WO2023078447A1 - 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒 - Google Patents
抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒 Download PDFInfo
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Images
Classifications
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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
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/567—Framework region [FR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- 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
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- G—PHYSICS
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- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present disclosure relates to the technical field of antibodies, in particular, to an anti-new coronavirus antibody, a reagent and a kit for detecting the new coronavirus.
- the structural proteins of the new coronavirus 2019-nCoV are divided into: spike glycoprotein (S protein), envelope glycoprotein (E protein), membrane glycoprotein (M protein) and nucleocapsid protein (N protein), these proteins include multiple antigenic epitopes.
- S protein spike glycoprotein
- E protein envelope glycoprotein
- M protein membrane glycoprotein
- N protein nucleocapsid protein
- the N protein and the viral genome RNA are intertwined to form the viral nucleocapsid, which plays an important role in the synthesis of viral RNA.
- the N protein is relatively conservative and accounts for the largest proportion of the structural proteins of the virus.
- the body can produce high-level antibodies against the N protein in the early stage of infection.
- the N protein is an important marker protein of the new coronavirus. Using the principle of specific combination of antigen and antibody, the presence of the antigen can be detected by the N protein monoclonal antibody, so as to directly prove that the sample contains the new coronavirus and realize the detection of the
- Antibodies detected are mainly divided into two classes: IgM and IgG.
- IgM antibody is produced early, and once infected, it is produced quickly, lasts for a short time, and disappears quickly.
- a positive test in the blood can reflect that the body is in an acute infection state, and can be used as an indicator of early infection.
- antibody detection samples are serum or plasma, which is less affected by sample sampling, which is conducive to early diagnosis and exclusion of suspicious cases. At the same time, the detection is fast, convenient, and suitable for large-scale screening.
- 2019-nCoV Since the outbreak of the novel coronavirus 2019-nCoV pneumonia, it has spread globally, seriously threatening human life and health. Respiratory droplets and close contact transmission are the main routes of transmission of novel coronavirus pneumonia, and there is a possibility of aerosol transmission in a relatively closed environment when exposed to high concentrations of aerosols for a long time. 2019-nCoV is highly contagious, and most patients develop clinical symptoms after infection, but some patients are asymptomatic. Common signs after a person is infected with a coronavirus include respiratory symptoms, fever, cough, shortness of breath, and dyspnea. In more severe cases, infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. Although there is currently no specific treatment for the disease caused by the new coronavirus, the treatment of mild or asymptomatic patients can greatly improve the cure rate and shorten the treatment time. Therefore, the detection or identification of patients becomes particularly important.
- nucleic acid detection and virus gene sequencing are mainly used as evidence for etiological diagnosis. Due to the influence of various factors such as sampling, operation, and reagents, nucleic acid detection may produce false negative results.
- the positive detection rate of viral nucleic acid in patients with highly suspected 2019 novel coronavirus (2019-nCoV) infection is only 30% to 50%.
- nucleic acid detection requires high equipment, testing sites and environmental conditions, and has disadvantages such as long detection time and low throughput, which is not convenient for large-scale detection of people under the current epidemic situation. Therefore, there is an urgent need to develop a rapid and convenient detection kit for clinical detection, so as to isolate infected people as soon as possible to block the spread of the virus. Therefore, antibody detection kits become more important.
- the present disclosure provides an antibody or a functional fragment thereof against a novel coronavirus or its N protein, the antibody or a functional fragment thereof includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1-HCDR3 include or It is an amino acid sequence consistent with HCDR1-HCDR3 of any one of the heavy chain variable regions shown in SEQ ID NO:15, 24, 25, and 26; The amino acid sequence consistent with LCDR1-LCDR3 of the light chain variable region is shown.
- the CDRs are defined by the Kabat, Chothia, IMGT, Lesk, AbM or Contact numbering system.
- the CDRs are defined by the Kabat, Chothia, IMGT or Lesk numbering system.
- the antibody or its functional fragment has the following complementarity determining regions:
- HCDR1 which comprises the amino acid sequence shown in SEQ ID NO.1, or consists of it;
- HCDR2 which comprises the amino acid sequence shown in SEQ ID NO.2 or 21, or consists of it;
- HCDR3 which comprises the amino acid sequence shown in SEQ ID NO.3, or consists of it;
- LCDR1 which comprises the amino acid sequence shown in SEQ ID NO.4, or consists of it;
- LCDR2 which comprises the amino acid sequence shown in SEQ ID NO.5 or 22, or consists of it;
- LCDR3 which comprises the amino acid sequence shown in SEQ ID NO.6, or consists of it.
- the antibody or its functional fragments also have the following framework regions:
- amino acid sequence of HFR1 is as shown in any of SEQ ID NO: 7, 23, or has at least 80% homology therewith;
- amino acid sequence of HFR2 is as shown in SEQ ID NO: 8 or has at least 80% homology therewith;
- amino acid sequence of HFR3 is as shown in SEQ ID NO:9 or has at least 80% homology therewith;
- the HFR4 amino acid sequence is as shown in SEQ ID NO: 10 or has at least 80% homology therewith;
- amino acid sequence of LFR1 is as shown in SEQ ID NO: 11 or has at least 80% homology therewith;
- amino acid sequence of LFR2 is as shown in SEQ ID NO: 12 or has at least 80% homology therewith;
- amino acid sequence of LFR3 is as shown in SEQ ID NO: 13 or has at least 80% homology therewith;
- the LFR4 amino acid sequence is as set forth in SEQ ID NO: 14 or has at least 80% homology thereto.
- the antibody or its functional fragment binds the novel coronavirus or its N protein with an affinity of K D ⁇ 10 -9 M.
- the antibody or its functional fragment binds the novel coronavirus or its N protein with an affinity of K D ⁇ 3.11*10 -10 M.
- the present disclosure provides an antibody or a functional fragment thereof against a novel coronavirus or its N protein, the antibody or a functional fragment thereof comprising a heavy chain variable region and/or a light chain variable region, and the heavy chain may be
- the variable region contains the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4
- the light chain variable region contains the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4
- amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO: 15, 24, 25, or 26;
- amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO: 16, 27.
- the antibody or functional fragment thereof further comprises a constant region.
- the constant region comprises a heavy chain constant region and/or a light chain constant region.
- the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the ⁇ -type or ⁇ -type light chain constant region .
- the species source of the constant region is bovine, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human .
- the species source of the constant region is mouse.
- the heavy chain constant region sequence is as shown in any of SEQ ID NO: 17, 32 or has at least 80% homology therewith, and the light chain constant region sequence is as shown in SEQ ID NO: 18 or with it have at least 80% homology.
- the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.
- the present disclosure also provides an antibody against a new type of coronavirus or its N protein, including a heavy chain and/or a light chain, the heavy chain including the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; the light chain
- the chain comprises the light chain variable region described above and the light chain constant region described above.
- amino acid sequence of the heavy chain is shown in any one of SEQ ID NO: 19, 28, 29, 30, 33; the amino acid sequence of the light chain is shown in any one of SEQ ID NO: 20, 31.
- the present disclosure also provides an antibody conjugate, which includes the above-mentioned antibody or a functional fragment thereof.
- the antibody or functional fragment thereof is labeled with a detectable label.
- the detectable label is selected from fluorescent dyes, enzymes that catalyze color development of substrates, radioactive isotopes, chemiluminescent reagents and nanoparticle-based labels.
- the fluorescent dye is selected from the group consisting of fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy series dyes and derivatives thereof, Alexa series dyes and derivatives thereof, protein dyes and derivatives thereof thing.
- the enzyme that catalyzes the color development of the substrate is selected from horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.
- the radioactive isotope is selected from 212 Bi, 131 I, 111 In, 90 Y, 186 Re, 211 At, 125 I, 188 Re, 153 Sm, 213 Bi, 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho, 105 Rh, 177Lu , 172Lu and 18F .
- the chemiluminescence reagent is selected from the group consisting of luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium esters and its derivatives, dioxane Ethane and its derivatives, lophine and its derivatives, and peroxalate and its derivatives.
- the nanoparticle marker is selected from nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.
- the colloid is selected from colloidal metals, disperse dyes, dye-labeled microspheres and latex.
- the colloidal metal is selected from colloidal gold, colloidal silver and colloidal selenium.
- the antibody or functional fragment thereof is coated onto a solid phase.
- the solid phase is selected from microspheres, plates and membranes.
- the solid phase is selected from magnetic microspheres, plastic microspheres, plastic particles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.
- the solid phase is selected from magnetic microspheres.
- the present disclosure also provides a reagent or kit, which includes the above-mentioned antibody or its functional fragment or the above-mentioned antibody conjugate.
- the present disclosure also provides a nucleic acid encoding the antibody or a functional fragment thereof of any one of the above.
- the present disclosure also provides a vector comprising a nucleic acid fragment encoding any of the above-mentioned antibodies or functional fragments thereof.
- the present disclosure also provides a recombinant cell containing the vector.
- the present disclosure also provides a method for preparing the antibody or its functional fragment described in any one of the above, which comprises: culturing the recombinant cells, and separating and purifying the antibody or its functional fragment from the culture product.
- the present disclosure also provides the use of the antibody or its functional fragment described in any one of the above, or the antibody, or the antibody conjugate, in the preparation of a reagent or kit, and the reagent or kit uses It is used to detect novel coronavirus or its N protein, or to diagnose novel coronavirus infection or diseases related to novel coronavirus infection.
- the present disclosure also provides the antibody or its functional fragment described in any one of the above, or the antibody, or the antibody conjugate, or the reagent or kit, for detecting novel coronavirus or its N Protein, or the use in diagnosing novel coronavirus infection or diseases related to novel coronavirus infection.
- the diseases associated with novel coronavirus infection include respiratory symptoms, fever, cough, shortness of breath, hypoxemia, dyspnea, pneumonia, acute respiratory distress syndrome, severe acute respiratory syndrome, renal failure or Septic shock.
- the present disclosure also provides a method for diagnosing a subject who is infected with a new coronavirus or a disease related to a new coronavirus infection, including:
- the diseases associated with novel coronavirus infection include respiratory symptoms, fever, cough, shortness of breath, hypoxemia, dyspnea, pneumonia, acute respiratory distress syndrome, severe acute respiratory syndrome, renal failure or Septic shock.
- the present disclosure also provides a method for detecting novel coronavirus or its N protein in a test sample, comprising:
- the novel coronavirus or its N protein antigen in the test sample is combined with the antibody or its functional fragment described in any one of the above, or the antibody, or said antibody conjugate, or said reagent or kit contacted to form an immune complex;
- the immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment.
- the immune complex further includes a second antibody that binds to the novel coronavirus or its N protein antigen.
- Fig. 1 is the result of reducing SDS-PAGE of the anti-new coronavirus antibody of embodiment 1.
- Some embodiments of the present disclosure provide an anti-new coronavirus or its N protein antibody or its functional fragment, the antibody or its functional fragment includes HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein, HCDR1, HCDR2 , HCDR3 include amino acid sequences consistent with HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in any one of SEQ ID NO:15, 24, 25, and 26; An amino acid sequence consistent with LCDR1, LCDR2, LCDR3 of the light chain variable region indicated.
- HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2, and HCDR3 of any one of the heavy chain variable regions shown in SEQ ID NO: 15, 24, 25, and 26; LCDR1, LCDR2, LCDR3 is an amino acid sequence consistent with LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in any one of SEQ ID NO: 16, 27.
- CDRs are defined by the Kabat, Chothia, IMGT or Lesk numbering system.
- CDRs are defined by the AbM or Contact numbering system.
- Some embodiments of the present disclosure provide an antibody or functional fragment thereof against novel coronavirus or its N protein, the antibody or functional fragment thereof includes the following complementarity determining regions:
- HCDR1 which comprises the amino acid sequence shown in SEQ ID NO.1, or consists of it;
- HCDR2 which comprises the amino acid sequence shown in SEQ ID NO.2 or 21, or consists of it;
- HCDR3 which comprises the amino acid sequence shown in SEQ ID NO.3, or consists of it;
- LCDR1 which comprises the amino acid sequence shown in SEQ ID NO.4, or consists of it;
- LCDR2 which comprises the amino acid sequence shown in SEQ ID NO.5 or 22, or consists of it;
- LCDR3 which comprises the amino acid sequence shown in SEQ ID NO.6, or consists of it.
- antibody is used in the broadest sense, which may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.
- CDRs complementarity determining regions
- CDRs complementarity determining regions
- CDRs refer to the hypervariable regions of the heavy and light chains of immunoglobulins, which contain one or more, or even all, of the antibodies or The region of major amino acid residues responsible for the binding affinity of an antigen-binding fragment to the antigen or epitope it recognizes.
- CDRs refer to the hypervariable regions of the heavy and light chains of antibodies.
- the heavy chain complementarity determining region is denoted by "HCDR", which includes HCDR1, HCDR2 and HCDR3;
- the light chain complementarity determining region is denoted by LCDR, which includes LCDR1, LCDR2 and LCDR3.
- CDR labeling methods in the field include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions.
- a "framework region” or "FR” region includes a heavy chain framework region and a light chain framework region and refers to an antibody heavy chain variable region (which may be denoted as VH) and a light chain variable region (which may be denoted as VL) Regions other than the CDRs in ; where the heavy chain framework region is denoted by "HFR” and can be further subdivided into contiguous regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework The regions are indicated by "LFR” and can be further subdivided into contiguous regions separated by CDRs, comprising the LFR1, LFR2, LFR3 and LFR4 framework regions.
- the heavy chain variable region is obtained by joining the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is composed of the following numbered CDRs and FRs The following combinations and permutations are obtained: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
- the terms "subject” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.
- the antibody or its functional fragment also has the following framework regions:
- amino acid sequence of HFR1 is as shown in any of SEQ ID NO: 7, 23, or has at least 80% homology therewith;
- amino acid sequence of HFR2 is as shown in SEQ ID NO: 8 or has at least 80% homology therewith;
- amino acid sequence of HFR3 is as shown in SEQ ID NO: 9 or has at least 80% homology therewith;
- the HFR4 amino acid sequence is as shown in SEQ ID NO: 10 or has at least 80% homology thereto;
- amino acid sequence of LFR1 is as shown in SEQ ID NO: 11 or has at least 80% homology therewith;
- amino acid sequence of LFR2 is as shown in SEQ ID NO: 12 or has at least 80% homology therewith;
- amino acid sequence of LFR3 is as shown in SEQ ID NO: 13 or has at least 80% homology therewith;
- the LFR4 amino acid sequence is as set forth in SEQ ID NO: 14 or has at least 80% homology thereto.
- the amino acid sequence of each framework region of the antibody or its functional fragment provided by the present disclosure may be identical to the above-mentioned corresponding framework region (SEQ ID NO: 7, 8, 9, 10, 11, 12 , 13, or 14) have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98% or 99% homology.
- the amino acid sequence of HFR1 can also be SEQ ID NO:23.
- the antibody or its functional fragment binds the novel coronavirus or its N protein with an affinity of K D ⁇ 10 ⁇ 9 M.
- the antibody or its functional fragment binds the novel coronavirus or its N protein with an affinity of K D ⁇ 3.11*10 -10 M;
- Some embodiments of the present disclosure also provide an antibody or functional fragment thereof against novel coronavirus or its N protein, the antibody or functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain
- the variable region contains the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, the light chain variable region contains the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, HCDR1, HCDR2, HCDR3,
- amino acid sequence of the heavy chain variable region is as shown in any of SEQ ID NO: 15, 24, 25, or 26.
- the light chain variable region amino acid sequence is as shown in any of SEQ ID NO: 16, 27.
- the antibody further comprises a constant region.
- the constant region comprises a heavy chain constant region and/or a light chain constant region.
- a heavy chain constant region may be denoted CH; a “light chain constant region” may be denoted CL.
- the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and the light chain constant region is selected from the ⁇ -type or ⁇ -type light chain constant region .
- the species source of the constant region is cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck , Goose, Turkey, Gamecock Or Man.
- the species source of the constant region is mouse.
- the heavy chain constant region sequence (CH) is shown in SEQ ID NO: 17
- the light chain constant region (CL) sequence is shown in SEQ ID NO: 18.
- the constant region sequence can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86% of the above constant region (SEQ ID NO: 17 or 18) %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.
- the heavy chain constant region can also be SEQ ID NO:32.
- the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.
- Functional fragments of the above-mentioned antibodies generally have the same binding specificity as the antibody from which they were derived.
- the functional fragments of the above-mentioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or methods of splitting disulfide bonds by chemical reduction.
- enzymatic digestion including pepsin or papain
- splitting disulfide bonds by chemical reduction.
- those skilled in the art can easily obtain the above-mentioned functional fragments.
- Some embodiments of the present disclosure also provide an antibody against novel coronavirus or its N protein, including a heavy chain and/or a light chain, the heavy chain including the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; the light chain Including the above-mentioned light chain variable region and the above-mentioned light chain constant region.
- amino acid sequence of the heavy chain is as shown in any of SEQ ID NO: 19, 28, 29, 30, 33; the amino acid sequence of the light chain is as shown in any of SEQ ID NO: 20, 31.
- amino acid sequence of the heavy chain is shown in any one of SEQ ID NO: 19, 28, 29, 30, 33; the amino acid sequence of the light chain is shown in SEQ ID NO: 20.
- amino acid sequence of the heavy chain is shown in any one of SEQ ID NO: 19, 28, 29, 30, 33; the amino acid sequence of the light chain is shown in SEQ ID NO: 31.
- Some embodiments of the present disclosure also provide an antibody conjugate, which includes the above-mentioned antibody or a functional fragment thereof.
- the antibody or its functional fragment in the above antibody conjugate is labeled with a detectable label.
- Detectable markers refer to a class of substances that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc., through which the corresponding target can be qualitatively or quantitatively detection.
- detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze color development of substrates, radioactive isotopes, chemiluminescent reagents, and nanoparticle-based labels.
- the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET ) etc. or their analogues), rhodamine dyes and their derivatives (for example including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC) etc. or their analogues) , Cy series dyes and derivatives thereof (such as including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc.
- fluorescein dyes and their derivatives for example including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET ) etc. or their analogues
- Alexa series dyes and derivatives thereof such as including but not limited to limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs
- protein dyes and their derivatives such as including but not limited to Phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinoxanthin-chlorophyll protein (preCP), etc.
- enzymes that catalyze color development of substrates include, but are not limited to, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphate glucose deoxygenase.
- radioactive isotopes include but are not limited to 212 Bi, 131 I, 111 In, 90 Y, 186 Re, 211 At, 125 I, 188 Re, 153 Sm, 213 Bi, 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn, 97 Ru, 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y, 88 Y, 121 Sn, 161 Tb, 166 Ho , 105 Rh, 177 Lu, 172 Lu and 18 F.
- chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium esters and its derivatives , dioxetane and its derivatives, lophine and its derivatives, and peroxyoxalate and its derivatives.
- nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
- colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
- colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
- the antibody or a functional fragment thereof in the above antibody conjugate is coated on a solid phase.
- the solid phase is selected from microspheres, plates and membranes.
- solid phases include, but are not limited to, magnetic microspheres, plastic microspheres, microplastics, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.
- the solid phase is magnetic microspheres.
- Some embodiments of the present disclosure also provide a reagent or a kit, which includes the above-mentioned antibody or a functional fragment thereof or the above-mentioned antibody conjugate.
- Some embodiments of the present disclosure also provide the use of the above reagent or kit in the detection of novel coronavirus or its N protein.
- Some embodiments of the present disclosure also provide a nucleic acid molecule encoding the above antibody or a functional fragment thereof.
- Some embodiments of the present disclosure also provide vectors comprising the nucleic acid molecules described above.
- Some embodiments of the present disclosure also provide recombinant cells containing the above vectors.
- Some embodiments of the present disclosure also provide a method for preparing an antibody or a functional fragment thereof, which includes: culturing the above-mentioned recombinant cells, and separating and purifying the above-mentioned antibody or a functional fragment thereof from the culture product.
- Some embodiments of the present disclosure also provide the use of the above-mentioned antibody or its functional fragment, or the above-mentioned antibody, or the above-mentioned antibody conjugate, in the preparation of a reagent or kit for detecting novel coronavirus or its N protein, and/or diagnose novel coronavirus infection or diseases related to novel coronavirus infection.
- Some embodiments of the present disclosure also provide the above-mentioned antibodies or functional fragments thereof, or the above-mentioned antibodies, or the above-mentioned antibody conjugates, or the above-mentioned reagents or kits, which are used to detect the new coronavirus or its N protein, and/or diagnose Use in novel coronavirus infection or diseases related to novel coronavirus infection.
- Some embodiments of the present disclosure also provide a method for diagnosing a subject infected with a new coronavirus or a disease related to a new coronavirus infection, including:
- diseases associated with novel coronavirus infection include respiratory symptoms, fever, cough, shortness of breath, hypoxemia, dyspnea, pneumonia, acute respiratory distress syndrome, severe acute respiratory syndrome, renal failure or septic shock.
- Some embodiments of the present disclosure also provide a method for detecting novel coronavirus or its N protein in a test sample, comprising:
- the novel coronavirus or its N protein antigen in the test sample is combined with the above-mentioned antibody or its functional fragment, or the above-mentioned antibody, or the above-mentioned antibody conjugate, or the above-mentioned reagent or the kit is contacted to form an immune complex;
- the immune complex further includes a second antibody that binds to the above-mentioned antibody or antigen-binding fragment.
- the immune complex further includes a second antibody that binds to the novel coronavirus or its N protein antigen.
- the antibody or its functional fragment can be prepared by using genetic engineering technology or other techniques (chemical synthesis, recombinant expression), for example, from It is easy for those skilled in the art to separate and purify the antibody or its functional fragment from the cultured product of recombinant cells capable of recombinantly expressing the above-mentioned antibody or its functional fragment. Based on this, no matter what technology is used Preparation of the antibody of the present disclosure or its functional fragments all belong to the protection scope of the present disclosure.
- the present disclosure provides antibodies against novel coronavirus, reagents and kits for detecting novel coronavirus, the antibody can specifically bind to the N protein of novel coronavirus, and has a high affinity to it, and the detection of novel coronavirus with the antibody has relatively high Good sensitivity and specificity.
- the present disclosure provides a richer selection of antibodies for the detection of novel coronaviruses.
- restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company.
- MagExtractor-RNA extraction kit was purchased from TOYOBO Company.
- BD SMART TM RACE cDNA Amplification Kit was purchased from Takara Company.
- the pMD-18T vector was purchased from Takara Company.
- Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.
- the mRNA was extracted from the hybridoma cell line that secretes the antibody against the N protein of the new coronavirus prepared in our laboratory, and the DNA product was obtained by RT-PCR method, and the product was inserted into pMD-18T after adding A reaction with rTaq DNA polymerase
- the vector was transformed into DH5 ⁇ competent cells, and after the colonies grew out, heavy chain (Heavy Chain) and light chain (Light Chain) gene clones were taken respectively, and each 4 clones were sent to a gene sequencing company for sequencing.
- the gene sequence obtained by the above sequencing was analyzed in the IMGT antibody database, and the VNTI11.5 software was used to analyze and confirm that the genes amplified by the primer pairs of the heavy chain and the light chain were correct, and the genes amplified by the light chain were correct.
- the light chain variable region (VL) gene sequence is 342bp, which belongs to the VkII gene family, and there is a 57bp leader peptide sequence in front of it; in the gene fragment amplified by the heavy chain primer pair, the heavy chain variable region (VH)
- the gene sequence is 336bp, belongs to the VH1 gene family, and there is a 57bp leader peptide sequence in front of it.
- pcDNA TM 3.4 Vector is a recombinant antibody eukaryotic expression vector constructed.
- the expression vector has introduced multiple cloning restriction sites such as HindIII, BamHI, EcoRI, etc., and named it pcDNA3.4A expression vector, which will be referred to as 3.4A expression vector in the future; according to the above pMD-18T
- the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases at both ends, and a 0.72kb light chain was amplified by PCR amplification gene fragment and a 1.41 kb heavy chain gene fragment.
- the heavy chain and light chain gene fragments were respectively digested with HindIII/EcoRI double enzymes, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. After the fragments and vectors were purified and recovered, the heavy chain genes and light chain genes were respectively connected to the 3.4A expression vectors, respectively. Recombinant expression plasmids for the heavy and light chains were obtained.
- the cells were first cultured in a 125ml shaker flask with an inoculation volume of 30ml, the medium was 100% Dynamis medium, and placed in a shaker with a rotation speed of 120r/min, a temperature of 37°C, and 8% carbon dioxide. Cultivate for 72 hours, inoculate expansion culture at a seeding density of 500,000 cells/ml, the expansion volume is calculated according to production requirements, and the medium is 100% Dynamis medium. Then every 72h expansion once. When the amount of cells meets the production requirements, strictly control the inoculation density to about 500,000 cells/ml for production.
- Shake flask parameters rotational speed 120r/min, temperature 37°C, carbon dioxide 8%.
- Fed-batch feeding Feed feeding starts every day when the shake flask is cultured for 72 hours.
- HyCloneTM Cell BoostTM Feed 7a feeds 3% of the initial culture volume every day, and Feed 7b feeds 1/1000 of the initial culture volume every day. Replenish until the 12th day (feeding on the 12th day).
- Glucose was supplemented with 3g/L on the sixth day. Receive samples on the 13th day.
- Affinity purification was performed with protein A (proteinA) affinity chromatography column. Take 4 ⁇ g of purified antibody for reducing SDS-PAGE, and 4 ⁇ g of foreign control antibody as a control.
- the electropherogram is shown in Figure 1 below. After reducing SDS-PAGE, two bands are displayed, and one Mr is 50KD (heavy chain, SEQ ID NO:19), the other Mr is 28KD (light chain,
- Example 1 On the basis of the anti-new coronavirus antibody (3E50-1) in Example 1, a single-point saturation mutation was performed on the complementarity determining region, and three mutant antibodies 3E50-2, 3E50-3, and 3E50-4 were obtained through activity screening. The above four antibody mutations were optimized, and 3E50-A, 3E50-B, 3E50-C, 3E50-D, and 3E50-E were obtained through activity screening.
- Antibody number heavy chain light chain 3E50-1 SEQ ID NO:19 SEQ ID NO:20 3E50-2 SEQ ID NO:29 SEQ ID NO:20 3E50-3 SEQ ID NO:29 SEQ ID NO: 31 3E50-4 SEQ ID NO:19 SEQ ID NO: 31 3E50-A SEQ ID NO:28 SEQ ID NO:20 3E50-B SEQ ID NO: 30 SEQ ID NO:20 3E50-C SEQ ID NO: 30 SEQ ID NO: 31 3E50-D SEQ ID NO:28 SEQ ID NO: 31 3E50-E SEQ ID NO:33 SEQ ID NO:20
- the purified antibody was diluted to 10 ⁇ g/mL with PBST, and the 2019-nCoV N protein antigen was serially diluted with PBST: 1.41 ⁇ g/mL, 0.70 ⁇ g/mL, 0.35 ⁇ g/mL, 0.18 ⁇ g/mL, 0.09 ⁇ g/mL, 0.04 ⁇ g/mL.
- 3E50-1 antibody and its mutants were used as the labeled antibody, and 6E8 was used as the coating antibody (purchased from Fipeng) to detect the new coronavirus.
- Colloidal gold preparation using the traditional sodium citrate reduction method, first heat the chloroauric acid solution to boiling, quickly add a certain proportion of trisodium citrate solution, stir evenly, and wait until the color of the solution turns wine red and does not change Stop heating at the time, be cooled to room temperature, obtain the colloidal gold solution that concentration is 4/10,000;
- Gold coating Dilute the concentrated gold obtained from the resuspension and spread it on a glass cellulose membrane, then freeze-dry it in a freeze dryer (1-2 hours) or dry it overnight in a drying room at 37°C.
- Quality control Lx-nCoVN, diluted with PBS to 25ng/ml, 2ng/ml, 0.5ng/ml (500pg/ml), 0.1ng/ml (100pg/ml), etc.
- 3E50-1, 3E50-2, 3E50-3, 3E50-4 are clearly detected at 100pg/mL (C7+ color development), and can be detected at 25pg/mL (C8 color development), the lowest detection limit can reach 10pg/mL ml.
- 3E50-A, 3E50-B, 3E50-C, 3E50-D, 3E50-E are clearly detected at 25pg/mL (C7+ color development), and can be detected at 10pg/mL (C8 color development), the lowest detection limit Up to 5pg/ml.
- the anti-new coronavirus antibody provided by the present disclosure has a good affinity to the N protein of the new coronavirus, and the detection of the new coronavirus by using the antibody has good sensitivity and specificity.
- This disclosure provides a more excellent antibody selection for the detection of novel coronavirus, therefore, the anti-new coronavirus antibody, reagent and kit for detecting novel coronavirus provided by this disclosure all have excellent practical performance and broad market application prospects .
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Abstract
本公开提供了一种抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒,涉及抗体技术领域。本公开的抗新型冠状病毒的抗体包括重链互补决定区和轻链互补决定区。该抗体对新型冠状病毒的N蛋白的亲和力较好,使用该抗体检测新型冠状病毒具有较好的灵敏度和特异性。本公开为新型冠状病毒的检测提供了更为优秀的抗体选择。
Description
相关申请的交叉引用
本公开要求于2021年11月08日提交中国专利局的申请号为CN202111315855.X、名称为“抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒”的中国专利申请的优先权,其全部内容通过引用结合在本公开中。
本公开涉及抗体技术领域,具体而言,涉及一种抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒。
新型冠状病毒2019-nCoV的结构蛋白分为:刺突糖蛋白(S蛋白)、包膜糖蛋白(E蛋白)、膜糖蛋白(M蛋白)和核衣壳蛋白(N蛋白),这些蛋白包括多个抗原表位。N蛋白与病毒基因组RNA相互缠绕形成病毒核衣壳,在病毒RNA的合成过程中发挥着重要的作用。同时,N蛋白相对保守,在病毒的结构蛋白中所占比例最大,感染早期机体就能产生抗N蛋白的高水平抗体。最后,N蛋白是新型冠状病毒重要的标志蛋白,利用抗原与抗体特异性结合的原理,可通过N蛋白单克隆抗体检测抗原的存在,从而直接证明样本中含有新型冠状病毒,实现新型冠状病毒的检测。
检测的抗体主要分为IgM和IgG两类。目前对新型冠状病毒的这两类抗体的产生和持续时间尚缺乏系统性研究。通常情况下,IgM抗体产生早,一经感染,快速产生,维持时间短,消失快,血液中检测阳性可反应机体处于急性感染状态,可作为早期感染的指标。与核酸检测方法相比,抗体检测样本为血清或血浆,受样本采样的影响较小,有利于早期诊断和排除可疑病例,同时检测快速、方便、适合大规模筛查。
新型冠状病毒2019-nCoV肺炎疫情发生以来,在全球蔓延,严重威胁着人类的生命安全和身体健康。呼吸道飞沫和密切接触传播是新型冠状病毒肺炎主要的传播途径,在相对封闭的环境中长时间暴露于高浓度气溶胶情况中存在经气溶胶传播的可能。2019-nCoV具有很强的传染性,多数患者感染后出现临床症状,但也有部分患者为无症状感染者。人感染了冠状病毒后常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等。在较严重病例中,感染可导致肺炎、严重急性呼吸综合征、肾衰竭,甚至死亡。虽然目前对于新型冠状病毒所致疾病没有特异治疗方法,但是轻症、或者无症状患者通过治疗可以大大的提高治愈率,缩短治疗时间。因此,对患者的检测或者鉴定变的尤为重要。
目前主要以核酸检测和病毒基因测序为病原学确诊证据,由于采样、操作以及试剂等多方面因素影响,核酸检测会出现假阴性结果。高度疑似2019新型冠状病毒(2019-nCoV)感染患者的病毒核酸阳性检出率仅为30%~50%。另外,核酸检测对仪器设备、检测场地及环境条件要求高,存在检 测时间长、通量低等缺点,不便于目前疫情下的人群大规模检测。因此,迫切需要研发出一种快速便捷检测试剂盒用于临床检测,从而尽快隔离感染人群以阻断病毒传播。因此,抗体检测试剂盒变得更为重要。
目前2019-nCoV N蛋白单克隆抗体产品较少,性能存在差异。
发明内容
本公开提供了一种抗新型冠状病毒或其N蛋白的抗体或其功能性片段,所述抗体或其功能性片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3,所述HCDR1~HCDR3包括或为与SEQ ID NO:15、24、25、26任一所示重链可变区的HCDR1~HCDR3一致的氨基酸序列;所述LCDR1~LCDR3包括或为与SEQ ID NO:16、27任一所示轻链可变区的LCDR1~LCDR3一致的氨基酸序列。
可选地,所述CDRs由Kabat、Chothia、IMGT、Lesk、AbM或Contact编号系统定义。
可选地,所述CDRs由Kabat、Chothia、IMGT或Lesk编号系统定义。
可选地,所述抗体或其功能性片段具有如下互补决定区:
HCDR1,其包含SEQ ID NO.1所示的氨基酸序列,或由其组成;
HCDR2,其包含SEQ ID NO.2或21所示的氨基酸序列,或由其组成;
HCDR3,其包含SEQ ID NO.3所示的氨基酸序列,或由其组成;
LCDR1,其包含SEQ ID NO.4所示的氨基酸序列,或由其组成;
LCDR2,其包含SEQ ID NO.5或22所示的氨基酸序列,或由其组成;
LCDR3,其包含SEQ ID NO.6所示的氨基酸序列,或由其组成。
可选地,所述抗体或其功能性片段还具有以下的框架区:
HFR1氨基酸序列如SEQ ID NO:7、23任一所示,或与其具有至少80%同源性;
HFR2氨基酸序列如SEQ ID NO:8所示或与其具有至少80%同源性;
HFR3氨基酸序列如SEQ ID NO:9所示或与其具有至少80%同源性;
HFR4氨基酸序列如SEQ ID NO:10所示或与其具有至少80%同源性;
LFR1氨基酸序列如SEQ ID NO:11所示或与其具有至少80%同源性;
LFR2氨基酸序列如SEQ ID NO:12所示或与其具有至少80%同源性;
LFR3氨基酸序列如SEQ ID NO:13所示或与其具有至少80%同源性;
LFR4氨基酸序列如SEQ ID NO:14所示或与其具有至少80%同源性。
可选地,所述抗体或其功能性片段以K
D≤10
-9M的亲和力结合新型冠状病毒或其N蛋白。
可选地,所述抗体或其功能性片段以K
D≤3.11*10
-10M的亲和力结合新型冠状病毒或其N蛋白。
本公开提供了一种抗新型冠状病毒或其N蛋白的抗体或其功能性片段,所述抗体或其功能性片段包含重链可变区和/或轻链可变区,所述重链可变区含有HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4的序列结构,所述轻链可变区区含有 LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4的序列结构,所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3的氨基酸序列为上述的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3的氨基酸序列,所述HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3、LFR4的氨基酸序列为上述的HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3、LFR4的氨基酸序列。
可选地,所述重链可变区氨基酸序列如SEQ ID NO:15、24、25、26任一所示;
所述轻链可变区氨基酸序列如SEQ ID NO:16、27任一所示。
可选地,所述抗体或其功能性片段还包含恒定区。
可选地,所述恒定区包括重链恒定区和/或轻链恒定区。
可选地,所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD的重链恒定区;所述轻链恒定区选自κ型或λ型轻链恒定区。
可选地,所述恒定区的种属来源为牛、马、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅或人。
可选地,所述恒定区的种属来源为小鼠。
可选地,所述重链恒定区序列如SEQ ID NO:17、32任一所示或与其具有至少80%同源性,所述轻链恒定区序列如SEQ ID NO:18所示或与其具有至少80%同源性。
可选地,所述功能性片段选自所述抗体的F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
本公开还提供了一种抗新型冠状病毒或其N蛋白的抗体,包括重链和/或轻链,所述重链包括上述的重链可变区和上述的重链恒定区;所述轻链包括上述的轻链可变区和上述的轻链恒定区。
可选地,所述重链的氨基酸序列如SEQ ID NO:19、28、29、30、33任一所示;所述轻链的氨基酸序列如SEQ ID NO:20、31任一所示。
本公开还提供了一种抗体偶联物,所述抗体偶联物包括上述的抗体或其功能性片段。
可选地,所述抗体或其功能性片段标记有可被检测的标记物。
可选地,所述可被检测的标记物选自荧光染料、催化底物显色的酶、放射性同位素、化学发光试剂和纳米颗粒类标记物。
可选地,所述荧光染料选自荧光素类染料及其衍生物、罗丹明类染料及其衍生物、Cy系列染料及其衍生物、Alexa系列染料及其衍生物和蛋白类染料及其衍生物。
可选地,所述催化底物显色的酶选自辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶。
可选地,所述放射性同位素选自
212Bi、
131I、
111In、
90Y、
186Re、
211At、
125I、
188Re、
153Sm、
213Bi、
32P、
94mTc、
99mTc、
203Pb、
67Ga、
68Ga、
43Sc、
47Sc、
110mIn、
97Ru、
62Cu、
64Cu、
67Cu、
68Cu、
86Y、
88Y、
121Sn、
161Tb、
166Ho、
105Rh、
177Lu、
172Lu和
18F。
可选地,所述化学发光试剂选自鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物。
可选地,所述纳米颗粒类标记物选自纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒。
可选地,所述胶体选自胶体金属、分散型染料、染料标记的微球和乳胶。
可选地,所述胶体金属选自胶体金、胶体银和胶体硒。
可选地,所述抗体或其功能性片段包被至固相。
可选地,所述固相选自微球、板和膜。
可选地,所述固相选自磁性微球、塑料微球、塑胶微粒、微孔板、玻璃、毛细管、尼龙和硝酸纤维素膜。可选地,所述固相选自磁性微球。
本公开还提供了一种试剂或试剂盒,所述试剂或试剂盒包括上述的抗体或其功能性片段或上述的抗体偶联物。
本公开还提供了一种核酸,其编码上文任一项所述的抗体或其功能性片段。
本公开还提供了一种载体,其含有编码上文任一项所述的抗体或其功能性片段的核酸片段。
本公开还提供了一种重组细胞,其含有所述载体。
本公开还提供了一种制备上文任一项所述的抗体或其功能性片段的方法,其包括:培养所述重组细胞,从培养产物中分离纯化得到所述抗体或其功能性片段。
本公开还提供了上文任一项所述的抗体或其功能性片段,或者所述抗体,或者所述抗体偶联物,在制备试剂或试剂盒中的用途,所述试剂或试剂盒用于检测新型冠状病毒或其N蛋白,或者诊断新型冠状病毒感染或与新型冠状病毒感染相关疾病。
本公开还提供了上文任一项所述的抗体或其功能性片段,或者所述抗体,或者所述抗体偶联物,或者所述试剂或试剂盒,用于检测新型冠状病毒或其N蛋白,或者诊断新型冠状病毒感染或与新型冠状病毒感染相关疾病中的用途。
可选地,所述与新型冠状病毒感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、低氧血症、呼吸困难、肺炎、急性呼吸窘迫综合征、严重急性呼吸综合征、肾衰竭或脓毒症休克。
本公开还提供了一种诊断受试者在感染新型冠状病毒或与新型冠状病毒感染相关疾病中的方法,包括:
A)在足以发生结合反应的条件下,使上文任一项所述的抗体或其功能性片段,或者所述抗体,或者所述抗体偶联物,或者所述试剂或试剂盒,与来自所述受试者的样品接触以进行结合反应;以及
B)检测结合反应产生的免疫复合物。
可选地,所述与新型冠状病毒感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、低氧血症、呼吸困难、肺炎、急性呼吸窘迫综合征、严重急性呼吸综合征、肾衰竭或脓毒症休克。
本公开还提供了一种检测测试样品中的新型冠状病毒或其N蛋白的方法,其包括:
a)在足以发生抗体/抗原结合反应的条件下,使所述测试样品中的新型冠状病毒或其N蛋白抗原与上文任一项所述的抗体或其功能性片段,或者所述抗体,或者所述抗体偶联物,或者所述试剂或 试剂盒接触以形成免疫复合物;和
b)检测所述免疫复合物的存在,所述复合物的存在指示所述测试样品中所述抗原的存在。
可选的,在步骤a)中,所述免疫复合物中还包括第二抗体,所述第二抗体与所述抗体或抗原结合片段结合。
可选的,在步骤a)中,所述免疫复合物中还包括第二抗体,所述第二抗体与所述新型冠状病毒或其N蛋白抗原结合。
为了更清楚地说明本公开实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本公开的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为实施例1的抗新型冠状病毒的抗体的还原性SDS-PAGE的结果。
本公开的一些实施方式提供了一种抗新型冠状病毒或其N蛋白的抗体或其功能性片段,抗体或其功能性片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3,其中,HCDR1、HCDR2、HCDR3包括与SEQ ID NO:15、24、25、26任一所示重链可变区的HCDR1、HCDR2、HCDR3一致的氨基酸序列;LCDR1、LCDR2、LCDR3包括与SEQ ID NO:16、27任一所示轻链可变区的LCDR1、LCDR2、LCDR3一致的氨基酸序列。
在可选的实施方式中,HCDR1、HCDR2、HCDR3为与SEQ ID NO:15、24、25、26任一所示重链可变区的HCDR1、HCDR2、HCDR3一致的氨基酸序列;LCDR1、LCDR2、LCDR3为与SEQ ID NO:16、27任一所示轻链可变区的LCDR1、LCDR2、LCDR3一致的氨基酸序列。
在可选的实施方式中,CDRs由Kabat、Chothia、IMGT或Lesk编号系统定义。
在可选的实施方式中,CDRs由AbM或Contact编号系统定义。
本公开的一些实施方式提供了一种抗新型冠状病毒或其N蛋白的抗体或其功能性片段,抗体或其功能性片段包括如下互补决定区:
HCDR1,其包含SEQ ID NO.1所示的氨基酸序列,或由其组成;
HCDR2,其包含SEQ ID NO.2或21所示的氨基酸序列,或由其组成;
HCDR3,其包含SEQ ID NO.3所示的氨基酸序列,或由其组成;
LCDR1,其包含SEQ ID NO.4所示的氨基酸序列,或由其组成;
LCDR2,其包含SEQ ID NO.5或22所示的氨基酸序列,或由其组成;
LCDR3,其包含SEQ ID NO.6所示的氨基酸序列,或由其组成。
如本文所用,术语“抗体”在最广义上使用,其可以包括全长单克隆抗体,双特异性或多特异性抗体,以及嵌合抗体,只要它们展示所需的生物学活性。
如本文所用,术语“互补性决定区”、“CDR”或“CDRs”是指免疫球蛋白的重链和轻链的高度可变区,指包含一种或多种或者甚至全部的对抗体或抗原结合片段与其识别的抗原或表位的结合亲和力起作用的主要氨基酸残基的区域。在本公开具体实施方式中,CDRs是指抗体的重链和轻链的高度可变区。
如本文所用,重链互补决定区用“HCDR”表示,其包括HCDR1、HCDR2和HCDR3;轻链互补决定区用LCDR表示,其包括LCDR1、LCDR2和LCDR3。本领域常用的CDR标示方法包括:Kabat编号方案、IMGT编号方案、Chothia和Lesk编号方案以及1997年Lefranc等人为免疫球蛋白超家族的所有蛋白质序列引入的新的标准化编号系统。Kabat等人是第一个为免疫球蛋白可变区提出标准化编号方案的人。在过去的几十年中,序列的积累导致了KABATMAN数据库的创建,Kabat编号方案通常被认为是编号抗体残基广泛采用的标准。本公开采用Kabat注释标准标示CDR区,但其他方法标示的CDR区也属于本公开的保护范围。如本文所用,“框架区”或“FR”区包括重链框架区和轻链框架区,是指抗体重链可变区(可以表示为VH)和轻链可变区(可以表示为VL)中除CDR之外的区域;其中,重链框架区用“HFR”表示,并且可以被进一步细分成被CDR分隔开的毗邻区域,包含HFR1、HFR2、HFR3和HFR4框架区;轻链框架区用“LFR”表示,并且可以被进一步细分成被CDR分隔开的毗邻区域,包含LFR1、LFR2、LFR3和LFR4框架区。
如本文所用,重链可变区由以下编号的CDR与FR按如下组合排列连接获得:HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4;轻链可变区由以下编号的CDR与FR按如下组合排列连接获得:LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4。
如本文所用,术语“受试者”和“患者”在本文可以互换使用,是指脊椎动物,优选地是哺乳动物,最优选地是人类。哺乳动物包括但不限于鼠类、猿类、人类、家畜、竞技动物和宠物。在体内获得的或在体外培养的生物实体的组织、细胞及其子代也包括在内。
在可选的实施方式中,抗体还或其功能性片段还具有以下的框架区:
HFR1氨基酸序列如SEQ ID NO:7、23任一所示,或与其具有至少80%同源性;
HFR2氨基酸序列如SEQ ID NO:8所示或与其具有至少80%同源性;
HFR3氨基酸序列如SEQ ID NO:9所示或与其具有至少80%同源性;
HFR4氨基酸序列如SEQ ID NO:10所示或与其具有至少80%同源性;和
LFR1氨基酸序列如SEQ ID NO:11所示或与其具有至少80%同源性;
LFR2氨基酸序列如SEQ ID NO:12所示或与其具有至少80%同源性;
LFR3氨基酸序列如SEQ ID NO:13所示或与其具有至少80%同源性;
LFR4氨基酸序列如SEQ ID NO:14所示或与其具有至少80%同源性。
需要说明的是,在其他的实施方式中,本公开提供的抗体或其功能性片段的各骨架区氨基酸序列可以与上述对应骨架区(SEQ ID NO:7、8、9、10、11、12、13或14)具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%的同源性。例如,HFR1的氨基酸序列还可以是SEQ ID NO:23。
在可选的实施方式中,抗体或其功能性片段以K
D≤10
-9M的亲和力结合新型冠状病毒或其N蛋白。
在可选的实施方式中,抗体或其功能性片段以K
D≤3.11*10
-10M的亲和力结合新型冠状病毒或其N蛋白;
K
D的检测参考本公开实施方式中的方法进行。本公开的一些实施方式还提供了一种抗新型冠状病毒或其N蛋白的抗体或其功能性片段,抗体或其功能性片段包含重链可变区和/或轻链可变区,重链可变区含有HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4的序列结构,轻链可变区区含有LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4的序列结构,HCDR1、HCDR2、HCDR3、LCDR 1、LCDR2、LCDR3的氨基酸序列为上述的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3的氨基酸序列,HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3、LFR4的氨基酸序列为上述的HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3、LFR4的氨基酸序列。
在可选的实施方式中,重链可变区氨基酸序列如SEQ ID NO:15、24、25、26任一所示。
在可选的实施方式中,轻链可变区氨基酸序列如SEQ ID NO:16、27任一所示。
在可选的实施方式中,抗体还包含恒定区。
在可选的实施方式中,恒定区包括重链恒定区和/或轻链恒定区。如本文所用,“重链恒定区”可以表示为CH;“轻链恒定区”可以表示为CL。
在可选的实施方式中,重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD的重链恒定区,轻链恒定区选自κ型或λ型轻链恒定区。
在可选的实施方式中,恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在可选的实施方式中,恒定区的种属来源为小鼠。
在可选的实施方式中,重链恒定区序列(CH)如SEQ ID NO:17所示,轻链恒定区(CL)序列如SEQ ID NO:18所示。
需要说明的是,在其他的实施方式中,恒定区序列可以与上述恒定区(SEQ ID NO:17或18)具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%的同源性。例如,重链恒定区还可以是SEQ ID NO:32。
在可选的实施方式中,功能性片段选自抗体的VHH、F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
上述抗体的功能性片段通常具有与其来源抗体相同的结合特异性。本领域技术人员根据本公开记载的内容容易理解到,上述抗体的功能性片段可以通过比如酶消化的方法(包括胃蛋白酶或木瓜蛋白酶)和/或通过化学还原分裂二硫键的方法获得。在本公开提供了完整抗体的结构基础上,本领域技术人员容易获得上述的功能性片段。
上述抗体的功能性片段还可以通过也是本领域技术人员所知的重组遗传学技术或通过例如自动肽合成仪,比如Applied BioSystems等销售的自动肽合成仪合成获得。
本公开的一些实施方式还提供一种抗新型冠状病毒或其N蛋白的抗体,包括重链和/或轻链,重链包括上述的重链可变区和上述的重链恒定区;轻链包括上述的轻链可变区和上述的轻链恒定区。
在可选的实施方式中,重链的氨基酸序列如SEQ ID NO:19、28、29、30、33任一所示;轻链的氨基酸序列如SEQ ID NO:20、31任一所示。
在可选的实施方式中,重链的氨基酸序列如SEQ ID NO:19、28、29、30、33任一所示;轻链的氨基酸序列如SEQ ID NO:20所示。
在可选的实施方式中,重链的氨基酸序列如SEQ ID NO:19、28、29、30、33任一所示;轻链的氨基酸序列如SEQ ID NO:31所示。
本公开的一些实施方式还提供一种抗体偶联物,抗体偶联物包括上述的抗体或其功能性片段。
在可选的实施方式中,上述抗体偶联物中抗体或其功能性片段标记有可被检测的标记物。
可被检测的标记物是指具有能够被肉眼直接观察或被仪器检测或探测到的特性例如发光、显色、放射性等特性的一类物质,通过该特性可以实现对相应目标物的定性或定量检测。
在可选的实施方式中,可被检测的标记物包括但不限于荧光染料、催化底物显色的酶、放射性同位素、化学发光试剂和纳米颗粒类标记物。
在实际的使用过程中,本领域技术人员可以根据检测条件或实际需要选择合适的标记物,无论使用何种标记物,其均属于本公开的保护范围。
在可选的实施方式中,荧光染料包括但不限于荧光素类染料及其衍生物(例如包括但不限于异硫氰酸荧光素(FITC)羟基光素(FAM)、四氯光素(TET)等或其类似物)、罗丹明类染料及其衍生物(例如包括但不限于红色罗丹明(RBITC)、四甲基罗丹明(TAMRA)、罗丹明B(TRITC)等或其类似物)、Cy系列染料及其衍生物(例如包括但不限于Cy2、Cy3、Cy3B、Cy3.5、Cy5、Cy5.5、Cy3等或其类似物)、Alexa系列染料及其衍生物(例如包括但不限于AlexaFluor350、405、430、488、532、546、555、568、594、610、33、647、680、700、750等或其类似物)和蛋白类染料及其衍生物(例如包括但不限于藻红蛋白(PE)、藻蓝蛋白(PC)、别藻蓝蛋白(APC)、多甲藻黄素-叶绿素蛋白(preCP)等)。
在可选的实施方式中,催化底物显色的酶包括但不限于辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶。
在可选的实施方式中,放射性同位素包括但不限于
212Bi、
131I、
111In、
90Y、
186Re、
211At、
125I、
188Re、
153Sm、
213Bi、
32P、
94mTc、
99mTc、
203Pb、
67Ga、
68Ga、
43Sc、
47Sc、
110mIn、
97Ru、
62Cu、
64Cu、
67Cu、
68Cu、
86Y、
88Y、
121Sn、
161Tb、
166Ho、
105Rh、
177Lu、
172Lu和
18F。
在可选的实施方式中,化学发光试剂包括但不限于鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物。
在可选的实施方式中,纳米颗粒类标记物包括但不限于纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒。
在可选的实施方式中,胶体包括但不限于胶体金属、分散型染料、染料标记的微球和乳胶。
在可选的实施方式中,胶体金属包括但不限于胶体金、胶体银和胶体硒。
在可选的实施方式中,上述抗体偶联物中抗体或其功能性片段包被至固相。
在可选的实施方式中,固相选自微球、板和膜。
在可选的实施方式中,固相包括但不限于磁性微球、塑料微球、塑胶微粒、微孔板、玻璃、毛细管、尼龙和硝酸纤维素膜。
在可选的实施方式中,固相为磁性微球。
本公开的一些实施方式还提供一种试剂或试剂盒,试剂或试剂盒包括上述的抗体或其功能性片段或上述的抗体偶联物。
本公开的一些实施方式还提供上述试剂或试剂盒在新型冠状病毒或其N蛋白检测中的用途。
本公开的一些实施方式还提供一种编码上述抗体或其功能性片段的核酸分子。
本公开的一些实施方式还提供含有上述核酸分子的载体。
本公开的一些实施方式还提供含有上述载体的重组细胞。
本公开的一些实施方式还提供一种制备抗体或其功能性片段的方法,其包括:培养上述的重组细胞,从培养产物中分离纯化得到上述抗体或其功能性片段。
本公开的一些实施方式还提供了上述抗体或其功能性片段,或者上述抗体,或者上述抗体偶联物,在制备试剂或试剂盒中的用途,所述试剂或试剂盒用于检测新型冠状病毒或其N蛋白,和/或诊断新型冠状病毒感染或与新型冠状病毒感染相关疾病。
本公开的一些实施方式还提供了上述抗体或其功能性片段,或者上述抗体,或者上述抗体偶联物,或者上述试剂或试剂盒,用于检测新型冠状病毒或其N蛋白,和/或诊断新型冠状病毒感染或与新型冠状病毒感染相关疾病中的用途。
本公开的一些实施方式还提供了一种诊断受试者在感染新型冠状病毒或与新型冠状病毒感染相关疾病中的方法,包括:
A)在足以发生结合反应的条件下,使上述抗体或其功能性片段,或者上述抗体,或者上述抗体偶联物,或上述试剂或试剂盒与来自受试者的样品接触以进行结合反应;以及
B)检测结合反应产生的免疫复合物。
在可选的实施方式中,与新型冠状病毒感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、低氧血症、呼吸困难、肺炎、急性呼吸窘迫综合征、严重急性呼吸综合征、肾衰竭或脓毒症休克。
本公开的一些实施方式还提供了一种检测测试样品中的新型冠状病毒或其N蛋白的方法,其包括:
a)在足以发生抗体/抗原结合反应的条件下,使测试样品中的新型冠状病毒或其N蛋白抗原与上述抗体或其功能性片段,或者上述抗体,或者上述抗体偶联物,或者上述试剂或试剂盒接触以形成免疫复合物;和
b)检测所述免疫复合物的存在,所述复合物的存在指示所述测试样品中所述抗原的存在。
在可选的实施方式中,在步骤a)中,所述免疫复合物中还包括第二抗体,所述第二抗体与上述抗体或抗原结合片段结合。
在可选的实施方式中,在步骤a)中,所述免疫复合物中还包括第二抗体,所述第二抗体与新型冠状病毒或其N蛋白抗原结合。
在本公开提供了抗体或其功能性片段的氨基酸序列的基础上,本领域技术人员容易想到采用基因工程技术或其他技术(化学合成、重组表达)制备得到该抗体或其功能性片段,例如从能够重组表达如上述的抗体或其功能性片段的重组细胞的培养产物中分离纯化得到该抗体或其功能性片段,这对本领域技术人员来说是容易实现的,基于此,无论采用何种技术制备本公开的抗体或其功能性片段,其均属于本公开的保护范围。
为使本公开实施方式的目的、技术方案和优点更加清楚,下面将对本公开实施方式中的技术方案进行清楚、完整地描述。实施方式中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
除非另有定义,否则本文使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试,但现在描述一些方法和材料。除非另有说明,否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
除非另外指明,否则实践本公开将采用细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术,所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术,如《分子克隆:实验室手册(Molecular Cloning:A Laboratory Manual)》,第二版(Sambrook等人,1989);《寡核苷酸合成(Oligonucleotide Synthesis)》(M.J.Gait编,1984);《动物细胞培养(Animal Cell Culture)》(R.I.Freshney编,1987);《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press,Inc.);《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir和C.C.Blackwell编);《哺乳动物细胞用基因转移载体(Gene Transfer Vectors for Mammalian Cells)》(J.M.Miller和M.P.Calos编,1987);《当代分子生物学方法(Current Protocols in Molecular Biology)》(F.M.Ausubel等人编,1987);《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis等人编,1994);以及《当代免疫学方法(Current Protocols in Immunology)》(J.E.Coligan等人编,1991),所述文献中的每个文献均通过引用明确并入本文中。
本公开提供了抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒,该抗体可以特异性结合新型冠状病毒的N蛋白,对其具有较高的亲和力,用该抗体检测新型冠状病毒具有较好的灵敏度和特异性。本公开为新型冠状病毒的检测提供了更为丰富的抗体选择。
实施例
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1抗体3E50-1的制备
本实施例中限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMART
TMRACE cDNA Amplification Kit试剂盒购自Takara公司。pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由Invitrogen公司完成。
1重组质粒的构建
(1)抗体基因制备
从本实验室制备的分泌抗新型冠状病毒N蛋白的抗体的杂交瘤细胞株中提取mRNA,通过RT-PCR方法获得DNA产物,该产物用rTaq DNA聚合酶进行加A反应后插入到pMD-18T载体中,转化到DH5α感受态细胞中,长出菌落后分别取重链(Heavy Chain)及轻链(Light Chain)基因克隆,各4个克隆送基因测序公司进行测序。
(2)抗体可变区基因的序列分析
将上述测序得到的基因序列放在IMGT抗体数据库中进行分析,并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的,其中轻链扩增出的基因片段中,轻链可变区(VL)基因序列为342bp,属于VkII基因家族,其前方有57bp的前导肽序列;重链引物对扩增出的基因片段中,重链可变区(VH)基因序列为336bp,属于VH1基因家族,其前方有57bp的前导肽序列。
(3)重组抗体表达质粒的构建
pcDNA
TM3.4
vector为构建的重组抗体真核表达载体,该表达载体已经引入HindIII、BamHI、EcoRI等多克隆酶切位点,并命名为pcDNA3.4A表达载体,后续简称3.4A表达载体;根据上述pMD-18T中抗体可变区基因测序结果,设计该抗体的VL和VH基因特异性引物,两端分别带有HindIII、EcoRI酶切位点和保护碱基,通过PCR扩增方法扩出0.72kb的轻链基因片段和1.41kb的重链基因片段。
重链和轻链基因片段分别采用HindIII/EcoRI双酶切,3.4A载体采用HindIII/EcoRI双酶切,将片段和载体纯化回收后重链基因和轻链基因分别连接3.4A表达载体中,分别得到重链和轻链的重组表达质粒。
2稳定细胞株筛选
(1)重组抗体表达质粒瞬时转染CHO细胞,确定表达质粒活性
质粒用超纯水稀释至400ng/ml,调节CHO细胞1.43×10
7个细胞/ml于离心管中,100μL质粒与700μL细胞混合,转入电转杯,电转,第3、5、7天取样计数,第7天收样检测。
包被液稀释2019-nCoV N蛋白抗原到1μg/ml,每孔100μL,4℃过夜;次日,洗涤液清洗2次,拍干;加入封闭液(20%BSA(牛血清白蛋白)+80%PBS),每孔120μL,37℃,1h,拍干;加入稀释后的细胞上清,100μL/孔,37℃,30min(部分上清1h);洗涤液清洗5次,拍干;加入羊抗鼠IgG-HRP(辣根过氧化物酶标记的IgG单抗),每孔100μL,37℃,30min;洗涤液清洗5次,拍干;加入显色液A液(50μL/孔,含柠檬酸+醋酸钠+乙酰苯胺+过氧化脲),加入显色液B液(50μL/孔,含柠檬酸+EDTA·2Na+TMB(3,3',5,5'-四甲基联苯胺)+浓HCL),10min;加入终止液(50μL/孔,含 EDTA·2Na+浓H
2SO
4);酶标仪上450nm(参考630nm)处读OD值。结果显示细胞上清稀释1000倍后反应OD仍大于1.0,未加细胞上清孔反应OD小于0.1,表明质粒瞬转后产生的抗体对2019-nCoVN蛋白抗原有活性。
(2)重组抗体表达质粒线性化
准备下述试剂:Buffer 50μL、DNA 100μg/管、Puv Ⅰ酶10μL、无菌水补至500μL,37℃水浴酶切过夜;先用等体积酚/氯仿/异戊醇(下层)25:24:1,再用氯仿(水相)依次进行抽提;0.1倍体积(水相)3M醋酸钠和2倍体积乙醇冰上沉淀,70%乙醇漂洗沉淀,去除有机溶剂,待乙醇挥发完全用适量的灭菌水进行复融,最后进行浓度的测定。
(3)重组抗体表达质粒稳定转染,加压筛选稳定细胞株
质粒用超纯水稀释至400ng/ml,调节CHO细胞1.43×10
7个细胞/ml于离心管中,100μL质粒与700μL细胞混合,转入电转杯,电转,次日计数;25μmol/L MSX 96孔加压培养约25天。
显微镜下观察标记长有细胞的克隆孔,并记录汇合度;取培养上清,送样检测;挑选抗体浓度、相对浓度高的细胞株转24孔,3天左右转6孔;3天后保种批培,调整细胞密度0.5×10
6个细胞/ml,2.2ml进行批培养,细胞密度0.3×10
6个细胞/ml,2ml进行保种;7天6孔批培上清送样检测,挑选抗体浓度及细胞直径较小的细胞株转TPP保种传代。
3重组抗体生产
(1)细胞扩培
细胞复苏之后先在125ml规格的摇瓶中培养,接种体积为30ml,培养基为100%Dynamis培养基,放置于转速120r/min,温度为37℃,二氧化碳为8%的摇床中。培养72h,以50万个细胞/ml接种密度接种扩培,扩培体积根据生产需求进行计算,培养基为100%Dynamis培养基。之后每72h扩培一次。当细胞量满足生产需求时,严格控制接种密度为50万个细胞/ml左右进行生产。
(2)摇瓶生产及纯化
摇瓶参数:转速120r/min,温度为37℃,二氧化碳为8%。流加补料:在摇瓶中培养至72h时开始每天补料,HyCloneTM Cell BoostTM Feed 7a每天流加初始培养体积的3%,Feed 7b每天流加量为初始培养体积的千分之一,一直补到第12天(第12天补料)。葡萄糖在第六天补加3g/L。第13天收样。用蛋白A(proteinA)亲和层析柱进行亲和纯化。取4μg纯化的抗体进行还原性SDS-PAGE,4μg外来对照抗体作为对照,电泳图如下图1所示,在还原性SDS-PAGE后显示两条带,1条Mr为50KD(重链,SEQ ID NO:19),另一条Mr为28KD(轻链,SEQ ID NO:20)。
在实施例1的抗新型冠状病毒抗体(3E50-1)基础上,对互补决定区进行单点饱和突变,经活性筛选得到3E50-2、3E50-3、3E50-4三个突变抗体,进一步对上述4个抗体突变优化,经活性筛选得到3E50-A、3E50-B、3E50-C、3E50-D、3E50-E。
表1抗体3E50-1及其突变体的序列
抗体编号 | 重链 | 轻链 |
3E50-1 | SEQ ID NO:19 | SEQ ID NO:20 |
3E50-2 | SEQ ID NO:29 | SEQ ID NO:20 |
3E50-3 | SEQ ID NO:29 | SEQ ID NO:31 |
3E50-4 | SEQ ID NO:19 | SEQ ID NO:31 |
3E50-A | SEQ ID NO:28 | SEQ ID NO:20 |
3E50-B | SEQ ID NO:30 | SEQ ID NO:20 |
3E50-C | SEQ ID NO:30 | SEQ ID NO:31 |
3E50-D | SEQ ID NO:28 | SEQ ID NO:31 |
3E50-E | SEQ ID NO:33 | SEQ ID NO:20 |
实施例2
抗体的性能检测
(1)抗体3E50-1及其突变体的活性检测
对表1中的抗体结合活性检测:
包被液(主要成分NaHCO
3)稀释2019-nCoV N蛋白抗原到1μg/ml进行微孔板包被,每孔100μl,4℃过夜;次日,洗涤液清洗2次,拍干;加入封闭液(20%BSA+80%PBS),每孔120μl,37℃,1h,拍干;加入稀释后的表1中的单克隆抗体,100μl/孔,37℃,30min-60min;洗涤液清洗5次,拍干;加入羊抗鼠IgG-HRP,每孔100μl,37℃,30min;洗涤液(PBS)清洗5次,拍干;加入显色液A液(50μl/孔,含2.1g/L柠檬酸、12.25g/L柠檬酸、0.07g/L乙酰苯胺和0.5g/L过氧化脲),加入显色液B液(50μl/孔,含1.05g/L柠檬酸、0.186g/LEDTA·2Na、0.45g/L TMB和0.2ml/L浓HCl),10min;加入终止液(50μl/孔,含0.75g/EDTA·2Na和10.2ml/L浓H
2SO
4);酶标仪上450nm(参考630nm)处读OD值。结果见下表2。
表2 3E50-1抗体及其突变体的活性数据
(2)抗体及其突变体的亲和力检测
亲和力分析
利用AMC传感器,将纯化出来的抗体用PBST稀释到10μg/mL,2019-nCoV N蛋白抗原用PBST进行梯度稀释:1.41μg/mL、0.70μg/mL、0.35μg/mL、0.18μg/mL、0.09μg/mL、0.04μg/mL。
运行流程:缓冲液1(PBST)中平衡60s,抗体溶液中固化抗体300s,缓冲液2(PBST)中孵育180s,抗原溶液中结合420s,缓冲液2中解离1200s,用10mM pH 1.69GLY溶液及缓冲液3进行传感器再生,输出数据。K
D表示平衡解离常数即亲和力;kon表示结合速率;kdis表示解离速率。结果见下表3。
表3亲和力检测数据
(3)稳定性考核
将上述抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天,取7天、14天、21天样品进行状态观察,并对21天样品进行活性检测,活性检测方法参见实施例2。
结果显示三种考核条件下抗体放置21天均未见明显蛋白状态变化,活性也未随考核温度的升高呈下降趋势,说明上述抗体稳定。下表4为3E50-A抗体考核21天的酶免活性检测OD结果。
表4
(4)新冠病毒的检测
将上述3E50-1抗体及其突变体作为标记抗体,6E8作为包被抗体(购自菲鹏),进行新冠病毒的检测。
1.标记
(1)胶体金制备:采用传统柠檬酸钠还原法,首先将氯金酸溶液加热至沸腾,迅速加入一定比例的柠檬酸三钠溶液,搅拌均匀,待溶液颜色变为酒红色且不再变化时停止加热,冷却至室温,得到浓度为万分之四的胶体金溶液;
(2)标记:向胶体金溶液中加入0.2M K
2CO
3溶液调节pH至6.0-7.5;
(3)离心:向调节pH后的胶体金溶液中加入标记抗体并混匀,后加入封闭剂,终止标记,离心10000rpm/7min/4℃,去上清;
(4)复溶:重悬至100uL,超声2-3次;
(5)铺金:将重悬得到的浓缩金稀释并铺于玻璃纤维素膜,然后放入冻干机冻干(1-2h)或者放入37℃干燥房干燥过夜。
2.包被
(1)将硝酸纤维素膜与胶体金PVC底板组装好备用;
(2)将包被抗体稀释至1.0-2.0mg/mL,用喷金画膜仪,在NC膜上均匀的画线,然后放入37℃恒温箱中进行干燥,至少干燥45min以上。组装切条,加样检测。
3.检测
(1)质控品:Lx-nCoVN,使用PBS稀释至25ng/ml,2ng/ml,0.5ng/ml(500pg/ml),0.1ng/ml(100pg/ml)等。
(2)检测方法:使用胶体金试纸条进行检测。
4.验证3E50-1抗体及其突变体的检测灵敏度
表5
表5中,字母B代表不显色(未检出),字母C后的数字代表显色,数字越大,显色越弱(活性越低)。
3E50-1、3E50-2、3E50-3、3E50-4在100pg/mL时有明显检出(C7+显色),对25pg/mL能检出(C8显色),最低检测限可达10pg/ml。
3E50-A、3E50-B、3E50-C、3E50-D、3E50-E在25pg/mL时有明显检出(C7+显色),对10pg/mL能检出(C8显色),最低检测限可达5pg/ml。
以上所述仅为本公开的可选的实施例而已,并不用于限制本公开,对于本领域的技术人员来说,本公开可以有各种更改和变化。凡在本公开的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本公开的保护范围之内。
本公开提供的抗新型冠状病毒的抗体,其对新型冠状病毒的N蛋白的亲和力较好,使用该抗体检测新型冠状病毒具有较好的灵敏度和特异性。本公开为新型冠状病毒的检测提供了更为优秀的抗体选择,因此,本公开提供的抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒均具备优异的实用性能和广阔的市场应用前景。
Claims (19)
- 一种抗新型冠状病毒或其N蛋白的抗体或其功能性片段,所述抗体或其功能性片段包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3,其特征在于,所述HCDR1~HCDR3包括与SEQ ID NO:15、24、25、26任一所示重链可变区的HCDR1~HCDR3一致的氨基酸序列;所述LCDR1~LCDR3包括与SEQ ID NO:16、27任一所示轻链可变区的LCDR1~LCDR3一致的氨基酸序列。
- 根据权利要求1所述的抗体或其功能性片段,其特征在于,所述CDRs由Kabat、Chothia、IMGT、Lesk、AbM或Contact编号系统定义;可选地,所述CDRs由Kabat、Chothia、IMGT或Lesk编号系统定义。
- 根据权利要求1所述的抗体或其功能性片段,其特征在于,所述抗体或其功能性片段包括如下互补决定区:HCDR1,其包含SEQ ID NO.1所示的氨基酸序列,或由其组成;HCDR2,其包含SEQ ID NO.2或21所示的氨基酸序列,或由其组成;HCDR3,其包含SEQ ID NO.3所示的氨基酸序列,或由其组成;LCDR1,其包含SEQ ID NO.4所示的氨基酸序列,或由其组成;LCDR2,其包含SEQ ID NO.5或22所示的氨基酸序列,或由其组成;LCDR3,其包含SEQ ID NO.6所示的氨基酸序列,或由其组成。
- 根据权利要求1-3任一项所述的抗体或其功能性片段,其特征在于,所述抗体或其功能性片段还具有以下的框架区:HFR1氨基酸序列如SEQ ID NO:7、23任一所示,或与其具有至少80%同源性;HFR2氨基酸序列如SEQ ID NO:8所示或与其具有至少80%同源性;HFR3氨基酸序列如SEQ ID NO:9所示或与其具有至少80%同源性;HFR4氨基酸序列如SEQ ID NO:10所示或与其具有至少80%同源性;LFR1氨基酸序列如SEQ ID NO:11所示或与其具有至少80%同源性;LFR2氨基酸序列如SEQ ID NO:12所示或与其具有至少80%同源性;LFR3氨基酸序列如SEQ ID NO:13所示或与其具有至少80%同源性;LFR4氨基酸序列如SEQ ID NO:14所示或与其具有至少80%同源性;可选地,所述抗体或其功能性片段以K D≤10 -9M的亲和力结合新型冠状病毒或其N蛋白;可选地,所述抗体或其功能性片段以K D≤3.11*10 -10M的亲和力结合新型冠状病毒或其N蛋白。
- 一种抗新型冠状病毒或其N蛋白的抗体或其功能性片段,其特征在于,所述抗体或其功能性片段包含重链可变区和/或轻链可变区,所述重链可变区含有HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4的序列结构,所述轻链可变区区含有LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4的序列结构,所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3的氨基酸序列为权利要求1-3任一项所述的HCDR1、HCDR2、HCDR3、LCDR1、 LCDR2、LCDR3的氨基酸序列,所述HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3、LFR4的氨基酸序列为权利要求4所述的HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3、LFR4的氨基酸序列;可选地,所述重链可变区氨基酸序列如SEQ ID NO:15、24、25、26任一所示;所述轻链可变区氨基酸序列如SEQ ID NO:16、27任一所示。
- 根据权利要求1-5任一所述的抗体或其功能性片段,其特征在于,所述抗体或其功能性片段还包含恒定区;可选地,所述恒定区包括重链恒定区和/或轻链恒定区;可选地,所述重链恒定区选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD的重链恒定区;所述轻链恒定区选自κ型或λ型轻链恒定区;可选地,所述恒定区的种属来源为牛、马、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅或人;可选地,所述恒定区的种属来源为小鼠;可选地,所述重链恒定区序列如SEQ ID NO:17、32任一所示或与其具有至少80%同源性,所述轻链恒定区序列如SEQ ID NO:18所示或与其具有至少80%同源性。
- 根据权利要求1-6任一所述的抗体或其功能性片段,其特征在于,所述功能性片段选自所述抗体的F(ab’)2、Fab’、Fab、Fv和scFv中的任意一种。
- 一种抗新型冠状病毒或其N蛋白的抗体,包括重链和/或轻链,其特征在于,所述重链包括权利要求5所述的重链可变区和权利要求6所述的重链恒定区;所述轻链包括权利要求5所述的轻链可变区和权利要求6所述的轻链恒定区;可选地,所述重链的氨基酸序列如SEQ ID NO:19、28、29、30、33任一所示;所述轻链的氨基酸序列如SEQ ID NO:20、31任一所示。
- 一种抗体偶联物,其特征在于,所述抗体偶联物包括权利要求1-8任一项所述的抗体或其功能性片段;可选地,所述抗体或其功能性片段标记有可被检测的标记物;可选地,所述可被检测的标记物选自荧光染料、催化底物显色的酶、放射性同位素、化学发光试剂和纳米颗粒类标记物;可选地,所述荧光染料选自荧光素类染料及其衍生物、罗丹明类染料及其衍生物、Cy系列染料及其衍生物、Alexa系列染料及其衍生物和蛋白类染料及其衍生物;可选地,所述催化底物显色的酶选自辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶;可选地,所述放射性同位素选自 212Bi、 131I、 111In、 90Y、 186Re、 211At、 125I、 188Re、 153Sm、 213Bi、 32P、 94mTc、 99mTc、 203Pb、 67Ga、 68Ga、 43Sc、 47Sc、 110mIn、 97Ru、 62Cu、 64Cu、 67Cu、 68Cu、 86Y、 88Y、 121Sn、 161Tb、 166Ho、 105Rh、 177Lu、 172Lu和 18F;可选地,所述化学发光试剂选自鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物;可选地,所述纳米颗粒类标记物选自纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒;可选地,所述胶体选自胶体金属、分散型染料、染料标记的微球和乳胶;可选地,所述胶体金属选自胶体金、胶体银和胶体硒;可选地,所述抗体或其功能性片段包被至固相;可选地,所述固相选自微球、板和膜;可选地,所述固相选自磁性微球、塑料微球、塑胶微粒、微孔板、玻璃、毛细管、尼龙和硝酸纤维素膜;可选为磁性微球。
- 一种试剂或试剂盒,其特征在于,所述试剂或试剂盒包括权利要求1-8任一项所述的抗体或其功能性片段或权利要求9所述的抗体偶联物。
- 一种核酸,其特征在于,其编码权利要求1-8任一项所述的抗体或其功能性片段。
- 一种载体,其特征在于,其含有编码权利要求1-8任一项所述的抗体或其功能性片段的核酸片段。
- 一种重组细胞,其特征在于,其含有权利要求12所述的载体。
- 一种制备如权利要求1-8任一项所述的抗体或其功能性片段的方法,其特征在于,其包括:培养权利要求13所述的重组细胞,从培养产物中分离纯化得到所述抗体或其功能性片段。
- 权利要求1-7任一项所述的抗体或其功能性片段,或者权利要求8所述的抗体,或者权利要求9所述的抗体偶联物在制备试剂或试剂盒中的用途,所述试剂或试剂盒用于检测新型冠状病毒或其N蛋白,或者诊断新型冠状病毒感染或与新型冠状病毒感染相关疾病。
- 权利要求1-7任一项所述的抗体或其功能性片段,或者权利要求8所述的抗体,或者权利要求9所述的抗体偶联物,或者权利要求10所述的试剂或试剂盒,用于检测新型冠状病毒或其N蛋白,或者诊断新型冠状病毒感染或与新型冠状病毒感染相关疾病中的用途。
- 根据权利要求15或16所述的用途,其中,所述与新型冠状病毒感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、低氧血症、呼吸困难、肺炎、急性呼吸窘迫综合征、严重急性呼吸综合征、肾衰竭或脓毒症休克。
- 一种诊断受试者在感染新型冠状病毒或与新型冠状病毒感染相关疾病中的方法,包括:A)在足以发生结合反应的条件下,使权利要求1-7任一项所述的抗体或其功能性片段,或者权利要求8所述的抗体,或者权利要求9所述的抗体偶联物,或者权利要求10所述的试剂或试剂盒与来自所述受试者的样品接触以进行结合反应;以及B)检测结合反应产生的免疫复合物;可选地,所述与新型冠状病毒感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、低氧血症、 呼吸困难、肺炎、急性呼吸窘迫综合征、严重急性呼吸综合征、肾衰竭或脓毒症休克。
- 一种检测测试样品中的新型冠状病毒或其N蛋白的方法,其包括:a)在足以发生抗体/抗原结合反应的条件下,使所述测试样品中的新型冠状病毒或其N蛋白抗原与使权利要求1-7任一项所述的抗体或其功能性片段,或者权利要求8所述的抗体,或者权利要求9所述的抗体偶联物,或者权利要求10所述的试剂或试剂盒接触以形成免疫复合物;和b)检测所述免疫复合物的存在,所述复合物的存在指示所述测试样品中所述抗原的存在;可选的,在步骤a)中,所述免疫复合物中还包括第二抗体,所述第二抗体与所述抗体或抗原结合片段结合;可选的,在步骤a)中,所述免疫复合物中还包括第二抗体,所述第二抗体与所述新型冠状病毒或其N蛋白抗原结合。
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