WO2023131317A1 - 一种鉴别新冠突变型抗原的抗体、试剂及方法 - Google Patents

一种鉴别新冠突变型抗原的抗体、试剂及方法 Download PDF

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WO2023131317A1
WO2023131317A1 PCT/CN2023/071102 CN2023071102W WO2023131317A1 WO 2023131317 A1 WO2023131317 A1 WO 2023131317A1 CN 2023071102 W CN2023071102 W CN 2023071102W WO 2023131317 A1 WO2023131317 A1 WO 2023131317A1
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antibody
seq
amino acid
acid sequence
optionally
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PCT/CN2023/071102
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French (fr)
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孟媛
李蔚芝
钟冬梅
游辉
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东莞市朋志生物科技有限公司
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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]
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • 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
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/56983—Viruses
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
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    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • 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 antibody, reagent and method for identifying mutant antigens of the novel 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 a state of acute infection, 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.
  • the present disclosure provides an antibody or a functional fragment thereof, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1, HCDR2, HCDR3 include any one of SEQ ID NO: 15, 27, 34 shown in the heavy
  • the LCDR1, LCDR2, and LCDR3 include LCDR1, LCDR2, and LCDR3 consistent with any one of the light chain variable regions shown in SEQ ID NO: 16, 28, and 35 amino acid sequence.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat, Chothia, IMGT, AbM or Contact system.
  • the antibody or its functional fragments include 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 consists of it;
  • HCDR3 which comprises the amino acid sequence shown in any of SEQ ID NO: 3, 21, 32, or consists of it;
  • LCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 4 or 22, or consists of it;
  • LCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 5 or 23, or consists of it;
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of it.
  • the present disclosure also provides an antibody or a functional fragment thereof, the antibody or a functional fragment thereof comprising 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 consists of it;
  • HCDR3 which comprises the amino acid sequence shown in any of SEQ ID NO: 3, 21, 32, or consists of it;
  • LCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 4 or 22, or consists of it;
  • LCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 5 or 23, or consists of it;
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of it.
  • the antibody or functional fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.
  • the antibody or its functional fragments also have the following framework regions:
  • the HFR1 amino acid sequence is shown in SEQ ID NO: 7, or has at least 80% homology therewith;
  • amino acid sequence of HFR2 is as shown in any of SEQ ID NO:8, 24, 33 or has at least 80% homology therewith;
  • the HFR3 amino acid sequence is as shown in SEQ ID NO: 9 or 25 or has at least 80% homology therewith;
  • the HFR4 amino acid sequence is as shown in SEQ ID NO: 10 or 26 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 to the non-Omicron novel coronavirus N antigen with an affinity of K D ⁇ 10 ⁇ 7 M.
  • the antibody or its functional fragments include HCDR3 shown in SEQ ID NO:3 and LCDR1 and LCDR2 shown in SEQ ID NO:4,5.
  • the antibody or its functional fragments include HCDR3 shown in SEQ ID NO: 21 and LCDR1 and LCDR2 shown in SEQ ID NO: 22 and 23.
  • the antibody or its functional fragments include HCDR3 shown in SEQ ID NO:32 and LCDR1 and LCDR2 shown in SEQ ID NO:4 and 23.
  • the antibody or a functional fragment thereof comprises an amino acid sequence such as a heavy chain variable region shown in any of SEQ ID NOs: 15, 27, and 34, and an amino acid sequence such as any of SEQ ID NOs: 16, 28, or 35.
  • a light chain variable region is indicated.
  • the present disclosure provides an antibody or a functional fragment thereof, the antibody or a functional fragment thereof comprising a heavy chain variable region and/or a light chain variable region, the amino acid sequence of the heavy chain variable region is as SEQ ID NO : shown in any of 15, 27, 34; the amino acid sequence of the light chain variable region is shown in any of SEQ ID NO: 16, 28, 35.
  • the present disclosure provides an antibody or a functional fragment thereof, the antibody or a functional fragment thereof comprising a heavy chain variable region and/or a light chain variable region, the heavy chain variable region containing HFR1-HCDR1-HFR2-
  • the sequence structure of HCDR2-HFR3-HCDR3-HFR4 contains the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3
  • the amino acid sequence is the amino acid sequence of the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and the amino acid sequence of the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4 is the above-mentioned HFR1, HFR2, HFR3, Amino acid sequences of HFR4, LFR1, LFR2, LFR3, LFR4.
  • the amino acid sequence of the heavy chain variable region is as shown in any of SEQ ID NO: 15, 27, 34; the amino acid sequence of the light chain variable region is as shown in any of SEQ ID NO: 16, 28, 35 one shown.
  • 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, donkey, deer, mink, chicken, duck, goose or human.
  • the species source of the constant region is mouse.
  • the sequence of the heavy chain constant region is as shown in SEQ ID NO: 17 or has at least 80% homology therewith, and the sequence of the light chain constant region is as shown in SEQ ID NO: 18 or 29 or with it have at least 80% homology.
  • the antibody or a functional fragment thereof comprises a heavy chain having an amino acid sequence as shown in any of SEQ ID NO: 19, 30, 36, and an amino acid sequence as shown in any of SEQ ID NO: 20, 31, 37 light chain.
  • 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, comprising a heavy chain and/or a light chain, the heavy chain comprising the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; the light chain comprising the above-mentioned light chain variable region and the light chain constant region described above.
  • amino acid sequence of the heavy chain is shown in any of SEQ ID NO:19, 30, 36; the amino acid sequence of the light chain is shown in any of SEQ ID NO: 20, 31, 37.
  • the present disclosure also provides an antibody, comprising a heavy chain and/or a light chain, the amino acid sequence of the heavy chain is as shown in any of SEQ ID NO: 19, 30, 36; the amino acid sequence of the light chain is as shown in SEQ ID NO: any one of 20, 31, 37.
  • the antibody or a functional fragment thereof binds to a non-Omicron novel coronavirus N antigen.
  • 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 label.
  • the label is selected from fluorescent dyes, enzymes, 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 is selected from horseradish peroxidase, alkaline phosphatase, beta-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 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 functional fragment thereof according to any one of the above, which comprises: culturing the recombinant cells.
  • the present disclosure provides a reagent or a kit, which includes the above-mentioned antibody or a functional fragment thereof or the above-mentioned antibody conjugate.
  • the reagent or kit is a reagent or kit for detecting non-Omicron novel coronaviruses or identifying new crown mutants.
  • the novel coronavirus mutant is an Omicron mutant.
  • the present disclosure also provides a method for identifying new coronavirus mutant antigens, using antibody 1, antibody 2 and antibody 3 to carry out immunodetection on the sample to be tested, the antibody 1 can bind to M new coronavirus antigens; the antibody 2 can Combining with M-1 novel coronavirus antigens except Omicron mutant strains, the antibody 2 is selected from the above-mentioned antibodies; the antibody 3 can bind to M novel coronavirus antigens, and M is an integer greater than or equal to 2.
  • the sandwich of antibody 1 and antibody 3 detects the novel coronavirus antigen; the sandwich of antibody 2 and antibody 3 detects the novel coronavirus antigen.
  • novel coronavirus antigen is novel coronavirus N antigen.
  • the method is selected from immunochromatography, enzyme-linked immunosorbent assay, chemiluminescent method, latex immunoturbidimetric method.
  • the present disclosure also provides an immunochromatographic test paper
  • the immunochromatographic test paper includes a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and a water-absorbing pad, and the detection line 1 and the detection line 2 are arranged on the nitrocellulose membrane , the above-mentioned antibody 3 is set on the binding pad, the above-mentioned antibody 1 is coated on the detection line 1, and the above-mentioned antibody 2 is coated on the detection line 2; the antibody 3 is labeled with a label.
  • the immunochromatographic test paper is an immunochromatographic test paper for identifying mutant antigens of the novel coronavirus.
  • a quality control line is also set on the nitrocellulose membrane.
  • the disclosure also provides the above-mentioned antibody or its functional fragment, the above-mentioned antibody conjugate, the above-mentioned reagent or kit, the above-mentioned method or the above-mentioned immunochromatographic test paper in the detection of Omicron mutant strains or their N proteins, and the detection of non-Omicron novel Application of coronavirus or its N protein and identification of new coronavirus mutants.
  • the novel coronavirus mutant is an Omicron mutant.
  • the present disclosure also provides a method for diagnosing a subject infected with a new coronavirus or a disease related to a new coronavirus infection, including:
  • the antibody 1 can bind to M novel coronavirus antigens; the antibody 2 can bind to M-1 novel coronavirus antigens except the Omicron mutant, and the antibody 2 is selected from the above-mentioned antibodies or functional fragments thereof ;
  • the antibody 3 can be combined with M novel coronavirus antigens, and M is an integer greater than or equal to 2.
  • the sandwich of antibody 1 and antibody 3 detects the novel coronavirus antigen; the sandwich of antibody 2 and antibody 3 detects the novel coronavirus antigen.
  • novel coronavirus antigen is novel coronavirus N antigen.
  • the method is selected from immunochromatography, enzyme-linked immunosorbent assay, chemiluminescent method, latex immunoturbidimetric method.
  • Fig. 1 is the result of reducing SDS-PAGE of the 19E3 monoclonal antibody of Example 1.
  • 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 active.
  • 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 regions of major amino acid residues that contribute to the binding affinity of an antibody or 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 represented by HCDR, which includes HCDR1, HCDR2 and HCDR3;
  • the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.
  • Embodiments of the present disclosure provide an antibody or a functional fragment thereof, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3 including any one of SEQ ID NO: 15, 27, 34
  • the amino acid sequence consistent with HCDR1, HCDR2, and HCDR3 of the heavy chain variable region; LCDR1, LCDR2, and LCDR3 include those consistent with LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in any one of SEQ ID NO: 16, 28, and 35 amino acid sequence.
  • CDR notation methods commonly used in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the 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. The accumulation of sequences over the past few decades has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues.
  • CDR CDR sequences defined by conventional systems in the art are within the protection scope of the present application.
  • the CDR definition method is referred to such as Kabat et al., U.S.Dept.of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) or Chothia et al., J Mol Biol 196:901-917 (1987).
  • Exemplary defined CDRs are listed in Table 1 below. Given the variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues comprise a particular CDR.
  • Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody.
  • One of ordinary skill in the art can unambiguously map this Kabat numbering system to any variable region sequence, without reliance on any experimental data other than the sequence itself.
  • Kabat numbering refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest” (1983).
  • the polypeptide sequences in the sequence listing are not numbered according to the Kabat numbering system. However, those of ordinary skill in the art are fully able to convert the sequence numbers of the sequence listing into Kabat numbers.
  • HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by Kabat, Chothia, IMGT, AbM or Contact system.
  • HCDR1, HCDR2, and HCDR3 sequentially comprise amino acid sequences at positions 31-35, 50-65, and 95-97 of the heavy chain variable region, or sequentially such as 31-35 of the heavy chain variable region. Position, 50-65, 95-97 amino acid sequence;
  • LCDR1, LCDR2 and LCDR3 sequentially comprise the 24-34, 50-56, 89-95 amino acid sequences of the light chain variable region, or such as the 24-34, 50-56, 89 ⁇ 95th amino acid sequence; and, the numbering of the amino acid positions is based on the Kabat numbering system.
  • HCDR1, HCDR2, and HCDR3 sequentially comprise amino acid sequences at positions 31-35, 50-65, and 95-98 of the heavy chain variable region, or sequentially such as 31-35 of the heavy chain variable region. position, 50-65 and 95-98 amino acid sequences;
  • LCDR1, LCDR2 and LCDR3 sequentially comprise the 24-34, 50-56, 89-95 amino acid sequences of the light chain variable region, or such as the 24-34, 50-56, 89 ⁇ 95th amino acid sequence; and, the numbering of the amino acid positions is based on the Kabat numbering system.
  • the antibody or functional fragment thereof comprises 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 consists of it;
  • HCDR3 which comprises the amino acid sequence shown in any of SEQ ID NO: 3, 21, 32, or consists of it;
  • LCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 4 or 22, or consists of it;
  • LCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 5 or 23, or consists of it;
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of it.
  • Embodiments of the present disclosure also provide an antibody or a functional fragment thereof, the antibody or a functional fragment thereof comprising 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 consists of it;
  • HCDR3 which comprises the amino acid sequence shown in any of SEQ ID NO: 3, 21, 32, or consists of it;
  • LCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 4 or 22, or consists of it;
  • LCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 5 or 23, 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 include HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 1-3 and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 4-6.
  • the antibody or its functional fragments include HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 1, 2, 21 and LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 22, 23, 6 .
  • the antibody or its functional fragments include HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 1, 2, 32 and LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 4, 23, 6 .
  • framework region or "FR” region includes the heavy chain framework region and the light chain framework region, and refers to the region except the CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein, heavy Chain framework regions can be further subdivided into contiguous regions separated by CDRs, comprising HFR1, HFR2, HFR3, and HFR4 framework regions; light chain framework regions can be further subdivided into contiguous regions separated by CDRs, comprising LFR1 , LFR2, LFR3 and LFR4 framework regions.
  • the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is composed of the following numbered CDRs and FRs Arrange and connect as follows: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
  • the antibody or a functional fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.
  • the antibody or functional fragment thereof further has the following framework regions:
  • the HFR1 amino acid sequence is shown in SEQ ID NO: 7, or has at least 80% homology therewith;
  • amino acid sequence of HFR2 is as shown in any of SEQ ID NO:8, 24, 33 or has at least 80% homology therewith;
  • the HFR3 amino acid sequence is as shown in SEQ ID NO: 9 or 25 or has at least 80% homology therewith;
  • the HFR4 amino acid sequence is as shown in SEQ ID NO: 10 or 26 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 amino acid sequence of each framework region of the antibody or its functional fragment provided by the present disclosure may have at least 80%, 81%, 82%, 83%, 84% of the above-mentioned corresponding framework region , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.
  • the antibody or functional fragment thereof binds to the non-Omicron novel coronavirus N antigen with an affinity of K D ⁇ 10 -7 M, for example, an affinity of K D ⁇ 4.90 ⁇ 10 -8 M.
  • the antibody or functional fragment thereof binds to the non-Omicron novel coronavirus N antigen with an affinity of KD ⁇ 10 -8 M, for example, binds with an affinity of KD ⁇ 4.02 ⁇ 10 -9 M.
  • the detection of KD is carried out with reference to the methods in the embodiments and examples of the present disclosure.
  • the antibody or a functional fragment thereof comprises an amino acid sequence such as a heavy chain variable region shown in any one of SEQ ID NO: 15, 27, 34, and an amino acid sequence such as SEQ ID NO: 16, 28, 35 any one of the indicated light chain variable regions.
  • Embodiments of the present disclosure also provide an antibody or a functional fragment thereof, the antibody or a functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the amino acid sequence of the heavy chain variable region is as SEQ ID NO: 15, 27, 34 shown in any one; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO: 16, 28, 35.
  • Embodiments of the present disclosure also provide an antibody or a functional fragment thereof, the antibody or a functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain variable region contains HFR1-HCDR1-HFR2-HCDR2 - the sequence structure of HFR3-HCDR3-HFR4, the light chain variable region region contains the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as above
  • the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, the amino acid sequence of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4 is the above-mentioned HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, Amino acid sequence of LFR4.
  • amino acid sequence of the heavy chain variable region is as shown in any of SEQ ID NO: 15, 27, 34.
  • amino acid sequence of the light chain variable region is as shown in any of SEQ ID NO: 16, 28, or 35.
  • the antibody 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, 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, donkey, deer, mink, chicken, duck, goose , Turkey, Fighting Cock Or Man.
  • the species source of the constant region is mouse.
  • sequence of the heavy chain constant region (CH) is shown in SEQ ID NO: 17
  • sequence of the light chain constant region (CL) is shown in SEQ ID NO: 18 or 29.
  • the constant region sequence may 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 antibody or a functional fragment thereof comprises an amino acid sequence such as a heavy chain shown in any of SEQ ID NO: 19, 30, 36, and an amino acid sequence such as SEQ ID NO: 20, 31, 37 Any of the light chains indicated.
  • 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.
  • an embodiment of the present disclosure also provides an antibody, including a heavy chain and/or a light chain, the heavy chain includes the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; the light chain includes 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, 30, 36; the amino acid sequence of the light chain is as shown in any of SEQ ID NO: 20, 31, 37.
  • Embodiments of the present disclosure also provide an antibody, including a heavy chain and/or a light chain, the amino acid sequence of the heavy chain is as shown in any of SEQ ID NO: 19, 30, and 36; the amino acid sequence of the light chain is as shown in SEQ ID NO: 20 , 31, 37 as shown in any one.
  • the antibody or functional fragment thereof binds to a non-Omicron novel coronavirus N antigen.
  • the embodiment of the present disclosure also provides 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 label.
  • a marker refers to a class of substances that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc. qualitative or quantitative detection.
  • labels include, but are not limited to, fluorescent dyes, enzymes, 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 (such as 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 include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate 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 antibodies or functional fragments thereof in the above-mentioned antibody conjugates are 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.
  • An embodiment of the present disclosure also provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.
  • Embodiments of the present disclosure also provide vectors containing the above-mentioned nucleic acid molecules.
  • Embodiments of the present disclosure also provide recombinant cells containing the above-mentioned vectors.
  • An embodiment of the present disclosure also provides a method for preparing an antibody or a functional fragment thereof, which includes: culturing the above-mentioned recombinant cells.
  • 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 culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described in any one of the above. Based on this, no matter Which technology is used to prepare the antibody or its functional fragments of the present disclosure belongs to the protection scope of the present disclosure.
  • 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.
  • the reagent or kit is a reagent or kit for detecting non-Omicron novel coronaviruses or identifying new crown mutants.
  • the new coronavirus mutant is an Omicron mutant.
  • Embodiments of the present disclosure also provide a method for identifying new coronavirus mutant antigens.
  • Antibody 1, antibody 2 and antibody 3 are used to perform immunodetection on the sample to be tested.
  • Antibody 1 can bind to M new coronavirus antigens;
  • Antibody 2 is selected from the above-mentioned antibodies for binding to M-1 novel coronavirus antigens outside the mutant strain;
  • antibody 3 can bind to M novel coronavirus antigens, and M is an integer greater than or equal to 2.
  • the "M kinds of new coronavirus antigens” in this disclosure include antigens of wild strains and mutant new coronaviruses, wherein the mutants are not limited to Omicron mutants, but can also be B.1.1.7 mutants, B1.351 mutants or B. 1.1.28 mutant strains.
  • the "M-1 new coronavirus antigens other than Omicron mutant strains” mentioned in this disclosure include wild strains, and optionally other mutant strains other than Omicron mutant strains, such as B.1.1.7 mutant strains, B1.351 Mutant strain or B.1.1.28 mutant strain.
  • antibody 1 and antibody 3 are sandwiched to detect novel coronavirus antigens; antibody 2 and antibody 3 are sandwiched to detect novel coronavirus antigens.
  • the SARS-CoV-2 antigen described here is not limited to the N antigen, or it can also be the S antigen, not limited to the wild strain, or a mutant strain. In some embodiments, the SARS-CoV-2 antigen is the SARS-CoV-2 antigen N antigen.
  • the novel coronavirus antigen is the N antigen of the novel coronavirus.
  • the method is selected from immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, latex immunoturbidimetry.
  • the embodiment of the present disclosure also provides an immunochromatographic test paper.
  • the immunochromatography test paper includes a bottom plate, a sample pad, a binding pad, a nitrocellulose membrane and an absorbent pad.
  • the detection line 1 and the detection line 2 are arranged on the nitrocellulose membrane, the above-mentioned antibody 3 is arranged on the binding pad, and the detection line 1 is coated with The above-mentioned antibody 1 and the detection line 2 are coated with the above-mentioned antibody 2; the antibody 3 is labeled with a marker.
  • the immunochromatographic test paper is brought into contact with the sample to be tested, and when the detection line 1 and the detection line 2 develop color at the same time or the detection line 1 and the detection line 2 have basically the same color (for example, the difference is within 1 color card), then it is determined
  • the sample does not contain the new coronavirus Omicron mutant strain; when the detection line 1 develops color and the detection line 2 does not develop color, or there is a significant difference in the color development between detection line 1 and 2 (for example, the difference is more than 3 color cards), it is determined to be a sample Contains the new coronavirus Omicron mutant strain.
  • the immunochromatographic test paper is an immunochromatographic test paper for identifying mutant antigens of the new coronavirus.
  • a quality control line is also provided on the nitrocellulose membrane.
  • Embodiments of the present disclosure also provide the above-mentioned antibodies or functional fragments thereof, antibody conjugates, reagents or kits, methods or test papers in the detection of Omicron mutant strains or their N proteins, and the detection of non-Omicron novel coronaviruses or their N proteins. Application in protein and identification of new crown mutants.
  • the novel coronavirus mutant is an Omicron mutant.
  • Embodiments of the present disclosure also provide the above-mentioned antibody or its functional fragment, antibody conjugate, reagent or kit, method or test paper in the preparation and detection of Omicron mutant strain or its N protein, detection of non-Omicron new coronavirus or its Application of N protein and identification of new crown mutant products.
  • Embodiments of the present disclosure also provide applications of the above-mentioned antibodies or functional fragments thereof, antibody conjugates, the above-mentioned reagents or kits, methods, or immunochromatographic test papers in the detection of Omicron mutant strains or the preparation of detection products for Omicron mutant strains.
  • 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:
  • Antibody 1 can be combined with M new coronavirus antigens; said antibody 2 can be combined with M-1 new coronavirus antigens except Omicron mutant strains, and said antibody 2 is selected from the above-mentioned antibodies or their functional Fragment; the antibody 3 can bind to M novel coronavirus antigens, and M is an integer greater than or equal to 2.
  • the sandwich of antibody 1 and antibody 3 detects the novel coronavirus antigen; the sandwich of antibody 2 and antibody 3 detects the novel coronavirus antigen.
  • the novel coronavirus antigen is novel coronavirus N antigen.
  • the method is selected from immunochromatography, enzyme-linked immunosorbent assay, chemiluminescent method, and latex immunoturbidimetric method.
  • 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 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 KABAT 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 gene amplified by the light chain
  • the VL gene sequence is 333bp (19E3), 333bp (2F18), 333bp (12H87), and there is a 57bp leader peptide sequence in front of it; in the gene fragment amplified by the heavy chain primer pair, the VH gene sequence is 342bp (19E3 ), 345bp (2F18), 345bp (12H87), belonging to the VH1 gene family, with a 57bp leader peptide sequence in front of it.
  • the heavy and light chain sequences of 19E3 are shown in SEQ ID NO: 19 and 20 respectively, the heavy and light chain sequences of 2F18 are shown in SEQ ID NO: 30 and 31 respectively, and the heavy and light chain sequences of 12H87 are shown in SEQ ID NO: 36 respectively , 37).
  • the vector is a recombinant antibody eukaryotic expression vector constructed.
  • the expression vector has introduced multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named as pcDNA3.4A expression vector, which will be referred to as 3.4A expression vector subsequently; according to the above
  • the VL and VH gene-specific primers of the antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, and 0.72kb was amplified by PCR amplification method The light chain gene fragment and the 1.40kb 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.
  • Dilute 2019-nCoVN protein (purchased from Fapon) to 3 ⁇ g/mL in coating solution, 100 ⁇ L per well, overnight at 4°C; the next day, wash twice with washing solution and pat dry; add blocking solution (20% BSA+80% PBS ), 120 ⁇ L per well, 37°C, 1h, and pat dry; add diluted cell supernatant, 100 ⁇ L/well, 37°C, 30min (partial supernatant 1h); wash 5 times with washing solution, pat dry; add goat anti-mouse IgG-HRP, 100 ⁇ L per well, 37°C, 30 min; wash with washing solution 5 times, and pat dry; Color solution B (50 ⁇ L/well, containing citric acid + EDTA 2Na + TMB + concentrated HCL), 10 min; add stop solution (50 ⁇ L/well, containing EDTA 2Na + concentrated H 2 SO 4 ); Read the OD value at 630nm).
  • the cells were first cultured in a 125mL shake 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 rotation 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 using a protein A affinity column. Take 6.6 ⁇ g of the purified antibody for reducing SDS-PAGE, and the electropherogram is shown in Figure 1.
  • Dilute 2019-nCoVN protein (purchased from Fapon) to 3 ⁇ g/mL in coating solution (main component NaHCO 3 ) for microplate coating, 100 ⁇ L per well, overnight at 4°C; the next day, wash twice with washing solution and pat dry ;
  • the purified antibody was diluted to 10 ⁇ g/mL with PBST, and the 2019-nCoVN protein (purchased from Fapon) was serially diluted with PBST.
  • 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.
  • the labeled antibody in this example is 15C8 or Mb117 (purchased from Fipeng), the T1 line coating antibody is Mb30 (purchased from Fipeng), and 15C8, Mb117 or Mb30 can all bind to the N antigen of the new coronavirus including the Omicron mutant strain ;
  • the T2 line coating antibody is the antibody 19E3, 2F18 or 12H87 obtained in Example 1.
  • Test items New coronavirus Omicron mutant N antigen, and new coronavirus wild strain N antigen (2019-nCoVN);
  • the 19E3, 2F18 or 12H87 antibody does not react to the N antigen of the Omicron mutant strain, but reacts to the N antigen of the wild strain of the new coronavirus, so that the rapid identification of the Omicron mutant strain can be realized.
  • the present disclosure provides an antibody, a reagent and a method for identifying a mutant antigen of the new crown, and the antibody, reagent or method of the disclosure can quickly and accurately identify the mutant antigen of a new crown, and the detection efficiency is high and the cost is low, so they all have excellent Practical performance.

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Abstract

本公开提供了一种鉴别新冠突变型抗原的抗体、试剂及方法。利用本公开抗体、试剂或方法能够快速、准确地鉴别新冠突变型抗原，且检测效率高、成本低。

Description

一种鉴别新冠突变型抗原的抗体、试剂及方法
相关申请的交叉引用
本公开要求于2022年01月10日提交中国专利局的申请号为CN202210021930.X、名称为“一种鉴别新冠突变型抗原的抗体、试剂及方法”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。
技术领域
本公开涉及抗体技术领域，具体而言，涉及一种鉴别新冠突变型抗原的抗体、试剂及方法。
背景技术
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。
2021年11月在南非发现一种新冠病毒突变株，世界卫生组织将该新冠病毒B.1.1.529变异株命名为Omicron(奥密克戎)。因其病毒量大、传播力高的特点。因此，针对该变异株的早筛早查极为重要。目前市场上针对新冠突变型检测试剂极少，尤其是Omicron突变株的检测。
发明内容
本公开提供了一种抗体或其功能性片段，包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，所述HCDR1、HCDR2、HCDR3包括与SEQ ID NO:15、27、34任一所示的重链可变区的HCDR1、HCDR2、HCDR3一致的氨基酸序列；所述LCDR1、LCDR2、LCDR3包括与SEQ ID NO:16、28、35任一所示的轻链可变区的LCDR1、LCDR2、LCDR3一致的氨基酸序列。
可选地，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由Kabat、Chothia、IMGT、AbM或Contact系统定义。
可选地，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所示的氨基酸序列，或由其组成；
HCDR3，其包含SEQ ID NO:3、21、32任一所示的氨基酸序列，或由其组成；
LCDR1，其包含SEQ ID NO:4或22所示的氨基酸序列，或由其组成；
LCDR2，其包含SEQ ID NO:5或23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。
本公开还提供了一种抗体或其功能性片段，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所示的氨基酸序列，或由其组成；
HCDR3，其包含SEQ ID NO:3、21、32任一所示的氨基酸序列，或由其组成；
LCDR1，其包含SEQ ID NO:4或22所示的氨基酸序列，或由其组成；
LCDR2，其包含SEQ ID NO:5或23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HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3和LFR4中的至少之一。
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所示，或与其具有至少80％同源性；
HFR2氨基酸序列如SEQ ID NO:8、24、33任一所示或与其具有至少80％同源性；
HFR3氨基酸序列如SEQ ID NO:9或25所示或与其具有至少80％同源性；
HFR4氨基酸序列如SEQ ID NO:10或26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 -7M的亲和力结合非Omicron新型冠状病毒N抗原。
可选地，所述抗体或其功能性片段包括SEQ ID NO:3所示的HCDR3及SEQ ID NO:4、5所示的LCDR1、LCDR2。
可选地，所述抗体或其功能性片段包括SEQ ID NO:21所示的HCDR3及SEQ ID NO:22、23所示的LCDR1、LCDR2。
可选地，所述抗体或其功能性片段包括SEQ ID NO:32所示的HCDR3及SEQ ID NO:4、23所示的LCDR1、LCDR2。
可选地，所述抗体或其功能性片段包括氨基酸序列如SEQ ID NO:15、27、34任一所示的重链可变区，和氨基酸序列如SEQ ID NO:16、28、35任一所示的轻链可变区。
本公开提供了一种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，所述重链可变区的氨基酸序列如SEQ ID NO:15、27、34任一所示；所述轻链可变区的氨基酸序列如SEQ ID NO:16、28、35任一所示。
本公开提供了一种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、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，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，所述重链可变区的氨基酸序列如SEQ ID NO:15、27、34任一所示；所述轻链可变区的氨基酸序列如SEQ ID NO:16、28、35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、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，所述重链恒定区的序列如SEQ ID NO:17所示或与其具有至少80％同源性，所述轻链恒定区的序列如SEQ ID NO:18或29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包括氨基酸序列如SEQ ID NO:19、30、36任一所示的重链，和氨基酸序列如SEQ ID NO:20、31、37任一所示的轻链。
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、30、36任一所示；所述轻链的氨基酸序列如SEQ ID NO:20、31、37任一所示。
本公开还提供了一种抗体，包括重链和/或轻链，所述重链的氨基酸序列如SEQ ID NO:19、30、36任一所示；所述轻链的氨基酸序列如SEQ ID NO:20、31、37任一所示。
可选地，所述抗体或其功能性片段结合非Omicron新型冠状病毒N抗原。
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标记物。
可选地，所述标记物选自荧光染料、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。
本公开还提供一种核酸，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。
本公开还提供一种重组细胞，其含有所述载体。
本公开还提供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。本公开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。
可选地，所述试剂或试剂盒为检测非Omicron新型冠状病毒或鉴别新冠突变型的试剂或试剂盒。
可选地，所述新冠突变型为Omicron突变株。
本公开还提供了一种鉴别新冠突变型抗原的方法，利用抗体1、抗体2和抗体3对待测样本进行免疫检测，所述抗体1能与M种新型冠状病毒抗原结合；所述抗体2能与除Omicron突变株外的M-1种新型冠状病毒抗原结合，所述抗体2选自上述的抗体；所述抗体3能与M种新型冠状病毒抗原结合，M为大于等于2的整数。
可选地，所述抗体1和抗体3夹心检测新型冠状病毒抗原；所述抗体2和抗体3夹心检测新型冠状病毒抗原。
可选地，所述新型冠状病毒抗原为新型冠状病毒N抗原。
可选地，所述方法选自免疫层析法、酶联免疫吸附法、化学发光法、胶乳免疫比浊法。
本公开还提供了一种免疫层析试纸，所述免疫层析试纸包括底板、样品垫、结合垫、硝酸纤维素膜和吸水垫，所述硝酸纤维素膜上设置检测线1和检测线2，所述结合垫上设置有上述的抗体3，所述检测线1上包被上述的抗体1、所述检测线2上包被上述的抗体2；所述抗体3用标记物进行标记。
可选地，所述免疫层析试纸为鉴别新冠突变型抗原的免疫层析试纸。
可选地，所述硝酸纤维素膜上还设置有质控线。
本公开还提供了一种上述的抗体或其功能性片段、上述抗体偶联物、上述试剂或试剂盒、上述方法或上述免疫层析试纸在检测Omicron突变株或其N蛋白、检测非Omicron新型冠状病毒或其N蛋白、鉴别新冠突变型中的应用。
可选地，所述新冠突变型为Omicron突变株。
本公开还提供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，使利用抗体1、抗体2和抗体3与来自所述受试者的样品接触以进行免疫检测；以及
b)检测结合反应产生的免疫复合物；
所述抗体1能与M种新型冠状病毒抗原结合；所述抗体2能与除Omicron突变株外的M-1种新型冠状病毒抗原结合，所述抗体2选自上述的抗体或其功能性片段；所述抗体3能与M种新型冠状病毒抗原结合，M为大于等于2的整数。
可选地，所述抗体1和抗体3夹心检测新型冠状病毒抗原；所述抗体2和抗体3夹心检测新型冠状病毒抗原。
可选地，所述新型冠状病毒抗原为新型冠状病毒N抗原。
可选地，所述方法选自免疫层析法、酶联免疫吸附法、化学发光法、胶乳免疫比浊法。
附图说明
为了更清楚地说明本公开实施方式和实施例的技术方案，下面将对实施方式和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方式和实施例，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19E3单克隆抗体的还原性SDS-PAGE的结果。
具体实施方式
在本公开中，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。
在本公开中，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。
在本公开中，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。
本公开的实施方式提供了一种抗体或其功能性片段，包括HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3，HCDR1、HCDR2、HCDR3包括与SEQ ID NO:15、27、34任一所示的重链可变区的HCDR1、HCDR2、HCDR3一致的氨基酸序列；LCDR1、LCDR2、LCDR3包括与SEQ ID NO:16、28、35任一所示的轻链可变区的LCDR1、LCDR2、LCDR3一致的氨基酸序列。
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。
对CDR进行定义的系统不受特别限制，本领域常规系统定义的CDR序列均在本申请的保护范围之内。例如CDR定义方法参见如Kabat等，U.S.Dept.of Health and Human Services,Sequences of Proteins of Immunological Interest(1983)或Chothia等，J Mol Biol 196：901-917(1987)。示例性的定义的CDR列于下表1中。在给定抗体的可变区氨基酸序列的情况下，本领域技术人员可以常规地确定哪些残基包含特定CDR。
表1：CDR定义 1
CDR Kabat AbM 2 IMGT
HCDR1 31-35 26-35 26-35
HCDR2 50-65 50-58 51-56
HCDR3 95-102 95-102 93-102
LCDR1 24-34 24-34 27-32
LCDR2 50-56 50-56 50-51
LCDR3 89-97 89-97 89-97
1表1中所有CDR定义的编号是依据Kabat编号系统(参见下文)。
2如表1中使用的“AbM”具有小写“b”，是指通过Oxford Molecular的“AbM”抗体建模软件定义的CDR。
Kabat等还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以明确地将该Kabat编号系统对应到任何可变区序列，而不依赖于序列本身之外的任何实验数据。如本文所述，“Kabat编号”是指Kabat等，U.S.Dept.of Health and HumanServices，“Sequence of Proteins of Immunological Interest”(1983)所述的编号系统。序列表中的多肽序列未根据Kabat编号系统编号。然而，本领域普通技术人员完全能够将序列表的序列编号转换为Kabat编号。
在可选的实施方式中，HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3(简称CDRs)由Kabat、Chothia、IMGT、AbM或Contact系统定义。
在可选的实施方式中，HCDR1、HCDR2和HCDR3依次包含重链可变区的31～35位、50～65位、95～97位氨基酸序列，或依次如重链可变区的31～35位、50～65位、95～97位氨基酸序列所示；
LCDR1、LCDR2和LCDR3依次包含轻链可变区的24～34位、50～56位、89～95位氨基酸序列，或依次如轻链可变区的24～34位、50～56位、89～95位氨基酸序列所示；且，所述氨基酸位点编号是依据Kabat编号系统。
在可选的实施方式中，HCDR1、HCDR2和HCDR3依次包含重链可变区的31～35位、50～65位、95～98位氨基酸序列，或依次如重链可变区的31～35位、50～65位、95～98位氨基酸序列所示；
LCDR1、LCDR2和LCDR3依次包含轻链可变区的24～34位、50～56位、89～95位氨基酸序列，或依次如轻链可变区的24～34位、50～56位、89～95位氨基酸序列所示；且，所述氨基酸位点编号是依据 Kabat编号系统。
在可选的实施方式中，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所示的氨基酸序列，或由其组成；
HCDR3，其包含SEQ ID NO:3、21、32任一所示的氨基酸序列，或由其组成；
LCDR1，其包含SEQ ID NO:4或22所示的氨基酸序列，或由其组成；
LCDR2，其包含SEQ ID NO:5或23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。
本公开的实施方式还提供了一种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所示的氨基酸序列，或由其组成；
HCDR3，其包含SEQ ID NO:3、21、32任一所示的氨基酸序列，或由其组成；
LCDR1，其包含SEQ ID NO:4或22所示的氨基酸序列，或由其组成；
LCDR2，其包含SEQ ID NO:5或23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。
在可选的实施方式中，抗体或其功能性片段包括SEQ ID NO:1-3所示的HCDR1、HCDR2、HCDR3及SEQ ID NO:4-6所示的LCDR1、LCDR2、LCDR3。
在可选的实施方式中，抗体或其功能性片段包括SEQ ID NO:1、2、21所示的HCDR1、HCDR2、HCDR3及SEQ ID NO:22、23、6所示的LCDR1、LCDR2、LCDR3。
在可选的实施方式中，抗体或其功能性片段包括SEQ ID NO:1、2、32所示的HCDR1、HCDR2、HCDR3及SEQ ID NO:4、23、6所示的LCDR1、LCDR2、LCDR3。
在本公开中，“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和轻链可变区中除CDR之外的区域；其中，重链框架区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。
在本公开中，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。
在可选的实施方式中，抗体或其功能性片段还具有HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3和LFR4中的至少之一。
在可选的实施方式中，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所示，或与其具有至少80％同源性；
HFR2氨基酸序列如SEQ ID NO:8、24、33任一所示或与其具有至少80％同源性；
HFR3氨基酸序列如SEQ ID NO:9或25所示或与其具有至少80％同源性；
HFR4氨基酸序列如SEQ ID NO:10或26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。
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。
在可选的实施方式中，抗体或其功能性片段以K D≤10 -7M的亲和力结合非Omicron新型冠状病毒N抗原，例如以K D≤4.90×10 -8M的亲和力结合。
在可选的实施方式中，抗体或其功能性片段以KD≤10 -8M的亲和力结合非Omicron新型冠状病毒N抗原，例如以KD≤4.02×10 -9M的亲和力结合。
K D的检测参考本公开实施方式和实施例中的方法进行。
在可选的实施方式中，抗体或其功能性片段包括氨基酸序列如SEQ ID NO:15、27、34任一所示的重链可变区，和氨基酸序列如SEQ ID NO:16、28、35任一所示的轻链可变区。
本公开实施方式还提供了一种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，重链可变区的氨基酸序列如SEQ ID NO:15、27、34任一所示；轻链可变区的氨基酸序列如SEQ ID NO:16、28、35任一所示。
本公开实施方式还提供了一种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、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，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，重链可变区的氨基酸序列如SEQ ID NO:15、27、34任一所示。
在可选的实施方式中，轻链可变区的氨基酸序列如SEQ ID NO:16、28、35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。
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、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，重链恒定区的序列(CH)如SEQ ID NO:17所示，轻链恒定区(CL)的序列如SEQ ID NO:18或29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。
在可选的实施方式中，所述抗体或其功能性片段包括氨基酸序列如SEQ ID NO:19、30、36任一所示的重链，和氨基酸序列如SEQ ID NO:20、31、37任一所示的轻链。
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。本公开实施方式还提供一种抗体，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、30、36任一所示；轻链的氨基酸序列如SEQ ID NO:20、31、37任一所示。
本公开实施方式还提供一种抗体，包括重链和/或轻链，重链的氨基酸序列如SEQ ID NO:19、30、36任一所示；轻链的氨基酸序列如SEQ ID NO:20、31、37任一所示。
在可选的实施方式中，抗体或其功能性片段结合非Omicron新型冠状病毒N抗原。
本公开实施方式还提供了一种抗体偶联物，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标记物。
在可选的实施方式中，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，标记物包括但不限于荧光染料、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，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。
本公开实施方式还提供含有上述核酸分子的载体。
本公开实施方式还提供含有上述载体的重组细胞。
本公开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：培养如上所述的重组细胞。
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任一项所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。
本公开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。
在可选的实施方式中，试剂或试剂盒为检测非Omicron新型冠状病毒或鉴别新冠突变型的试剂或试剂盒。
在可选的实施方式中，新冠突变型为Omicron突变株。
本公开实施方式还提供一种鉴别新冠突变型抗原的方法，利用抗体1、抗体2和抗体3对待测样本进行免疫检测，抗体1能与M种新型冠状病毒抗原结合；抗体2能与除Omicron突变株外的M-1种新型冠状病毒抗原结合，抗体2选自上述的抗体；抗体3能与M种新型冠状病毒抗原结合，M为大于等于2的整数。本公开所述“M种新冠病毒抗原”包括野生株和突变株新冠病毒的抗原，其中突变株不限于Omicron突变株，还可以是B.1.1.7突变株、B1.351突变株或B.1.1.28突变株。本公开所述“除Omicron 突变株外的M-1种新冠病毒抗原”包括野生株，可选地还包括除Omicron突变株外的其他突变株，如B.1.1.7突变株、B1.351突变株或B.1.1.28突变株。相关检测原理参见申请号：CN202110315361.5的专利申请。
在可选的实施方式中，抗体1和抗体3夹心检测新型冠状病毒抗原；抗体2和抗体3夹心检测新型冠状病毒抗原。这里所述的新冠病毒抗原不限于N抗原，或者也可以是S抗原，不限于野生株，也可以是突变株。在一些实施方式中，新冠病毒抗原为新冠病毒抗原N抗原。
在可选的实施方式中，新型冠状病毒抗原为新型冠状病毒的N抗原。
在可选的实施方式中，方法选自免疫层析法、酶联免疫吸附法、化学发光法、胶乳免疫比浊法。
本公开实施方式还提供一种免疫层析试纸。免疫层析试纸包括底板、样品垫、结合垫、硝酸纤维素膜和吸水垫，硝酸纤维素膜上设置检测线1和检测线2，结合垫上设置有上述的抗体3，检测线1上包被上述的抗体1、检测线2上包被上述的抗体2；抗体3用标记物进行标记。
在一些实施方式中，将免疫层析试纸与待测样本接触，当检测线1和检测线2同时显色或检测线1和2显色基本一致(例如相差1个色卡以内)，则判定为样品中不含有新冠病毒Omicron突变株；当检测线1显色，检测线2不显色时或者检测线1和2显色存在明显差异(例如相差3个色卡以上)，则判定为样品中含有新冠病毒Omicron突变株。
在可选的实施方式中，所述免疫层析试纸为鉴别新冠突变型抗原的免疫层析试纸。
在可选的实施方式中，硝酸纤维素膜上还设置有质控线。
本公开实施方式还提供了一种上述的抗体或其功能性片段、抗体偶联物、试剂或试剂盒、方法或试纸在检测Omicron突变株或其N蛋白、检测非Omicron新型冠状病毒或其N蛋白、鉴别新冠突变型中的应用。
在可选的实施方式中，所述新冠突变型为Omicron突变株。
本公开实施方式还提供了一种上述的抗体或其功能性片段、抗体偶联物、试剂或试剂盒、方法或试纸在制备检测Omicron突变株或其N蛋白、检测非Omicron新型冠状病毒或其N蛋白、鉴别新冠突变型产品中的应用。
本公开实施方式还提供上述抗体或其功能性片段、抗体偶联物、上述的试剂或试剂盒、方法或免疫层析试纸在检测Omicron突变株或制备Omicron突变株检测产品中的应用。
本公开实施方式还提供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，使利用抗体1、抗体2和抗体3与来自所述受试者的样品接触以进行免疫检测；以及
b)检测结合反应产生的免疫复合物；
抗体1能与M种新型冠状病毒抗原结合；所述抗体2能与除Omicron突变株外的M-1种新型冠状病毒抗原结合，所述抗体2选自上文所述的抗体或其功能性片段；所述抗体3能与M种新型冠状病毒抗原结合，M为大于等于2的整数。
在可选的实施方式中，所述抗体1和抗体3夹心检测新型冠状病毒抗原；所述抗体2和抗体3夹心 检测新型冠状病毒抗原。
在可选的实施方式中，所述新型冠状病毒抗原为新型冠状病毒N抗原。
在可选的实施方式中，所述方法选自免疫层析法、酶联免疫吸附法、化学发光法、胶乳免疫比浊法。
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例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。
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19E3、2F18或12H87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19E3、2F18或12H87单克隆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KABAT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，VL基因序列为333bp(19E3)、333bp(2F18)、333bp(12H87)，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，VH基因序列为342bp(19E3)、345bp(2F18)、345bp(12H87)，属于VH1基因家族，其前方有57bp的前导肽序列。(19E3的重轻链序列分别如SEQ ID NO:19、20所示，2F18的重轻链序列分别如SEQ ID NO:30、31所示，12H87的重轻链序列分别如SEQ ID NO:36、37所示)。
(3)重组抗体表达质粒的构建
pcDNA TM3.4
Figure PCTCN2023071102-appb-000001
载体(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1.40kb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40μg/100μL，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-nCoVN蛋白(购自菲鹏)至3μg/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+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，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+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、PuvⅠ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(体积比)，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40μg/100μL，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；25umol/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(protein)A亲和层析柱进行亲和纯化。取6.6μg纯化的抗体进行还原性SDS-PAGE，电泳图如图1所示。
实施例2
抗体的性能检测
(1)活性检测
包被液(主要成分NaHCO 3)稀释2019-nCoVN蛋白(购自菲鹏)至3μg/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；加入稀释后的实施例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活性数据
浓度(ng/mL) 2500.00 1250.00 625.00 312.50 156.25 0.00
19E3 2.372 1.866 0.894 0.311 0.113 0.032
2F18 2.274 1.521 0.831 0.400 0.223 0.043
浓度(ng/mL) 3000.00 1000.00 333.33 111.11 37.04 0.00
12H87 2.317 2.163 1.918 1.320 0.663 0.038
(2)亲和力检测
利用AMC传感器，将纯化出来的抗体用PBST稀释到10μg/mL，2019-nCoVN蛋白(购自菲鹏)用 PBST进行梯度稀释。
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
样品名称 KD(M) kon(1/Ms) kdis(1/s)
19E3 4.02E-09 3.56E+04 1.43E-04
2F18 2.22E-09 7.43E+04 1.65E-04
12H87 4.90E-08 6.37E+03 3.12E-04
(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，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为考核21天的酶免活性检测OD结果。
表4
样品浓度(ng/mL) 3000.00 2500.00 2500.00 625.00 625.00 37.04 0 0 0
样品名称 12H87 19E3 2F18 19E3 2F18 12H87 19E3 2F18 12H87
4℃，21天样品 2.373 2.357 2.257 0.843 0.793 0.693 0.032 0.044 0.037
-80℃，21天样品 2.352 2.355 2.244 0.827 0.789 0.649 0.035 0.048 0.039
37℃，21天样品 2.399 2.362 2.259 0.893 0.799 0.657 0.037 0.049 0.035
(4)鉴定突变型抗原
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100μL，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1.0-1.5mg/mL，用喷金画膜仪在NC膜上均匀地划T1线；再将19E3、2F18或12H87抗体稀释至1.0-1.5mg/mL，用喷金画膜仪在NC膜上均匀地划T2线；放37℃恒温箱进行干燥， 至少干燥45min以上。组装切条，加样检测。
本实施例的标记抗体为15C8或Mb117(购自菲鹏)，T1线包被抗体为Mb30(购自菲鹏)，15C8、Mb117或Mb30均能结合包括Omicron突变株在内的新冠病毒N抗原；T2线包被抗体为实施例1获得的抗体19E3、2F18或12H87。
3.检测
(1)检测品：新冠病毒Omicron突变株N抗原，和新冠病毒野生株N抗原(2019-nCoVN)；
(2)检测方法：根据色卡比对，肉眼判读显色读值。
4.结果：详见下表5
表5
Figure PCTCN2023071102-appb-000002
以上结果可以看出，19E3、2F18或12H87抗体对Omicron突变株N抗原不反应，对新冠病毒野生株N抗原反应，由此可以实现对Omicron突变株的快速鉴定。
以上所述仅为本公开的优选实施例而已，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。
本申请涉及的部分氨基酸序列如下表6所示：
表6
序列编号 序列片段
SEQ ID NO:1 DYSMH
SEQ ID NO:2 WINTETGEPTYADDFKG
SEQ ID NO:3 GLF
SEQ ID NO:4 KASQSVDYDGDSYMN
SEQ ID NO:5 AASNLES
SEQ ID NO:6 QQSNEDP
SEQ ID NO:7 QIQLVQSGPELKKPGETVKISCKASGYTFT
SEQ ID NO:8 WVKQAPGKGLKWMG
SEQ ID NO:9 RFAFSLETSASTAYLQINNLKNEDTATYFCGR
SEQ ID NO:10 DYWGQGTTLTVSS
SEQ ID NO:11 DIVLTQSPASLAVSLGQRATISC
SEQ ID NO:12 WYQQKPGQPPKLLIY
SEQ ID NO:13 GIPARFSGSGSGTDFTLNIHPVEEEDAATYYC
SEQ ID NO:14 LTFGAGTKLELK
SEQ ID NO:21 GGAS
SEQ ID NO:22 KASQSVDYDGHSYMN
SEQ ID NO:23 TASNLES
SEQ ID NO:24 WVKQAPGKGLRWMG
SEQ ID NO:25 RFAFSLETSASTAYLQINNLKNEDTATYFCAR
SEQ ID NO:26 GYWGQGTTLTVSS
SEQ ID NO:32 GAGT
SEQ ID NO:33 WVKQATGKGLKWMG
工业实用性
本公开提供了一种鉴别新冠突变型抗原的抗体、试剂及方法，利用本公开抗体、试剂或方法能够快速、准确地鉴别新冠突变型抗原，且检测效率高、成本低，因此均具有优异的实用性能。

Claims (22)

  1. 一种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、HCDR2、HCDR3包括与SEQ ID NO:15、27、34任一所示的重链可变区的HCDR1、HCDR2、HCDR3一致的氨基酸序列；所述LCDR1、LCDR2、LCDR3包括与SEQ ID NO:16、28、35任一所示的轻链可变区的LCDR1、LCDR2、LCDR3一致的氨基酸序列。
  2. 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HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由Kabat、Chothia、IMGT、AbM或Contact系统定义。
  3. 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所示的氨基酸序列，或由其组成；
    HCDR3，其包含SEQ ID NO:3、21、32任一所示的氨基酸序列，或由其组成；
    LCDR1，其包含SEQ ID NO:4或22所示的氨基酸序列，或由其组成；
    LCDR2，其包含SEQ ID NO:5或23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。
  4. 一种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所示的氨基酸序列，或由其组成；
    HCDR3，其包含SEQ ID NO:3、21、32任一所示的氨基酸序列，或由其组成；
    LCDR1，其包含SEQ ID NO:4或22所示的氨基酸序列，或由其组成；
    LCDR2，其包含SEQ ID NO:5或23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。
  5. 根据权利要求1-4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HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3和LFR4中的至少之一；
    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所示，或与其具有至少80％同源性；
    HFR2氨基酸序列如SEQ ID NO:8、24、33任一所示或与其具有至少80％同源性；
    HFR3氨基酸序列如SEQ ID NO:9或25所示或与其具有至少80％同源性；
    HFR4氨基酸序列如SEQ ID NO:10或26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 -7M的亲和力结合非Omicron新型冠状病毒N抗原；
    可选地，所述抗体或其功能性片段包括SEQ ID NO:3所示的HCDR3及SEQ ID NO:4、5所示的 LCDR1、LCDR2；
    可选地，所述抗体或其功能性片段包括SEQ ID NO:21所示的HCDR3及SEQ ID NO:22、23所示的LCDR1、LCDR2；
    可选地，所述抗体或其功能性片段包括SEQ ID NO:32所示的HCDR3及SEQ ID NO:4、23所示的LCDR1、LCDR2；
    可选地，所述抗体或其功能性片段包括氨基酸序列如SEQ ID NO:15、27、34任一所示的重链可变区，和氨基酸序列如SEQ ID NO:16、28、35任一所示的轻链可变区。
  6. 一种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，所述重链可变区的氨基酸序列如SEQ ID NO:15、27、34任一所示；所述轻链可变区的氨基酸序列如SEQ ID NO:16、28、35任一所示。
  7. 一种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1-4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5所述的HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3、LFR4的氨基酸序列；
    可选地，所述重链可变区的氨基酸序列如SEQ ID NO:15、27、34任一所示；所述轻链可变区的氨基酸序列如SEQ ID NO:16、28、35任一所示。
  8. 根据权利要求1-7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、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，所述重链恒定区的序列如SEQ ID NO:17所示或与其具有至少80％同源性，所述轻链恒定区的序列如SEQ ID NO:18或29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包括氨基酸序列如SEQ ID NO:19、30、36任一所示的重链，和氨基酸序列如SEQ ID NO:20、31、37任一所示的轻链。
  9. 根据权利要求1-8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。
  10. 一种抗体，包括重链和/或轻链，其特征在于，所述重链包括权利要求5-7任一项所述的重链可变区和权利要求8所述的重链恒定区；所述轻链包括权利要求5-7任一项所述的轻链可变区和权利要求 8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、30、36任一所示；所述轻链的氨基酸序列如SEQ ID NO:20、31、37任一所示。
  11. 一种抗体，包括重链和/或轻链，其特征在于，所述重链的氨基酸序列如SEQ ID NO:19、30、36任一所示；所述轻链的氨基酸序列如SEQ ID NO:20、31、37任一所示。
  12. 根据权利要求1-11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结合非Omicron新型冠状病毒N抗原。
  13. 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1-12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标记物；
    可选地，所述标记物选自荧光染料、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。
  14. 一种核酸，其特征在于，其编码权利要求1-12任一项所述的抗体或其功能性片段。
  15. 一种载体，其特征在于，其含有编码权利要求1-12任一项所述的抗体或其功能性片段的核酸片段。
  16. 一种重组细胞，其特征在于，其含有权利要求15所述的载体。
  17. 一种制备如权利要求1-12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16所述的重组细胞。
  18. 一种试剂或试剂盒，所述试剂或试剂盒包括权利要求1-12任一项所述的抗体或其功能性片段或权利要求13所述的抗体偶联物；
    可选地，所述试剂或试剂盒为检测非Omicron新型冠状病毒或鉴别新冠突变型的试剂或试剂盒；
    可选地，所述新冠突变型为Omicron突变株。
  19. 一种鉴别新冠突变型抗原的方法，其特征在于，利用抗体1、抗体2和抗体3对待测样本进行免疫检测，所述抗体1能与M种新型冠状病毒抗原结合；所述抗体2能与除Omicron突变株外的M-1种新型冠状病毒抗原结合，所述抗体2选自权利要求1-12任一所述的抗体；所述抗体3能与M种新型冠状病毒抗原结合，M为大于等于2的整数；
    可选地，所述抗体1和抗体3夹心检测新型冠状病毒抗原；所述抗体2和抗体3夹心检测新型冠状病毒抗原；
    可选地，所述新型冠状病毒抗原为新型冠状病毒N抗原；
    可选地，所述方法选自免疫层析法、酶联免疫吸附法、化学发光法、胶乳免疫比浊法。
  20. 一种免疫层析试纸，其特征在于，所述免疫层析试纸包括底板、样品垫、结合垫、硝酸纤维素膜和吸水垫，所述硝酸纤维素膜上设置检测线1和检测线2，所述结合垫上设置有权利要求19所述的抗体3，所述检测线1上包被权利要求19所述的抗体1、所述检测线2上包被权利要求19所述的抗体2；所述抗体3用标记物进行标记；
    可选地，所述免疫层析试纸为鉴别新冠突变型抗原的免疫层析试纸；
    可选地，所述硝酸纤维素膜上还设置有质控线。
  21. 权利要求1-12任一所述的抗体或其功能性片段、权利要求13所述的抗体偶联物、权利要求18所述的试剂或试剂盒、权利要求19所述的方法或权利要求20所述的免疫层析试纸在检测Omicron突变株或其N蛋白、检测非Omicron新型冠状病毒或其N蛋白、鉴别新冠突变型中的应用，
    可选地，所述新冠突变型为Omicron突变株。
  22. 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，使利用抗体1、抗体2和抗体3与来自所述受试者的样品接触以进行免疫检测；以及
    b)检测结合反应产生的免疫复合物；
    所述抗体1能与M种新型冠状病毒抗原结合；所述抗体2能与除Omicron突变株外的M-1种新型冠状病毒抗原结合，所述抗体2选自权利要求1-12任一项所述的抗体或其功能性片段；所述抗体3能与M种新型冠状病毒抗原结合，M为大于等于2的整数；
    可选地，所述抗体1和抗体3夹心检测新型冠状病毒抗原；所述抗体2和抗体3夹心检测新型冠状病毒抗原；
    可选地，所述新型冠状病毒抗原为新型冠状病毒N抗原；
    可选地，所述方法选自免疫层析法、酶联免疫吸附法、化学发光法、胶乳免疫比浊法。
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