进行测定条件下进行ELISA反应，复孔检测。
从图5可以看出，含有0.2-5μg/ml的CR3022抗体的阴性样本为最优标准阳性对照。
实施例4 假病毒生产
SARS-CoV-2冠状病毒S蛋白ORF DNA序列进行基因合成后使用限制性DNA内切酶HindIII和XbaI酶切，同时利用相同限制性内切酶酶切质粒载体p3XFLAG-CMV14(Sigma，货号E4901)，酶切后获得的带有粘性末端的S蛋白ORF和质粒载体片段使用T4连接酶连接，转化大肠杆菌感受态细胞，获得质粒p3XFLAG-CMV14-2019nCoV-S(DOI：https://doi.org/10.1371/journal.pone.0076469)。
在10cm细胞培养皿中接种4x10 6HEK293FT细胞(Thermo Fisher Scientific,货号R70007)，接种后第二天吸去原有培养基，加入10ml新鲜DMEM完全培养基。2小时后，将5μg表达NCP冠状病毒S蛋白的质粒p3XFLAG-CMV14-2019nCoV-S与20μg HIV-Luc质粒混合，加入500μl OptiMEM无血清培养基中，同时将50μl PEI分别加入500μl OptiMEM无血清培养基中，室温静置5分钟。将含有PEI的OptiMEM无血清培养基与500μl含有质粒的OptiMEM无血清培养基混合，室温静置8分钟，将1ml混合物加入HEK293FT细胞中。转染后24小时用10ml新鲜DMEM完全培养基替换含有转染混合物的培养基。转染后48小时收获含有假病毒的培养上清，用0.45μm孔径的滤器过滤后-80℃冻存。
实施例5 SARS-CoV-2病毒抗原夹心ELISA方法的建立
5.1 病毒抗原夹心ELISA检测试剂盒组成
1)SARS-CoV-2病毒抗原捕获板：包被抗SARS-CoV抗体(CR3022抗体)的96孔酶标板；
2)阴性对照：PBS缓冲液(8.5g氯化钠，1.4g磷酸氢二钠，0.2g磷酸二氢钠，0.5ml Tween 20，，1000ml ddH2O，pH 7.4)，10g BSA；
3)阳性对照：添加SARS-CoV-2 S RBD蛋白或SARS-CoV-2 S假病毒的阴性对照；
4)生物素标记的抗SARS-CoV-2 S RBD抗体：如CR3022抗体；
5)Streptavidin-HRP工作液：用样本稀释液1：50 000Streptavidin-HRP(金斯瑞，货号M00091)；
6)样本稀释液：PBS缓冲液(8.5g氯化钠，1.4g磷酸氢二钠，0.2g磷酸二氢钠，0.5ml Tween 20，1000ml ddH 2O，pH 7.4)，10g BSA；
7)洗涤液：PBS缓冲液(8.5g氯化钠，1.4g磷酸氢二钠，0.2g磷酸二氢钠，0.5ml Tween 20，1000ml ddH 2O，pH 7.4)；
8)底物反应液：Thermofisher货号34028；
9)终止液：8.3ml 12mol/L盐酸，91.7ml ddH 2O。
5.2 SARS-CoV-2病毒抗原捕获板制备
1)用碳酸盐包被缓冲液(8.5g氯化钠，1.4g磷酸氢二钠，0.2g磷酸二氢钠1000ml ddH 2O，pH 7.4)配制2μg/ml SARS-CoV-2 S RBD抗体溶液(如CR3022抗体溶液)；
2)每孔中加入100μlSARS-CoV-2 S RBD抗体，4℃包被18小时；
3)弃去孔内液体，PBS洗板一次；
4)每孔加100ul的封闭液(1*PBST中包含1％脱脂奶粉+2％蔗糖)，37℃孵育1小时；
5)每孔加入200ul的洗液，洗板3次拍干，拍干用铝膜真空封闭保存、备用。
5.3 试剂盒检测操作步骤
1)每孔加100ulSARS-CoV-2 S RBD重组蛋白标准品(250.00ng/ml，125.00ng/ml，62.50ng/ml，31.25ng/ml，15.63ng/ml，7.81ng/ml，3.91ng/ml，1.95ng/ml，0.00)、阴性对照和待测样品，37℃孵育1小时；或者每孔加100μl SARS-CoV-2假病毒标准品(浓度梯度为125ng/ml，62.5ng/ml，31.25ng/ml,15.625ng/ml,7.813ng/ml，3.906ng/ml，1.953ng/ml，0ng/ml)、阴性对照和待测样品，37℃孵育1小时。；
2)每孔加入200ul洗涤液，洗板3次拍干；
3)每孔中加入100ul生物素标记的SARS-CoV-2 SRBD抗体(南京金斯瑞，货号A02053，浓度为1ug/ml)溶液，37℃孵育1小时；
4)每孔加入200ul洗涤液，洗板3次拍干；
5)每孔加入100ul的Streptavidin-HRP工作液，37℃孵育10分钟；
6)每孔加入200ul的洗涤液，洗板3次拍干；
7)每孔加入100ul底物显色液，37℃孵育10分钟；
8)每孔加入50ul终止液反应；
9)用酶标仪测定OD450nm吸光值。
SARS-CoV-2抗原检测试剂盒RBD蛋白标准曲线见图6。SARS-CoV-2抗原检测试剂盒假病毒标准曲线见图7。SARS-CoV-2抗原检测试剂盒在检测病毒抗原和病毒上具有很好的线性关系，在SARS-CoV-2病毒检测具有潜在的应用价值。
序列信息：
SEQ ID NO：1 SARS-CoV-2 S蛋白
Figure PCTCN2021078179-appb-000002
SEQ ID NO：2 SARS-CoV-2 S1蛋白
Figure PCTCN2021078179-appb-000003
SEQ ID NO：3 SARS-CoV-2 S RBD蛋白
Figure PCTCN2021078179-appb-000004
Figure PCTCN2021078179-appb-000005
SEQ ID NO：4 CR3022抗体重链可变区
Figure PCTCN2021078179-appb-000006
SEQ ID NO：5 CR3022抗体人单抗轻链可变区
Figure PCTCN2021078179-appb-000007
SEQ ID NO：6 CR3022抗体重链
Figure PCTCN2021078179-appb-000008
SEQ ID NO：7 CR3022抗体轻链
Figure PCTCN2021078179-appb-000009

Claims (43)

  1. 一种检测样品中是否存在SARS-CoV-2病毒或其抗原的方法，所述方法包括：
    (1)将特异性结合SARS-CoV-2病毒抗原的抗体与样品接触，在合适条件下，所述抗体与样品中所述病毒或其抗原形成免疫复合物；
    (2)检测所述免疫复合物的存在。
  2. 根据权利要求1所述方法，所述抗体为抗SARS-CoV-2 S蛋白抗体，优选地，所述抗体为抗SARS-CoV-2 S RBD蛋白抗体。
  3. 根据权利要求1或2所述方法，所述抗体的重链可变区包含与SEQ ID NO:4所示序列至少80％一致性的氨基酸序列，所述抗体的轻链可变区包含与SEQ ID NO:5所示序列至少80％一致性的氨基酸序列。
  4. 根据权利要求1-3中任一项所述方法，所述抗体的重链序列包含与SEQ ID NO:6所示序列至少80％一致性氨基酸序列，所述抗体的轻链序列包含与SEQ ID NO:7所示序列至少80％一致性的氨基酸序列。
  5. 根据权利要求1-4中任一项所述方法，所述接触步骤包括先将所述抗体包被在固相支持物上，再将样品加入到包被抗体的固相支持物上孵育。
  6. 根据权利要求1-5中任一项所述方法，所述合适条件是指所述抗体与所述样品在25-37℃孵育15分钟到1.5小时。
  7. 根据权利要求1-6中任一项所述方法，所述检测免疫复合物步骤包括加入第二抗体，所述第二抗体为抗SARS-CoV-2病毒抗原的抗体。
  8. 根据权利要求7所述方法，所述第二抗体是生物素、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶标记的。
  9. 一种检测样品中SARS-CoV-2病毒或其抗原的试剂盒，所述试剂盒包括：
    (1)特异性结合SARS-CoV-2病毒抗原的抗体；
    (2)固相支持物；
    (3)检测所述样品中可能存在的病毒抗原与所述抗体形成免疫复合物的工具。
  10. 根据权利要求9所述的试剂盒，所述抗体为抗SARS-CoV-2 S蛋白抗体，优选地，所述抗体为抗SARS-CoV-2 S RBD蛋白抗体。
  11. 根据权利要求9或10所述的试剂盒，所述抗体的重链可变区包含与SEQ ID NO:4所示序列至少80％一致性氨基酸序列，和所述抗体的轻链可变区包含与SEQ ID NO:5所示序列至少80％一致性的氨基酸序列。
  12. 根据权利要求9-11中任一项所述试剂盒，所述抗体包被在固相支持物上。
  13. 根据权利要求9-12中任一项所述试剂盒，所述固相支持物包括无机和有机多聚物，优选地，所述固相支持物为孔板内壁。
  14. 根据权利要求9-13中任一项所述试剂盒，其中所述检测免疫复合物的工具包括第二抗体。
  15. 根据权利要求14所述的试剂盒，所述第二抗体为抗SARS-CoV-2病毒抗原的抗体。
  16. 根据权利要求14-15中任一项所述的试剂盒，所述第二抗体是生物素、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶标记的。
  17. 根据权利要求9-16中任一项所述的试剂盒，选自ELISA检测试剂盒、化学发光检测试剂盒、胶体金检测试剂盒或时间分辨免疫层析检测试剂盒，优选为ELISA检测试剂盒。
  18. 一种检测样品中SARS-CoV-2病毒或其抗原的ELISA试剂盒，所述试剂盒包括：
    (1)第一抗体包被的固相支持物，所述第一抗体为特异性结合SARS-CoV-2病毒抗原的抗体；
    (2)生物素或辣根过氧化物酶标记的第二抗体，所述第二抗体为特异性结合SARS-CoV-2病毒抗原的抗体；
    (3)阴性对照和阳性对照；
    (4)样本稀释液、洗涤液、可选择的辣根过氧化物酶标记链霉亲和素、底物反应液以及终止反应液。
  19. 根据权利要求18所述的ELISA试剂盒，所述第一抗体和第二抗体分别选自如下抗体序列：所述第一/第二抗体的重链可变区包含SEQ ID NO:4所示的氨基酸序列，所述第一/第二抗体的轻链可变区包含SEQ ID NO:5所示的氨基酸序列。
  20. 根据权利要求18或19所述的ELISA试剂盒，所述阳性对照包括SARS-CoV-2 S RBD蛋白或SARS-CoV-2 S假病毒。
  21. 根据权利要求1-8中任一项所述方法或权利要求9-20中任一项所述试剂盒，其中所述样品是来源于感染或疑似感染SARS-CoV-2病毒的血浆、血清、全血、痰液、口腔/鼻咽分泌物或洗液、尿液、粪便、胸腹腔积液、脑脊液、组织标本或非生物学样品。
  22. 一种检测样品中是否存在抗SARS-CoV-2病毒的抗体的方法，所述方法包括：
    (1)将SARS-CoV-2病毒抗原与样品接触，在合适条件下，所述抗原与所述样品中SARS-CoV-2病毒抗体形成免疫复合物；
    (2)检测所述免疫复合物的存在。
  23. 根据权利要求22所述方法，所述SARS-CoV-2病毒抗原为SARS-CoV-2 S RBD蛋白。
  24. 根据权利要求22或23所述方法，所述SARS-CoV-2病毒抗原包含与SEQ ID NO:2或3所示序列至少80％一致性的氨基酸序列。
  25. 根据权利要求22-24中任一项所述方法，所述接触步骤包括先将所述病毒抗原包被在固相支持物上，再将样品加入到包被抗原的固相支持物上孵育。
  26. 根据权利要求22-25中任一项所述方法，所述合适条件为所述病毒抗原与所述样品在25-37℃孵育15分钟到1.5小时。
  27. 根据权利要求22-26中任一项所述方法，所述检测步骤包括加入第二抗体，所述第二抗体为抗人抗体。
  28. 根据权利要求22-26中任一项所述的方法，所述第二抗体是生物素、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶标记的。
  29. 一种检测样品中抗SARS-CoV-2病毒的抗体的试剂盒，所述试剂盒包括：
    (1)SARS-CoV-2病毒抗原，所述抗原为SARS-CoV-2 S RBD蛋白；
    (2)固相支持物；
    (3)检测所述病毒抗原与所述样品中可能存在的抗体形成免疫复合物的工具。
  30. 根据权利要求29所述的试剂盒，所述SARS-CoV-2病毒抗原包含与SEQ ID NO:2或3所示序列至少80％一致性的氨基酸序列。
  31. 根据权利要求29或30所述的试剂盒，所述病毒抗原包被在固相支持物上。
  32. 根据权利要求29-31中任一项所述的试剂盒，所述固相支持物包括无机和有机多聚物，优选地，所述固相支持物为孔板内壁。
  33. 根据权利要求29-32中任一项所述的试剂盒，所述检测免疫复合物的工具包括一种检测抗体，所述抗体是生物素、放射性同位素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶标记的抗人抗体。
  34. 根据权利要求33所述的试剂盒，所述检测抗体为辣根过氧化物酶标记的抗IgG、IgM和/或IgA抗体。
  35. 根据权利要求29-34任一项所述的试剂盒，选自ELISA检测试剂盒、化学发光检测试剂盒、胶体金检测试剂盒或时间分辨免疫层析检测试剂盒，优选为ELISA检测试剂盒。
  36. 一种检测样品中抗SARS-CoV-2病毒的抗体的ELISA试剂盒，所述试剂盒包括：
    (1)包被SARS-CoV-2病毒抗原的孔板；
    (2)辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶标记的抗人抗体；
    (3)阴性对照及阳性对照；
    (4)样本稀释液、洗涤液、底物反应液以及终止液。
  37. 根据权利要求36所述的试剂盒，所述SARS-CoV-2病毒抗原为SARS-CoV-2 S RBD蛋白。
  38. 根据权利要求36或37所述的试剂盒，所述阳性对照包括特异性结合SARS-CoV-2病毒抗原的抗体。
  39. 根据权利要求38所述的试剂盒，所述阳性对照包含的抗体的重链可变区包含SEQ ID NO:4所示序列，和所述抗体的轻链可变区包含SEQ ID NO:5所示序列。
  40. 一种特异性结合SARS-CoV-2病毒的抗体在检测试剂盒中的应用。
  41. 根据权利要求40所述的应用，所述抗体的重链序列如SEQ ID NO:6所示，和所述抗体的轻链序列如SEQ ID NO:7所示。
  42. 根据权利要求40或41所述的应用，将所述抗体添加至阴性对照作为阳性对照。
  43. 根据权利要求22-28中任一项所述方法或权利要求29-39中任一项所述试剂盒，其中所述样品是来源于感染或疑似感染SARS-CoV-2病毒的血浆、血清、全血、胸腹腔积液、脑脊液或组织标本。
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