WO2022262321A1 - 嵌合的免疫球蛋白 - Google Patents

嵌合的免疫球蛋白 Download PDF

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WO2022262321A1
WO2022262321A1 PCT/CN2022/078975 CN2022078975W WO2022262321A1 WO 2022262321 A1 WO2022262321 A1 WO 2022262321A1 CN 2022078975 W CN2022078975 W CN 2022078975W WO 2022262321 A1 WO2022262321 A1 WO 2022262321A1
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polypeptide
region
igg
optionally
igm
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PCT/CN2022/078975
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English (en)
French (fr)
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孟媛
钟冬梅
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东莞市朋志生物科技有限公司
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Priority to CN202280041529.0A priority Critical patent/CN117545767A/zh
Priority to BR112023026352A priority patent/BR112023026352A2/pt
Priority to EP22823813.5A priority patent/EP4342908A1/en
Priority to CA3224182A priority patent/CA3224182A1/en
Priority to KR1020247000829A priority patent/KR20240019312A/ko
Publication of WO2022262321A1 publication Critical patent/WO2022262321A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C07K16/205—Plasmodium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • 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
    • G01N33/56905—Protozoa
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components

Definitions

  • This application relates to the field of antibody engineering, in particular, to a chimeric immunoglobulin.
  • Immunoglobulin is composed of four polypeptide chains, and each polypeptide chain is connected by a different number of interchain disulfide bonds. Ig can have a "Y"-shaped structure, called Ig monomer, which is the basic unit that constitutes an antibody.
  • Natural Ig molecules contain four heterologous polypeptide chains, of which the two chains with larger molecular weight are called heavy chains (H) and the two chains with smaller molecular weight are called light chains (L). The amino acid composition of the two heavy chains and the two light chains in the same Ig molecule are identical.
  • the heavy chain has a molecular weight of 50 000-75 000 and consists of 450-550 amino acid residues.
  • Ig can be divided into five classes (classes), namely IgM, IgD, IgG, IgA and IgE. Among these five classes, IgG, IgD, and IgE are all monomeric structures.
  • IgM is a basic antibody secreted by B cells. In the secreted form, it is a pentameric form composed of five Y-shaped monomers, which has a very high affinity. IgM has an extremely large molecular weight and is therefore very effective in antigen agglutination reactions. IgM is a polyvalent antibody.
  • IgA can exist as a monomer or as a dimer, trimer or tetramer.
  • Secretory IgA mostly exists in the form of dimers and trimers, and is also a multivalent antibody. Since a monomeric antibody has 2 antigen-binding sites, IgA usually has 4 or 6 antigen-binding sites, while IgM has 10 or 12 antigen-binding sites, all of which are multivalent antibodies.
  • Antibodies are mainly synthesized by B lymphocytes. Each B lymphocyte has the genetic code for synthesizing an antibody. When the body is stimulated by an antigen, many determinants on the antigen molecule activate B cells with different genes respectively. Activated B cells divide and proliferate to form polyclones and synthesize various antibodies.
  • B lymphocytes sheep red blood cell immunized mouse splenocytes (B lymphocytes), and found that the formed hybrid cells could proliferate infinitely in vitro, It can also continuously secrete specific antibodies, and the pure cells obtained through cloning culture can produce monoclonal antibodies, thus inventing monoclonal antibody technology.
  • monoclonal antibody technology is a major breakthrough in the field of immunology.
  • monoclonal antibody technology had been perfected day by day and began to be widely used in many fields such as biomedical research, biotechnology, and clinical diagnosis and treatment.
  • monoclonal antibodies give full play to their advantages.
  • the specificity of the monoclonal antibody is strong, which greatly improves the specificity of the antigen-antibody reaction, reduces the possibility of cross-reaction with other substances, and makes the test results more reliable.
  • the homogeneity and singleness of biological activity of monoclonal antibody make it easy to control the results of antigen-antibody reaction, which is conducive to standardization and standardization.
  • Immunodiagnostic reagents are a subdivision of in vitro diagnostic reagents.
  • Immunodiagnosis is the application of the theory, technology and methods of immunology to diagnose various diseases and determine the immune status.
  • Immunodiagnostic methods include radioimmunoassay, enzyme-linked immunoassay, chemiluminescence, fluorescence chromatography or colloidal gold chromatography, etc. The rationale for these methods is described below.
  • select a suitable marker and antigen or antibody to form an antigen or antibody-labeled complex by chemical method.
  • the test specimen determining the antibody or antigen in it
  • the antigen or antibody-labeled complex react with the antigen or antibody on the surface of the solid phase carrier in different steps. Washing is used to separate the antigen-antibody complex formed on the solid-phase carrier from other substances, and the final labeled complex bound to the solid-phase carrier is in a certain proportion to the amount of the substance to be tested in the sample.
  • the substrate is catalyzed into a colored product. Because the amount of the colored product is directly related to the amount of the tested substance in the specimen, qualitative or quantitative analysis can be carried out according to the depth of the color reaction.
  • the improvement of antibody titer for immunodiagnosis still depends on the improvement of antibody immunization and screening process, and the effect has great randomness, which cannot well meet the detection needs.
  • the first aspect of the present application relates to an immunoglobulin obtained by fusion of a first polypeptide and a second polypeptide, wherein
  • the first polypeptide includes an IgG variable region at the N-terminus;
  • the second polypeptide includes an IgA CH2-CH3 region at the C-terminus, or an IgM CH3-CH4 region.
  • the second aspect of the present application relates to a multimer, which is obtained by polymerizing the above-mentioned immunoglobulin as a monomer.
  • a third aspect of the present application relates to an isolated nucleic acid encoding an immunoglobulin as described above.
  • the fourth aspect of the present application relates to a vector comprising the nucleic acid as described above.
  • the fifth aspect of the present application relates to a host cell containing the nucleic acid as described above or transformed with the vector as described above.
  • the sixth aspect of the present application relates to a preparation method of immunoglobulin, comprising expressing the above-mentioned host cell or performing fusion expression of the above-mentioned first polypeptide and the second polypeptide, and the immunoglobulin prepared by the above-mentioned preparation method globulin.
  • the seventh aspect of the present application relates to a method for preparing a multimer, comprising polymerizing the immunoglobulin prepared by the above-mentioned immunoglobulin preparation method as a monomer, and polymerizing the multimer prepared by the above-mentioned preparation method body.
  • the eighth aspect of the present application relates to a solid phase carrier, the surface of which is coated with the polymer as described above.
  • the ninth aspect of the present application relates to a kit or a test strip containing the above-mentioned polymer, or the above-mentioned solid phase carrier.
  • the tenth aspect of the present application relates to a method for detecting an antigen, comprising contacting the above-mentioned polymer or the above-mentioned solid phase carrier with the antigen to form a conjugate, and detecting the conjugate;
  • the antigen is an antigen capable of specifically binding to the IgG variable region.
  • the eleventh aspect of the present application relates to the application of the polymer, solid phase carrier, kit or test strip in immunoassay.
  • the IgG and IgM/IgA chimeric immunoglobulins provided in this application have higher potency, and when coated on a solid phase, still have a higher titer than IgG monomers with the same valence , so it can be widely used in the field of detection.
  • Figure 1 shows the electrophoresis results of MA-9G7TB1 and MA-9G7TB2 IgG/IgM hybrid recombinant antibodies in one embodiment of the present application;
  • Figure 2 shows the electrophoresis results of the 3E50TB2 hybrid recombinant antibody in one embodiment of the present application
  • Figure 3 shows the electrophoresis results of the 5F27TB2 hybrid recombinant antibody in one embodiment of the present application
  • Figure 4 shows the electrophoresis results of the 21C5TB2 hybrid recombinant antibody in one embodiment of the present application.
  • the inventors of the present application unexpectedly found that normal IgG antibodies would aggregate when storage conditions such as temperature and buffer conditions changed.
  • the emergence of multimeric antibodies is directly reflected in the fact that the activity of antibodies during the application of diagnostic reagents is much higher than that of monomeric antibodies.
  • naturally occurring IgG multimers are unstable.
  • the inventors found that the chimeric antibodies of IgG/IgA and IgG/IgM constructed were effective in the detection of diagnostic reagents. The performance in the process is much higher than that of IgG antibodies, and the homogeneity and expression of chimeric antibodies have very good advantages.
  • IgG/IgA and IgG/IgM chimeric antibodies were constructed and applied to the coated or labeled ends of in vitro diagnostic detection reagents.
  • the IgG/IgA and IgG/IgM chimeric antibodies of the present application can replace existing IgG antibodies, thereby obtaining a detection kit with better activity.
  • the embodiment of the present application provides an immunoglobulin obtained by fusion of a first polypeptide and a second polypeptide, wherein
  • the first polypeptide includes an IgG variable region at the N-terminus;
  • the second polypeptide comprises an IgA CH2-CH3 region at the C-terminus, or an IgM CH3-CH4 region.
  • the IgG in the first polypeptide also has a CH1 region.
  • the IgA or IgM in the second polypeptide also has a CH1 region.
  • the first polypeptide further comprises an IgG hinge region.
  • the second polypeptide further comprises an IgA or IgM hinge region.
  • the IgG in the first polypeptide also has a CH1-CH2 region.
  • the IgG in the first polypeptide also has a CH1-CH2-CH3 region.
  • the IgM in the second polypeptide also has a CH1-CH2 region.
  • the IgG is IgGl, IgG2, IgG3 or IgG4.
  • At least one of the first polypeptide and the second polypeptide comprises a CH1 region.
  • At least one of the first polypeptide and the second polypeptide comprises a hinge region.
  • IgG, IgA and IgM can be independently selected from the same or different species sources, eg murine (mouse, rat), rabbit, sheep, goat, horse, chicken, bovine, canine and human.
  • murine mouse, rat
  • rabbit sheep, goat
  • horse chicken
  • bovine canine and human.
  • the embodiment of the present application provides a multimer, which is obtained by polymerizing the above-mentioned immunoglobulin as a monomer.
  • the multimer is obtained by polymerizing 2, 3 or 4 immunoglobulin monomers with IgA CH2-CH3 regions.
  • the multimer is obtained by polymerizing 5 immunoglobulin monomers with IgM CH3-CH4 regions.
  • the multimer is also conjugated with a signaling substance.
  • embodiments of the present application provide an isolated nucleic acid encoding an immunoglobulin as described above.
  • the embodiment of the present application further provides a vector containing the above-mentioned nucleic acid.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • Phage such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40).
  • the vectors described herein contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or multiple adenylation signal and poly U sequence, etc.).
  • the embodiment of the present application also provides a host cell, which contains the above-mentioned nucleic acid or is transformed by the above-mentioned vector.
  • the term "host cell” refers to cells that can be used to introduce vectors, including but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, such as S2 Drosophila cells or Sf9 Insect cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells etc. animal cells.
  • the host cells are preferably eukaryotic cells, more preferably mammalian cells.
  • the embodiment of the present application provides a method for preparing immunoglobulin, comprising expressing the above-mentioned host cell.
  • the embodiment of the present application also provides a method for preparing an immunoglobulin, comprising performing fusion expression of the first polypeptide and the second polypeptide, wherein,
  • the first polypeptide includes an IgG variable region at the N-terminus;
  • the second polypeptide comprises an IgA CH2-CH3 region at the C-terminus, or an IgM CH3-CH4 region.
  • the IgG in the first polypeptide of the immunoglobulin also has a CH1 region.
  • the IgA or IgM in the second polypeptide also has a CH1 region.
  • the first polypeptide further comprises an IgG hinge region.
  • the second polypeptide further comprises an IgA or IgM hinge region.
  • the IgG in the first polypeptide also has a CH1-CH2 region.
  • the IgG in the first polypeptide also has a CH1-CH2-CH3 region.
  • the IgM in the second polypeptide also has a CH1-CH2 region.
  • the IgG is IgGl, IgG2, IgG3 or IgG4.
  • At least one of the first polypeptide and the second polypeptide comprises a CH1 region
  • At least one of the first polypeptide and the second polypeptide comprises a hinge region.
  • the embodiment of the present application also provides an immunoglobulin prepared by the above immunoglobulin preparation method.
  • the embodiment of the present application provides a method for preparing a multimer, comprising polymerizing the immunoglobulin prepared by the method for preparing the immunoglobulin described above as a monomer.
  • 2, 3 or 4 immunoglobulin monomers having an IgA CH2-CH3 region are polymerized.
  • 5 immunoglobulin monomers having an IgM CH3-CH4 region are polymerized.
  • the embodiment of the present application also provides a multimer prepared by the above multimer preparation method.
  • the embodiment of the present application provides a solid phase carrier, the surface of which is coated with the polymer as described above.
  • the solid phase carrier is a plastic carrier, a microparticle or a membrane carrier;
  • the plastic carrier can be a polystyrene carrier;
  • the microparticle can be a magnetic particle;
  • the membrane carrier can be a nitrocellulose membrane, a glass fiber Plain film or nylon film.
  • the solid phase carrier is selected from the wells of test tubes, EP tubes, multi-well plates, chromatography columns, and micro-reaction plates.
  • the term "particle” may be spherical, nearly spherical, cubic, polyhedral or irregular in shape.
  • the diameter of the microparticles may be 10 nm to 1 mm, such as 100 nm, 500 nm, 1 ⁇ m, 10 ⁇ m, 100 ⁇ m, 500 ⁇ m; preferably 400 nm to 10 ⁇ m.
  • the microparticles are preferably magnetic microparticles containing a magnetic substance in their composition.
  • the magnetic substance can be metal (single metal or alloy), nonmetal, or a compound formed of metal and nonmetal.
  • Metals such as iron, alnico metal, etc.; nonmetals such as ferrite nonmetals (preferably Fe 2 O 3 or Fe 3 O 4 magnetic nanoparticles); metal and nonmetal composites such as NdFeB rubber magnets composite material.
  • the multiwell plate is preferably an ELISA plate, which may have 8 wells, 16 wells, 32 wells, 48 wells, 64 wells, 96 wells or more wells.
  • the embodiment of the present application provides a kit or a test strip containing the above-mentioned polymer, or the above-mentioned solid-phase carrier.
  • the test strip includes a sample pad, a binding pad, a reaction membrane and an absorption pad, and the reaction membrane is provided with a detection area and a quality inspection area;
  • the multimer is coated on the detection area and/or dropped on the binding pad.
  • the antigen epitopes bound by the IgG variable regions of the polymers are different, and the second polypeptide terminal domains can be the same or different.
  • the kit may also include sample pretreatment reagents (such as sample purification and enrichment reagents, lysate, etc.), cleaning solutions (such as water, etc.), buffers (such as PBS or Tris, etc.), Chromogenic reagents for substances (such as ECL or DAB for horseradish peroxidase as a signal substance), etc.
  • sample pretreatment reagents such as sample purification and enrichment reagents, lysate, etc.
  • cleaning solutions such as water, etc.
  • buffers such as PBS or Tris, etc.
  • Chromogenic reagents for substances such as ECL or DAB for horseradish peroxidase as a signal substance
  • the embodiments of the present application provide a method for detecting an antigen, comprising:
  • the antigen is an antigen capable of specifically binding to the IgG variable region.
  • the conjugate can be detected by any method known to those skilled in the art, such as a double-antibody sandwich method, that is, after the antigen is contacted with the solid phase carrier, an additional antibody against the antigen ( It is usually labeled with a signal substance, or it is detected with a secondary antibody labeled with a signal substance) to detect the conjugate.
  • a double-antibody sandwich method that is, after the antigen is contacted with the solid phase carrier, an additional antibody against the antigen ( It is usually labeled with a signal substance, or it is detected with a secondary antibody labeled with a signal substance) to detect the conjugate.
  • a conventional antibody is used as an antibody coated on a solid phase carrier, and it is co-incubated with the antigen to be detected, and the multimer is used as a free detection antibody (which is usually labeled with a signal substance, or with a labeled Secondary antibody with signal substance to detect it) for detection.
  • the antigen is detected by coordinating the polymer-coated solid phase carrier with another polymer, and at this time, the polymer coated on the solid phase carrier and the other polymer are paired antibodies.
  • the above-mentioned signal substances can be fluorescent substances, quantum dots, digoxin-labeled probes, biotin, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescence markers, ultrasonic contrast agents, photosensitizers , colloidal gold or enzymes.
  • the signal substance is colloidal gold, fluorescein, fluorescent microspheres, acridinium ester, horseradish peroxidase, alkaline phosphatase or ⁇ -galactosidase.
  • the embodiments of the present application provide the application of the above-mentioned polymer, solid phase carrier, kit or test strip in immunoassay.
  • Embodiments of the present application also provide the above-mentioned polymer, solid phase carrier, kit or test strip for immunoassay.
  • the embodiment of the present application also provides an immunoassay method, which includes using the polymer, solid phase carrier, kit or test strip as described above for immunoassay.
  • the embodiment of the present application provides an immunodiagnostic kit comprising IgG-IgM or IgG-IgA chimeric immunoglobulin multimers.
  • the immunoglobulin is fused from a first polypeptide to a second polypeptide, wherein
  • the first polypeptide includes an IgG variable region at the N-terminus;
  • the second polypeptide comprises an IgA CH2-CH3 region at the C-terminus, or an IgM CH3-CH4 region.
  • the IgG in the first polypeptide of the immunoglobulin also has a CH1 region.
  • the IgA or IgM in the second polypeptide also has a CH1 region.
  • the first polypeptide further comprises an IgG hinge region.
  • the second polypeptide further comprises an IgA or IgM hinge region.
  • the IgG in the first polypeptide also has a CH1-CH2 region.
  • the IgG in the first polypeptide also has a CH1-CH2-CH3 region.
  • the IgM or IgA in the second polypeptide also has a CH1-CH2 region.
  • the IgG is IgGl, IgG2, IgG3 or IgG4.
  • At least one of the first polypeptide and the second polypeptide comprises a CH1 region.
  • At least one of the first polypeptide and the second polypeptide comprises a hinge region.
  • the multimer is the polymerization of 2, 3 or 4 immunoglobulin monomers with IgA CH2-CH3 regions; or the polymerization of 5 immunoglobulin monomers with IgM CH3-CH4 regions .
  • the multimer is also conjugated with a signaling substance.
  • the kit further includes sample pretreatment reagents, washing solutions, buffers and chromogenic reagents.
  • the kit also includes a sample pad, a binding pad, a reaction membrane and an absorption pad, and the reaction membrane is provided with a detection area and a quality inspection area.
  • the kit can also include another antibody, which can be multimeric or monomeric.
  • the multimer can be immobilized on a solid phase support, and the solid phase support includes nitrocellulose membrane, latex, magnetic beads or ELISA plate, etc.
  • the multimer can be labeled with signal substances such as nanoparticles, chemiluminescent substances, fluorescent substances, radioactive substances, colloids, and enzymes.
  • signal substances such as nanoparticles, chemiluminescent substances, fluorescent substances, radioactive substances, colloids, and enzymes.
  • restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company.
  • the pMD-18T vector was purchased from Takara Company; the plasmid extraction kit was purchased from Tiangen Company; primer synthesis and gene sequencing were completed by Invitrogen Company.
  • the polynucleotide encoding the CH region of the mouse antibody can be obtained from the DNA of peripheral blood cells of the mouse by PCR, or from the hybridoma cell line secreting the corresponding type of monoclonal antibody by PCR.
  • the polynucleotide encoding the mouse IgM-CH region was obtained by PCR from the genome of the hybridoma cell line 6M9 (available from Fapon Biotech) secreting IgM-type mouse monoclonal antibodies.
  • the primers used were as follows:
  • Reverse primer sequence (5' ⁇ 3'): CCC GAA TTC TTA TCA ATA GCA GGT GCC ACC TGT GTC AGA CAT GAT CAG (SEQ ID NO: 2).
  • This polynucleotide encodes the mouse IgM-CH region, and its amino acid sequence is as follows from N-terminal to C-terminal:
  • the polynucleotide encoding the CH region of IgA was obtained from the DNA of isolated mouse peripheral blood cells (available from Faipeng Biotech) by means of PCR.
  • the primers used were as follows:
  • Reverse primer sequence (5' ⁇ 3'): CCC GAA TTC TCA GTA GCA GAT GCC ATC TCC CTC TGA CAT GAT (SEQ ID NO: 5).
  • An EcoRI restriction site was introduced by the reverse primer followed by the stop codon TGA (anticodon TCA).
  • a polynucleotide of about 1 kb was obtained by PCR technology, which was introduced into the pMD-18T vector for sequencing to confirm the sequence.
  • the polynucleotide encodes the mouse IgA-CH region, and its amino acid sequence is as follows from N-terminal to C-terminal:
  • a polynucleotide encoding the CH1, CH2, CH3 and CH4 domains of the IgM CH region or a combination thereof, or a polynucleotide encoding the CH1, CH2 and CH3 domains of the IgA CH region or a combination thereof is prepared in a similar manner.
  • the IgG antibody used in this example is anti-Pan-PLDH 9G7 monoclonal antibody (hereinafter referred to as MA 9G7).
  • MA 9G7 monoclonal antibody
  • the sequence of this monoclonal antibody is described in Chinese Patent Publication No. CN111363044A, which is incorporated herein by reference.
  • the antibody can be selected from any one of antibodies with any group of mutations in paragraphs [0087] to [0089] of CN111363044A, mutation combination 1 to mutation combination 56, such as mutation combination 32, which have similar properties.
  • VH heavy chain variable region
  • pcDNA3.4A-9G7TB1VCH a polynucleotide of about 1.8kb by PCR, and digest it with HindIII/EcoRI double enzymes, and connect it to pcDNA TM 3.4 cut with the corresponding restriction enzymes
  • the resulting vector is named pcDNA3.4A-9G7TB1VCH.
  • the polynucleotide encoding the VH region segment 2 of MA 9G7 was selected to be connected with the polynucleotide encoding the CH2 start to the C end of the IgM CH region by means of bridge PCR.
  • the amino acid sequence of the VH region segment 2 of MA 9G7 is as follows:
  • a DNA fragment of about 1.8 KB was obtained by PCR, digested by HindIII/EcoRI, and ligated to pcDNA TM 3.4 Vector, referred to as pcDNA3.4A-9G7TB2VCH.
  • the light chain clone of the antibody in this example was constructed using the light chain plasmid construction method disclosed in Chinese Patent Publication No. CN111363044A.
  • pcDNA3.4A-9G7TB1VCH plasmid and light chain plasmid were diluted to 40ug/100ul with ultrapure water at a ratio of 1:1, took a new centrifuge tube, added and adjusted the concentration of CHO cells to 1.43 ⁇ 107 cells/ml in the tube, and 100ul
  • the plasmid was mixed with 700ul cells, and transferred into an electroporation cup for electrotransfection. After transfection, the cells were continued to be cultured and counted the next day; 25umol/L MSX 96-well pressurized culture for about 25 days.
  • the pcDNA3.4A-9G7TB2VCH plasmid and the light chain plasmid were transfected and screened according to the method described above for the pcDNA3.4A-9G7TB1VCH plasmid, and a cell line expressing MA-9G7TB2 IgG/IgM hybrid recombinant antibody was obtained.
  • the cells were first inoculated in a 125ml shake flask containing 100% Dynamis medium, with an inoculation volume of 30ml, and cultured in a shaker with a rotation speed of 120r/min, a temperature of 37°C, and 8% carbon dioxide. After culturing for 72 hours, the expanded culture was inoculated at a seeding density of 5 ⁇ 10 5 cells/ml. The volume of the expanded culture was calculated according to production requirements, and the medium used was 100% Dynamis medium. Afterwards, the culture was expanded every 72 hours. When the amount of cells meets the production requirements, strictly control the inoculation density to about 5 ⁇ 10 5 cells/ml for production.
  • Shake flask production parameters shaker speed 120r/min, temperature 37°C, carbon dioxide content 8%.
  • Feed feeding Start feeding every day when the shake flask is cultured for 72 hours, add 3% of the initial culture volume of HyClone TM Cell Boost TM Feed 7a every day, and the daily feed amount is 1/1000 of the initial culture volume HyClone TM Cell Boost TM Feed 7b, fed until day 12 (Day 12 feed). Add 3g/L glucose on the sixth day. Receive samples on the 13th day.
  • the multimeric antibodies of MA-9G7TB1 and MA-9G7TB2 IgG/IgM hybrid recombinant antibodies were obtained by two-step purification of captoL (cytiva) and CHT (Bio-Rad) by affinity chromatography. Take 10 ul of the purified antibody (concentration: 1 mg/ml) for reducing SDS-PAGE, and the electrophoresis results are shown in Figure 1.
  • the left lane is the band of MA-9G7TB1 IgG/IgM recombinant antibody
  • the right lane is the band of MA-9G7TB2 IgG/IgM recombinant antibody.
  • Two bands are shown on each lane, one with a relative molecular mass (Mr) of 70-75 kD (heavy chain) and the other with a Mr of 20-35 kD (light chain).
  • nitrocellulose membrane Dilute the MA-9G7TB1 recombinant antibody to 1-5 mg/ml with coating buffer, draw the T line, which is the detection line, and the T line is close to the colloidal gold end; Goat anti-mouse IgG antibody (manufactured by Feipeng Biotechnology Co., Ltd., product number BA-PAB-MU0001) was diluted to 1-5 mg/ml, and the line C was drawn, which was the control line, and the line C was close to the absorbent pad. Dry at 37°C and package for later use.
  • the prepared colloidal gold has a pure, translucent appearance, no precipitation and floating matter, and is valid for one week.
  • the sample pad After soaking the sample pad in the blocking solution (including BSA) for 30 minutes, dry it at 37°C, package it, and store it at 4°C for later use.
  • the blocking solution including BSA
  • the MA protein uses the assembled detection test strip to detect whether the tested material contains Pan-PLDH protein (hereinafter referred to as MA protein), so as to determine the activity of MA antibody on MA protein detection.
  • the MA protein first combines with the colloidal gold-labeled MA antibody to form a MA-colloidal gold-labeled-MA antibody complex. Due to capillary action, the MA-colloidal gold-labeled-MA antibody complex moves forward along the nitrocellulose membrane. When reaching the detection line, the MA-colloidal gold label-MA antibody complex will combine with the streaked MA-coated antibody to form a complex of MA antibody-MA-colloidal gold label-MA antibody, which will be enriched on the detection line. A red precipitate line formed. Table 1 is the corresponding test data.
  • the depth of the red precipitation line indicates the strength of the reaction. The darker the red, the stronger the reactivity, and vice versa.
  • the reaction strength is represented by a combination of letters C and numbers. The smaller the number behind C, the stronger the reactivity, and vice versa. The weaker the reactivity, the red precipitation line is not formed, which is represented by B.
  • the IgG antibody used in this example is the 3E50 antibody, which is described in Chinese Patent Publication No. CN112239501A, which is incorporated herein by reference.
  • the antibody can be selected from any one of antibodies with any group of mutations in paragraphs [0049] to [0052] of CN112239501A, mutation combination 1 to mutation combination 68, such as mutation combination 50, which have similar properties.
  • the IgG antibody used in this example is the 5F27 antibody, which is described in Chinese patent CN112239500A, which is incorporated herein by reference.
  • the antibody can be selected from any one of antibodies with any group of mutations in paragraphs [0048] to [0049] of CN112239500A, mutation combination 1 to mutation combination 42, such as mutation combination 20, which have similar properties.
  • the expression plasmids of SARS-CoV-2 3E50TB2 IgG/IgM and 5F27TB2 IgG/IgM recombinant antibodies were constructed according to the construction method of the MA-9G7TB2 expression plasmid in Example 2.
  • the expressed SARS-CoV-2 3E50TB2 and 5F27TB2 IgG/IgM recombinant antibodies will be antibodies formed by linking the respective VH+CH1 segments (including the hinge region) of 3E50 and 5F27 IgG and the above CH2-CH3-CH4 segments of IgM .
  • SARS-CoV-2 3E50TB2 and 5F27TB2 IgG/IgM recombinant antibodies were prepared in the manner of expressing MA-9G7TB2 recombinant antibodies in Example 3.
  • Figure 2 and Figure 3 respectively show the electrophoresis results of 3E50TB2 and 5F27TB2 IgG/IgM hybrid recombinant antibodies.
  • the vaccine refers to the inactivated sample of the new coronavirus.
  • Anti-cTnI-21C5 monoclonal antibody (hereinafter referred to as CTNI 21C5) was used as the IgG antibody in this example.
  • CTNI 21C5 monoclonal antibody was used as the IgG antibody in this example.
  • the sequence of this monoclonal antibody is described in Chinese Patent Publication No. CN111018983A, which is incorporated herein by reference.
  • the antibody can be selected from any one of the antibodies with any group of mutations from [0080] to [0084] paragraphs [0080] to [0084] of CN111018983A, mutation combination 1 to mutation combination 56, such as mutation combination 37, which have similar properties.
  • the expression plasmid of CTNI 21C5TB2 IgG/IgM recombinant antibody was constructed referring to the construction method of the MA-9G7TB2 expression plasmid in Example 2.
  • CTNI 21C5TB2 IgG/IgM recombinant antibody will be an antibody formed by linking the VH+CH1 segment (including the hinge region) of CTNI 21C5 IgG and the above CH2-CH3-CH4 segment of IgM.
  • the expression plasmid of CTNI 21C5TB1 IgG/IgM recombinant antibody was constructed referring to the construction method of MA-9G7TB1 expression plasmid in Example 2.
  • CTNI 21C5TB1 IgG/IgM recombinant antibody will be an antibody formed by splicing the heavy chain variable region (VH) segment of CTNI 21C5 IgG with the above CH segment of IgM.
  • VH heavy chain variable region
  • the expression plasmids of CTNI 21C5TB3 and CTNI-TBQ IgG/IgM recombinant antibodies were constructed.
  • the expressed CTNI-TBQ IgG/IgM recombinant antibody will be an antibody formed by splicing the CTNI 21C5 full-length antibody (including the complete constant region) and the CH3-CH4 segment of IgM.
  • the expressed CTNI 21C5TB3 IgG/IgM recombinant antibody will be an antibody formed by linking the VH+CH1-CH2 segment of IgG (including the hinge region) and the CH3-CH4 segment of IgM.
  • CTNI 21C5TB2, CTNI 21C5TB3, full-length antibody CTNI-TBQ and CTNI 21C5TB1 IgG/IgM recombinant antibody were prepared according to the preparation method for expressing MA-9G7TB2 recombinant antibody in Example 3.
  • Fig. 4 is the electrophoresis result of the 21C5TB2 hybrid recombinant antibody.
  • CTNI 21C5TB2 Preparation of CTNI 21C5TB2, CTNI 21C5TB3, full-length antibody CTNI-TBQ, CTNI 21C5TB1 and CTNI 21C5 fluorescence chromatography detection reagent cards
  • nitrocellulose membrane Dilute CTNI 21C5TB2 recombinant antibody to 1-2mg/ml with coating buffer, draw T line (detection line), T line is close to the end of the gold standard pad, about 5mm away from the end of the gold standard pad; Dilute goat anti-mouse IgG antibody (manufactured by Feipeng Biological Co., Ltd., product number BA-PAB-MU0001) to 1-2 mg/ml in coating buffer, and draw line C (control line). Pad about 3mm. Dry at 37°C and package for later use.
  • CTNI 21C5TB3 full-length antibody CTNI-TBQ, CTNI 21C5TB1 and CTNI 21C5 fluorescence chromatography detection reagent cards to prepare fluorescence chromatography detection reagent cards according to the method described for the preparation of CTNI 21C5TB2 fluorescence chromatography detection reagent cards; CTNI 21C5TB1, full The mass concentration of the long antibody CTNI-TBQ, CTNI 21C5TB2, CTNI 21C5TB3 IgG/IgM hybrid recombinant antibody T-line hybrid recombinant antibody was the same as that of the control antibody.
  • the purchased CTNI antigen was used as the test sample. Tear open the aluminum foil bag and take out the test card. For each test sample, draw 50 ⁇ L of sample, add the sample to the sample diluent, mix well, draw 50 ⁇ L and add it to the sample hole of the test card; the reaction time of the test card after adding the sample is 15 minutes; the fluorescence detector will Automatically run tests and read results. At the same time, 30 clinically rated specimens collected from the hospital were used as test samples to detect the specimen correlation r of 21C5TB2 or 21C5 antibody. The specific test results are shown in Table 5, which shows that the detection sensitivity of the 21C5TB2 antibody is greater than that of 21C5.
  • the correlation r of 30 samples detected with 21C5TB1, 21C5TB3 and 21C5TBQ antibodies were all above 0.9, and the sensitivity was 21C5TB2>21C5TB3>21C5TBQ>21C5TB1.
  • Anti-CKMB 10C5 monoclonal antibody (hereinafter referred to as CKMB 10C5) was used as the IgG antibody in this example.
  • the sequence of this monoclonal antibody is described in Chinese Patent Publication No. CN111349168A, which is incorporated herein by reference.
  • Antibodies can randomly select any antibody with any mutation combination 1 to mutation combination 56 of paragraphs [0096] to [0098] of CN111349168A, such as mutation combination 11, which have similar properties.
  • the polynucleotide encoding the VH region + part of the CH1 segment of CKMB 10C5 was connected with the polynucleotide encoding the fragment from the hinge region to the C-terminus of the IgA CH region by means of bridge PCR.
  • the expression plasmid of CKMB 10C5TB1 IgG/IgM recombinant antibody was constructed according to the construction method of the expression plasmid of MA-9G7TB1 in Example 2.
  • the expressed CKMB 10C5TB1 IgG/IgM recombinant antibody is an antibody formed by linking the VH segment of CKMB 10C5IgG with the above CH segment of IgM.
  • CTNI-TBQ IgG/IgM recombinant antibody was constructed.
  • the expressed CTNI-TBQ IgG/IgM recombinant antibody is an antibody formed by linking the CKMB 10C5 full-length antibody (including the complete constant region) and the above-mentioned fragment of the same IgA.
  • the light chain clone of the antibody in this example was constructed using the light chain plasmid construction method disclosed in Chinese Patent Publication No. CN111349168A.
  • CKMB 10C5TB4, CKMB 10C5TB1 and CTNI-TBQ IgG/IgM recombinant antibodies were prepared according to the method of expressing MA-9G7TB2 recombinant antibodies in Example 3, using captoL (cytiva) and CHT (Bio-Rad) two kinds of fillers, by profiling and chromatography to obtain antibodies in two steps.
  • the purchased CKMB antigen was used as the test sample. Tear open the aluminum foil bag and take out the test card. For each test sample, draw 50 ⁇ L of sample, add the sample to the sample diluent, mix well, draw 50 ⁇ L and add it to the sample hole of the test card; the reaction time of the test card after adding the sample is 15 minutes; the fluorescence detector will Automatically run tests and read results.
  • the correlation r of 10C5TB4 or 10C5 antibody was detected by using 30 clinically rated samples collected from the hospital as test samples. The specific test results are shown in Table 6. It can be seen that the detection sensitivity of the 10C5TB4 antibody is greater than that of 10C5, and the correlation r of the 30 samples detected are all above 0.9.
  • the correlation r of 30 samples detected with 10C5TB1 and 10C5TBQ antibodies were all above 0.9, and the sensitivity was 10C5TB4>10C5TBQ>10C5TB1.

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Abstract

提供一种嵌合的免疫球蛋白。其由第一多肽和第二多肽融合得到：所述第一多肽包括位于N端的IgG可变区；以及所述第二多肽包括位于C端的IgA CH2- CH3区，或IgM CH3-CH4区。该免疫球蛋白具有更高的效价，且包被于固相时相比于价态相同的IgG单体仍然具有更高的效价，因此可广泛应用于检测领域。

Description

嵌合的免疫球蛋白
相关申请的交叉引用
本申请要求2021年6月17日申请的，申请号为202110674660.8，名称为“嵌合的免疫球蛋白”的中国专利申请的优先权，在此将其全文引入作为参考。
技术领域
本申请涉及抗体工程领域，具体而言，涉及一种嵌合的免疫球蛋白。
背景技术
免疫球蛋白(immunoglobulin，Ig)由四条多肽链组成，各多肽链之间由数量不等的链间二硫键连接。Ig可呈“Y”字型结构，称为Ig单体，是构成抗体的基本单位。天然Ig分子含有四条异源性多肽链，其中，分子量较大的两条链称为重链(heavy chain，H)，而分子量较小的两条链称为轻链(light chain，L)。同一Ig分子中的两条重链和两条轻链的氨基酸组成完全相同。重链分子量为50 000～75 000，由450～550个氨基酸残基组成。由于重链恒定(CH)区的氨基酸组成和排列顺序不同，其抗原性也不同。据此，可将Ig分为五类(class)，即IgM、IgD、IgG、IgA和IgE。在这五类中，IgG、IgD、IgE均为单体结构。IgM是由B细胞分泌的一种基本抗体，在分泌形态中则是由五个Y型单体构成的五聚体形式，具有极高的亲和力。IgM的分子量极大，因此在抗原凝集反应中非常有效。IgM是一种多价态抗体。IgA可以单体或二聚体、三聚体或四聚体形式存在。分泌型IgA多以二聚体、三聚体形式存在，也是一种多价态抗体。由于一个单体抗体具有2个抗原结合位点，因此，IgA通常具有4个或6个抗原结合位点，而IgM则具有10或12个抗原结合位点，均为多价态抗体。
抗体主要由B淋巴细胞合成。每个B淋巴细胞有合成一种抗体的遗传基因。当机体受抗原刺激时，抗原分子上的许多决定簇分别激活各个具有不同基因的B细胞。被激活的B细胞分裂增殖形成多克隆，并合成多种抗体。1975年，英国的科学家Kohler和Milstein合作将已适应于体外培养的小鼠骨髓瘤细胞与绵羊红细胞免疫小鼠脾细胞(B淋巴细胞)进行融合，发现形成的杂交细胞既能在体外无限增殖，又能持续的分泌特异性的抗体，通过克隆化的培养可获得的纯的细胞可以产生单克隆抗体，由此发明了单克隆抗体技术。单克隆抗体技术的出现，是免疫学领域的重大突破。到二十世纪八十年代中期，单克隆抗体技术已日臻完善，开始广泛应用于生物医学研究和生物技术以及临床诊断和治疗等许多领域。
随着科学技术进步，体外诊断已经成为临床医学诊断的重要帮手，扮演着医生眼睛的角色。单克隆抗体作为临床医学体外诊断试剂中的核心原材料之一充分发挥其优势。单克隆抗体的特异性强，大大提高了抗原—抗体反应的特异性，减少了和其它物质发生交叉反应的可能性，使试验结果可信度更大。单克隆抗体的均一性和生物活性的单一性，使抗原—抗体反应结果便于控制，利于标准化和规范化。
免疫诊断试剂是体外诊断试剂的细分领域。免疫诊断(immunodiagnosis)是应用免疫学的理论、技术和方法诊断各种疾病和测定免疫状态。免疫诊断的方法包括放射免疫、酶联免疫、化学发光、荧光层析或胶体金层析等。这些方法的基本原理如下描述。使抗原或抗体结合到某 种固相载体表面，并保持其免疫活性，选择合适的标记物与抗原或抗体通过化学法形成抗原或抗体标记复合物。在测定时，把受检标本(测定其中的抗体或抗原)和抗原或抗体标记复合物按不同的步骤与固相载体表面的抗原或抗体起反应。用洗涤的方法使固相载体上形成的抗原抗体复合物与其他物质分开，最终结合在固相载体上的标记复合物与标本中受检物质的量成一定的比例。加入与标记复合物反应的底物后，底物被催化变为有色产物。因为有色产物的量与标本中受检物质的量直接相关，故可根据颜色反应的深浅来进行定性或定量分析。然而，目前对免疫诊断用抗体效价的提升还多依赖于抗体免疫及筛选过程的改进，其效果具有很大的随机性，不能很好地满足检测需要。
发明内容
本申请的第一方面涉及一种免疫球蛋白，其由第一多肽和第二多肽融合得到，其中
所述第一多肽包括位于N端的IgG可变区；以及
所述第二多肽包括位于C端的IgA CH2-CH3区，或IgM CH3-CH4区。
本申请的第二方面涉及多聚体，其以如上所述的免疫球蛋白为单体聚合得到。
本申请的第三方面涉及分离的核酸，其编码如上所述的免疫球蛋白。
本申请的第四方面涉及载体，其含有如上所述的核酸。
本申请的第五方面涉及宿主细胞，其含有如上所述的核酸或被如上所述的载体所转化。
本申请的第六方面涉及一种免疫球蛋白的制备方法，包括表达如上所述的宿主细胞或将上述的第一多肽和第二多肽进行融合表达，及由上述制备方法制备得到的免疫球蛋白。
本申请的第七方面涉及一种多聚体的制备方法，包括将以如上所述免疫球蛋白的制备方法制备得到的免疫球蛋白作为单体进行聚合，及由上述制备方法制备得到的多聚体。
本申请的第八方面涉及固相载体，其表面包被有如上所述的多聚体。
本申请的第九方面涉及含有如上所述的多聚体，或如上所述固相载体的试剂盒或试纸条。
本申请的第十方面涉及检测抗原的方法，包括用如上所述的多聚体，或如上所述的固相载体与所述抗原接触形成缀合物，并对所述缀合物进行检测；
所述抗原为能够与所述IgG可变区发生特异性结合的抗原。
本申请的第十一方面涉及所述的多聚体、固相载体、试剂盒或试纸条在免疫检测中的应用。
本申请所提供的IgG与IgM/IgA嵌合的免疫球蛋白具有更高的效价，并且当包被于固相时，相比于价态相同的IgG单体，仍然具有更高的效价，因此可广泛应用于检测领域。
本申请的一个或多个实施方式的细节在下面的附图和描述中提出。本申请的其它特征、目的和优点将从说明书、附图以及权利要求书变得明显。
附图说明
为了更清楚地说明本申请具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本申请的一些实施方式，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的附图。
图1示出本申请一个实施例中MA-9G7TB1和MA-9G7TB2 IgG/IgM杂合重组抗体的电泳结果；
图2示出本申请一个实施例中3E50TB2杂合重组抗体的电泳结果；
图3示出本申请一个实施例中5F27TB2杂合重组抗体的电泳结果；
图4示出本申请一个实施例中21C5TB2杂合重组抗体的电泳结果。
具体实施方式
现将详细地提供本申请实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本申请。实际上，对本领域技术人员而言，显而易见的是，可以对本申请进行多种修改和变化而不背离本申请的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本申请的发明人在实验过程中意外发现正常IgG抗体随着温度、缓冲条件等保存条件的变化，会出现聚合的现象。多聚抗体的出现直接体现为，在诊断试剂应用的过程中抗体活性表现出远远高于单体抗体的活性。然而，自然出现的IgG多聚体是不稳定的。结合IgA和IgM抗体的特点以及其恒定区亲疏水的特性以及得到均一的、高表达的多聚体抗体的目标，发明人发现构建的IgG/IgA、IgG/IgM的嵌合抗体在诊断试剂检测过程中的性能远远高于IgG类抗体，并且嵌合抗体的均一性及表达量方面都有很好的优势。
由此，构建了IgG/IgA、IgG/IgM嵌合抗体并将其应用于体外诊断检测试剂的包被端或标记端。本申请的IgG/IgA、IgG/IgM嵌合抗体可替代现有的IgG抗体，从而得到活性更优的检测试剂盒。
第一方面，本申请的实施方式提供一种免疫球蛋白，其由第一多肽和第二多肽融合得到，其中
第一多肽包括位于N端的IgG可变区；以及
第二多肽包括位于C端的IgA CH2-CH3区，或IgM CH3-CH4区。
在一些实施方式中，第一多肽中的IgG还具有CH1区。
在一些实施方式中，第二多肽中的IgA或IgM还具有CH1区。
在一些实施方式中，第一多肽还包含IgG的铰链区。
在一些实施方式中，第二多肽还包含IgA或IgM的铰链区。
在一些实施方式中，第一多肽中IgG还具有CH1-CH2区。
在一些实施方式中，第一多肽中IgG还具有CH1-CH2-CH3区。
在一些实施方式中，第二多肽中IgM还具有CH1-CH2区。
在一些实施方式中，所述IgG为IgG1、IgG2、IgG3或IgG4。
在一些实施方式中，第一多肽和第二多肽中的至少一个包含CH1区。
在一些实施方式中，第一多肽和第二多肽中的至少一个包含铰链区。
IgG、IgA和IgM可以独立地选自相同或者不同的种属来源，例如鼠(小鼠、大鼠)、兔、绵羊、山羊、马、鸡、牛、犬以及人。
第二方面，本申请的实施方式提供多聚体，其以如上所述的免疫球蛋白为单体聚合得到。
在一些实施方式中，所述的多聚体为2、3或4个具有IgA CH2-CH3区的免疫球蛋白单体聚合得到。
在一些实施方式中，所述的多聚体为5个具有IgM CH3-CH4区的免疫球蛋白单体聚合得到。
在一些实施方式中，所述多聚体还缀合有信号物质。
第三方面，本申请的实施方式提供分离的核酸，其编码如上所述的免疫球蛋白。
第四方面，本申请的实施方式还提供载体，其含有如上所述的核酸。
术语“载体(vector)”是指，可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。在一些实施方式中，本申请所述载体中包含基因工程中常用的调控元件，例如增强子、启动子、内部核糖体进入位点(IRES)和其他表达控制元件(例如转录终止信号，或者多腺苷酸化信号和多聚U序列等)。
第五方面，本申请的实施方式还提供宿主细胞，其含有如上所述的核酸或被如上所述的载体所转化。
术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。宿主细胞优选为真核细胞，更优选为哺乳动物细胞。
第六方面，本申请的实施方式提供一种免疫球蛋白的制备方法，包括表达上述的宿主细胞。
本申请的实施方式还提供一种免疫球蛋白的制备方法，包括将第一多肽和第二多肽进行融合表达，其中，
第一多肽包括位于N端的IgG可变区；以及
第二多肽包括位于C端的IgA CH2-CH3区，或IgM CH3-CH4区。
在一些实施方式中，所述的免疫球蛋白，第一多肽中IgG还具有CH1区。
在一些实施方式中，第二多肽中IgA或IgM还具有CH1区。
在一些实施方式中，第一多肽还包含IgG的铰链区。
在一些实施方式中，第二多肽还包含IgA或IgM的铰链区。
在一些实施方式中，第一多肽中IgG还具有CH1-CH2区。
在一些实施方式中，第一多肽中IgG还具有CH1-CH2-CH3区。
在一些实施方式中，第二多肽中IgM还具有CH1-CH2区。
在一些实施方式中，所述IgG为IgG1、IgG2、IgG3或IgG4。
在一些实施方式中，第一多肽和第二多肽中的至少一个包含CH1区；
在一些实施方式中，第一多肽和第二多肽中的至少一个包含铰链区。
本申请的实施方式还提供一种由上述免疫球蛋白的制备方法制备得到的免疫球蛋白。
第七方面，本申请的实施方式提供一种多聚体的制备方法，包括将以如上所述免疫球蛋白的制备方法制备得到的免疫球蛋白作为单体进行聚合。
在一些实施方式中，将2、3或4个具有IgA CH2-CH3区的免疫球蛋白单体进行聚合。
在一些实施方式中，将5个具有IgM CH3-CH4区的免疫球蛋白单体进行聚合。
本申请的实施方式还提供一种由上述多聚体的制备方法制备得到的多聚体。
第八方面，本申请的实施方式提供一种固相载体，其表面包被有如上所述的多聚体。
在一些实施方式中，所述固相载体是塑料载体、微粒或膜载体；所述塑料载体可以是聚苯乙烯载体；微粒可以是磁微粒；所述膜载体可以是硝酸纤维素膜、玻璃纤维素膜或尼龙膜。
在一些实施方式中，所述固相载体选自试管、EP管、多孔板、层析柱、微量反应板的孔。
在本申请中，术语“微粒”可以为球体、近球体、立方体、多面体或不规则形状。微粒的直径可以为10nm～1mm，例如100nm、500nm、1μm、10μm、100μm、500μm；优选为400nm～10μm。
微粒优选为磁微粒，其成分中含有磁性物质。磁性物质可以为金属(金属单质或合金)、非金属，或金属与非金属所形成的复合物。金属例如铁、铝镍钴金属等；非金属例如铁氧体非金属(优选为Fe 2O 3或Fe 3O 4磁性纳米粒子)；金属与非金属所形成的复合物例如钕铁硼橡胶磁复合材料。
多孔板优选为酶标板，其可以具有8孔、16孔、32孔、48孔、64孔、96孔或更多孔。
第九方面，本申请的实施方式提供含有如上所述的多聚体，或如上所述固相载体的试剂盒或试纸条。
在一些实施方式中，所述试纸条包括样品垫、结合垫、反应膜和吸收垫，所述反应膜上设置有检测区和质检区；
所述多聚体包被于所述检测区和/或滴加在所述结合垫。
容易理解，当所述检测区和所述结合垫均含有多聚体的时候，多聚体的IgG可变区所结合的抗原表位不同，第二多肽端结构域可以相同也可以不同。
在一些实施方式中，所述试剂盒中还可以包含样品预处理试剂(如样品纯化富集试剂，裂解液等)、清洗液(如水等)、缓冲液(如PBS或者Tris等)、针对信号物质的显色试剂(如针对辣根过氧化物酶作为信号物质的ECL或DAB)等。
第十方面，本申请的实施方式提供一种检测抗原的方法，包括：
用如上所述的多聚体或如上所述的固相载体与所述抗原接触形成缀合物，和
对所述缀合物进行检测，
其中，所述抗原为能够与所述IgG可变区发生特异性结合的抗原。
可以通过本领域技术人员公知的任何方法对所述缀合物进行检测，例如双抗夹心法，即，在抗原与所述固相载体接触后，用额外的针对所述抗原的另一抗体(其通常标记有信号物质，或者用标记有信号物质的二抗对其进行检测)对所述缀合物进行检测。
可替代地，用常规抗体作为包被在固相载体上的抗体，并将其与待检测抗原共孵育，以所述多聚体为游离的检测抗体(其通常标记有信号物质，或者用标记有信号物质的二抗对其进行检测)进行检测。
可替代地，用多聚体包被的固相载体与另一多聚体配合检测所述抗原，此时固相载体上包被的多聚体与另一多聚体二者为配对抗体。
上述信号物质可以为荧光物质、量子点、地高辛标记探针、生物素、放射性同位素、放射性造影剂、顺磁离子荧光微球、电子致密物质、化学发光标记物、超声造影剂、光敏剂、胶体金或酶中的任一种。在一些实施方式中，所述信号物质是胶体金、荧光素、荧光微球、吖啶酯、辣根过氧化物酶、碱性磷酸酶或β-半乳糖苷酶。
第十一方面，本申请的实施方式提供如上所述的多聚体、固相载体、试剂盒或试纸条在免疫检测中的应用。
本申请的实施方式还提供用于免疫检测的如上所述的多聚体、固相载体、试剂盒或试纸条。
本申请的实施方式还提供一种免疫检测的方法，该方法包括使用如上所述的多聚体、固相载体、试剂盒或试纸条进行免疫检测。
再一方面，本申请的实施方式提供一种免疫诊断试剂盒，其包含IgG-IgM或IgG-IgA嵌合免疫球蛋白的多聚体。
在一些实施方式中，所述免疫球蛋白由第一多肽和第二多肽融合得到，其中
第一多肽包括位于N端的IgG可变区；以及
第二多肽包括位于C端的IgA CH2-CH3区，或IgM CH3-CH4区。
在一些实施方式中，所述免疫球蛋白，第一多肽中IgG还具有CH1区。
在一些实施方式中，第二多肽中IgA或IgM还具有CH1区。
在一些实施方式中，第一多肽还包含IgG的铰链区。
在一些实施方式中，第二多肽还包含IgA或IgM的铰链区。
在一些实施方式中，第一多肽中IgG还具有CH1-CH2区。
在一些实施方式中，第一多肽中IgG还具有CH1-CH2-CH3区。
在一些实施方式中，第二多肽中IgM或IgA还具有CH1-CH2区。
在一些实施方式中，所述IgG为IgG1、IgG2、IgG3或IgG4。
在一些实施方式中，第一多肽和第二多肽中的至少一个包含CH1区。
在一些实施方式中，第一多肽和第二多肽中的至少一个包含铰链区。
在一些实施方式中，所述的多聚体为2、3或4个具有IgA CH2-CH3区的免疫球蛋白单体聚合；或为5个具有IgM CH3-CH4区的免疫球蛋白单体聚合。
在一些实施方式中，所述多聚体还缀合有信号物质。
在一些实施方式中，所述试剂盒还包括样品预处理试剂、清洗液、缓冲液以及显色试剂。
在一些实施方式中，所述试剂盒还包括样品垫、结合垫、反应膜和吸收垫，所述反应膜上 设置有检测区和质检区。
在一些实施方式中，所述试剂盒还可以包括另一种抗体，另一种抗体可以是多聚体或单体。
在一些实施方式中，所述多聚体可以固定在固相载体上，所述固相载体包括硝酸纤维素膜、乳胶、磁珠或ELISA板等。
在一些实施方式中，所述多聚体可以用纳米颗粒、化学发光物质、荧光物质、放射性物质、胶体、酶等信号物质进行标记。
下面将结合实施例对本申请的实施方案进行详细描述。
实施例
以下实施例中，限制性内切酶、Prime Star DNA聚合酶购自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。
实施例1编码IgM、IgA重链恒定区的多核苷酸的制备
本申请中，编码鼠抗体CH区的多核苷酸可以通过PCR的方式从鼠外周血细胞DNA中获取，也可以通过PCR的方式从分泌相应类型的单克隆抗体的杂交瘤细胞株获取。
编码IgM CH区的多核苷酸的获得
通过PCR的方式从分泌IgM类型的鼠单克隆抗体的杂交瘤细胞株6M9(可获自菲鹏生物)的基因组中获取编码鼠IgM-CH区的多核苷酸。所使用的引物如下：
正向引物序列(5’→3’)：GAG AGT CAG TCC TTC CCA AAT GTC TTC CCC CTC GTC TC(SEQ ID NO:1)；
反向引物序列(5’→3’)：CCC GAA TTC TTA TCA ATA GCA GGT GCC ACC TGT GTC AGA CAT GAT CAG(SEQ ID NO:2)。
通过反向引物紧跟终止密码子TGA TAA(反密码子TTA TCA)之后导入EcoRI限制性位点(GAATTC)。该处采用双终止密码子。通过PCR技术获得约1.4kb的多核苷酸，并将其导入pMD-18T载体中，用于测序确认。
该多核苷酸编码鼠IgM-CH区，其氨基酸序列从N端到C端如下所示：
Figure PCTCN2022078975-appb-000001
编码IgA CH区的多核苷酸的获得
编码IgA CH区的多核苷酸通过PCR的方式从分离的鼠外周血细胞(可获自菲鹏生物)DNA中获取。所使用的引物如下：
正向引物序列(5’→3’)：GAG TCT GCG AGA AAT CCC A CC ATC TAC CCA CTG ACA CTC(SEQ ID NO:4)；
反向引物序列(5’→3’)：CCC GAA TTC TCA GTA GCA GAT GCC ATC TCC CTC TGA CAT GAT(SEQ ID NO:5)。
通过反向引物紧跟终止密码子TGA(反密码子TCA)之后导入EcoRI限制性位点。通过PCR技术获得约1kb的多核苷酸，将其导入pMD-18T载体中，用于测序确认序列。
该多核苷酸编码鼠IgA-CH区，其氨基酸序列从N端到C端如下所示：
Figure PCTCN2022078975-appb-000002
通过类似的方式制备编码IgM CH区的CH1、CH2、CH3和CH4结构域或其组合的多核苷酸，或编码IgA CH区的CH1、CH2和CH3结构域或其组合的多核苷酸。
实施例2 IgG/IgM杂合重组抗体的表达质粒的构建
本实施例中IgG抗体采用anti-Pan-PLDH 9G7单克隆抗体(以下称之为MA 9G7)，该单克隆抗体的序列记载于中国专利公开号CN111363044A，其通过引用并入本文。抗体可以选自任意一种具有CN111363044A中【0087】～【0089】段突变组合1～突变组合56的任意一组突变的抗体，例如突变组合32，它们具有相似的性质。
IgM本身序列具有的多聚体特点。首先将编码MA 9G7的重链可变区(VH)区段1的多核苷酸通过桥式PCR的方式和编码IgM CH区的多核苷酸进行连接。MA 9G7的VH区区段1的氨基酸序列如下：
Figure PCTCN2022078975-appb-000003
通过PCR获得1.8kb左右的多核苷酸，并通过HindIII/EcoRI双酶切，连接至用相应限制酶切割后的pcDNA TM3.4
Figure PCTCN2022078975-appb-000004
vector中，所得的载体命名为pcDNA3.4A-9G7TB1VCH。
然后，选择编码MA 9G7的VH区区段2的多核苷酸通过桥式PCR的方式和编码IgM CH区的CH2起始至C末端的的多核苷酸进行连接。MA 9G7的VH区区段2的氨基酸序列如下：
Figure PCTCN2022078975-appb-000005
通过PCR获得约1.8KB的DNA片段，并通过HindIII/EcoRI双酶切，连接至pcDNA TM 3.4
Figure PCTCN2022078975-appb-000006
vector中，简称pcDNA3.4A-9G7TB2VCH。
由于IgM的多聚特性取决于重链，故本实施例中的抗体的轻链克隆使用中国专利公开号CN111363044A公开的轻链质粒的构建方法进行构建。
实施例3 MA-9G7TB1和MA-9G7TB2 IgG/IgM杂合重组抗体表达制备
1.重组抗体表达质粒线性化
制备如下酶切体系：限制酶Buffer 50ul、重组质粒100ug/管、PvuⅠ酶10ul、无菌水补至500ul，37℃水浴酶切过夜；先用与酶切体系等体积的酚/氯仿/异戊醇(25:24:1)(下层)，再用氯仿(水相)依次进行抽提；用0.1倍体积(水相)的3M醋酸钠和2倍体积乙醇冰上静置沉淀DNA，70％乙醇漂洗DNA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溶，最后进行浓度的测定。
2.重组抗体表达质粒稳定转染，加压筛选稳定细胞株
pcDNA3.4A-9G7TB1VCH质粒和轻链质粒以1:1比例用超纯水稀释至40ug/100ul，取一新离心管，在管内添加并调节CHO细胞浓度至1.43×10 7cells/ml，将100ul质粒与700ul细胞混合，转入电转杯进行电转染，转染后继续培养细胞，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至24孔板，约3天后转至6孔板；3天后保种进行批量培养，调整细胞密度0.5×10 6cells/ml，以2.2ml体积进行批量培养，以细胞密度0.3×10 6cells/ml，2ml进行保种；7天后6孔板中的批量培养上清送样检测，挑选抗体浓度及细胞直径较小的细胞株转TPP管保种传代，得到表达MA-9G7TB1 IgG/IgM杂合重组抗体的细胞株。
pcDNA3.4A-9G7TB2VCH质粒及轻链质粒按如上针对pcDNA3.4A-9G7TB1VCH质粒描述的方法进行转染和筛选，得到表达MA-9G7TB2 IgG/IgM杂合重组抗体的细胞株。
3.细胞扩大培养
细胞复苏之后先接种在含有100％Dynamis培养基的125ml规格的摇瓶中，接种体积为30ml，放置于转速120r/min，温度为37℃，含8％二氧化碳的摇床中培养。培养72h后，以5×10 5cells/ml接种密度接种扩大培养，扩大培养体积根据生产需求进行计算，使用的培养基为100％Dynamis培养基。之后每72h扩大培养一次。当细胞量满足生产需求时，严格控制接种密度为5×10 5cells/ml左右进行生产。
4.摇瓶生产及纯化
摇瓶生产参数：摇床转速120r/min，温度为37℃，二氧化碳含量为8％。流加补料：在摇瓶中培养至72h时开始每天补料，每天流加初始培养体积的3％的HyClone TM Cell Boost TM Feed 7a，每天流加量为初始培养体积的千分之一的HyClone TM Cell Boost TM Feed 7b，一直补到第12天(第12天补料)。在第六天补加3g/L葡萄糖。第13天收样。利用captoL(cytiva)和CHT(Bio-Rad)两种填料，通过亲和层析分两步纯化获得MA-9G7TB1和MA-9G7TB2 IgG/IgM杂合重组抗体的多聚体抗体。取10ul纯化的抗体(浓度为1mg/ml)进行还原性SDS-PAGE，电泳结果如图1所示。图1中左侧泳道为MA-9G7TB1 IgG/IgM重组抗体的条带，右侧泳道为MA-9G7TB2 IgG/IgM重组抗体的条带。每个泳道上显示两条带，一条条带的相对分子质量(Mr)为70-75kD(重链)，另一条Mr为20-35kD(轻链)。
实施例4 MA-9G7TB1和MA-9G7TB2 IgG/IgM杂合重组抗体的应用
MA-9G7TB1胶体金检测试纸的制备
1.硝酸纤维素膜的制备
硝酸纤维素膜的制备：用包被缓冲液将MA-9G7TB1重组抗体稀释到1～5mg/ml，划线为T线，即为检测线，T线靠近胶体金端；用包被缓冲液将羊抗鼠IgG抗体(菲鹏生物股份有限公司生产，货号BA-PAB-MU0001)稀释到1～5mg/ml，划线为C线，即为控制线，C线靠近吸收垫。37℃烘干，封装备用。
2.胶体金、金标记单克隆抗体的制备
2.1胶体金的制备
用双蒸去离子水将1％氯金酸稀释成0.01％，置电炉煮沸，每100ml 0.01％氯金酸加入2ml 1％柠檬酸三钠，继续煮沸，直到液体呈亮红色即停止加热，冷却至室温后补足失水。制备好的胶体金外观纯净、透亮、无沉淀和漂浮物，有效期一周。
2.2胶体金标记抗体的制备
用0.1M碳酸钾调胶体金的pH值至8.2，按8～10μg抗体/ml胶体金加入另一株MA标记抗体(可获自菲鹏生物)，磁力搅拌器混匀30min，搅拌下加入BSA至终浓度为1％静置1小时。将液体在13000rpm、4℃离心30min，弃上清，沉淀用标记洗涤保存液洗涤两次，用十分之一初始胶体金体积的标记洗涤保存液将沉淀重悬，置4℃备用，有效期一周。
3.金标垫的制备
将金标垫浸泡于封闭液中30min后，于37℃烘干。然后将制备好的胶体金标记抗体均匀的铺在金标垫上，每毫升溶液铺20平方厘米，冷冻干燥，封装，置4℃备用。
4.试纸条样品垫的制备
将样品垫浸泡于封闭液(含BSA)中30min后，于37℃烘干，封装，置4℃备用。
5.检测试纸的组装
将吸收垫(购自Millipore公司)、硝酸纤维素膜、金标垫、样品垫设置在不吸水的支撑薄片上，切成3mm宽的小条。每十小条一包，加入干燥剂，真空封装，得到MA-9G7TB1胶体金检测试纸。
使用MA-9G7TB2重组抗体和MA-9G7重组抗体按照针对制备MA-9G7TB1胶体金检测试纸所描述的方法分别制备胶体金检测试纸，T线杂合重组抗体的质量浓度与对照抗体相同。
6.胶体检测试纸条的应用
利用组装好的检测试纸条检测被检材料中是否含有Pan-PLDH蛋白(以下称之为MA蛋白)，从而确定MA抗体对MA蛋白检测的作用性。检测时，MA蛋白先和胶体金标记的MA抗体结合形成MA-胶体金标记-MA抗体复合物，由于毛细管作用，MA-胶体金标记-MA抗体复合物沿硝酸纤维素膜向前泳动，到达检测线时，MA-胶体金标记-MA抗体复合物会与划线的MA包被抗体结合，形成MA抗体-MA-胶体金标记-MA抗体的复合物，从而富集在检测线上，形成红色沉淀线。表1为相应的检测数据。红色沉淀线的深浅表示反应的强弱，红色越深表示反应性越强，反之越弱，同时反应强弱用字母C和数字组合表示，C后面的数字越小表示反应性越强，反之表示反应性越弱，没有形成红色沉淀线用B表示。
表1
Figure PCTCN2022078975-appb-000007
实施例5 SARS-CoV-2 3E50TB2和5F27TB2 IgG/IgM杂合重组抗体的构建、制备及胶体金平台应用
本实施例中IgG抗体使用3E50抗体，该抗体记载于中国专利公开号CN112239501A，其通过引用并入本文。抗体可以选自任意一种具有CN112239501A中【0049】～【0052】段突变组合1～突变组合68的任意一组突变的抗体，例如突变组合50，它们具有相似的性质。本实施例中IgG抗体使用5F27抗体，该抗体记载于中国专利CN112239500A，其通过引用并入本文。抗体可以选自任意一种具有CN112239500A中【0048】～【0049】段突变组合1～突变组合42的任意一组突变的抗体，例如突变组合20，它们具有相似的性质。
1.SARS-CoV-2 3E50TB2 IgG/IgM和5F27TB2 IgG/IgM重组抗体的表达质粒按实施例2中MA-9G7TB2表达质粒的构建方式进行构建。
所表达的SARS-CoV-2 3E50TB2和5F27TB2 IgG/IgM重组抗体将是3E50和5F27 IgG各自的VH+CH1段(含铰链区)和上文的IgM的CH2-CH3-CH4段衔接而成的抗体。
2.SARS-CoV-2 3E50TB2和5F27TB2 IgG/IgM重组抗体按实施例3中表达MA-9G7TB2重组抗体的方式进行制备。
图2和图3分别依次为3E50TB2和5F27TB2 IgG/IgM杂合重组抗体的电泳结果。
3.SARS-CoV-2 3E50TB2和5F27TB2 IgG/IgM重组抗体的应用按实施例4的方法进行性能评估，具体性能评估结果如下表：
表2 3E50与3E50TB2分别作为胶体金包被与5F27标记配套使用的检测数据
Figure PCTCN2022078975-appb-000008
Figure PCTCN2022078975-appb-000009
注：疫苗是指新冠病毒灭活后的样品。
表3 5F27与5F27TB2分别作为胶体金包被与3E50标记配套使用的检测数据
Figure PCTCN2022078975-appb-000010
表4 3E50TB2和5F27TB2分别作为包被和标记相互搭配使用的检测结果
Figure PCTCN2022078975-appb-000011
实施例6 CTNI 21C5TB2 IgG/IgM杂合重组抗体的构建、制备及荧光层析平台应用
本实施例中IgG抗体采用Anti-cTnI-21C5单克隆抗体(以下称之为CTNI 21C5)，该单克隆抗体的序列记载于中国专利公开号CN111018983A，其通过引用并入本文。抗体可以选自任意一种具有CN111018983A的【0080】～【0084】段突变组合1～突变组合56的任意一组突变的抗体，例如突变组合37，它们具有相似的性质。
1.CTNI 21C5TB2 IgG/IgM重组抗体的表达质粒参照实施例2中MA-9G7TB2表达质粒的构建方式进行构建。
所表达的CTNI 21C5TB2 IgG/IgM重组抗体将是CTNI 21C5 IgG的VH+CH1段(含铰链区)段与上文的IgM的CH2-CH3-CH4段衔接而成的抗体。
CTNI 21C5TB1 IgG/IgM重组抗体的表达质粒参照实施例2中MA-9G7TB1表达质粒的构建方式进行构建。
所表达的CTNI 21C5TB1 IgG/IgM重组抗体将是CTNI 21C5 IgG的重链可变区(VH)段与上文的IgM的CH段衔接而成的抗体。
另外，构建了CTNI 21C5TB3和CTNI-TBQ IgG/IgM重组抗体的表达质粒。具体地，所表达的CTNI-TBQ IgG/IgM重组抗体将是CTNI 21C5全长抗体(包含完整恒定区)和IgM的CH3-CH4段衔接而成的抗体。具体地，所表达的CTNI 21C5TB3 IgG/IgM重组抗体将是IgG的VH+CH1-CH2段(含铰链区)和IgM的CH3-CH4段衔接而成的抗体。
2.CTNI 21C5TB2、CTNI 21C5TB3、全长抗体CTNI-TBQ和CTNI 21C5TB1 IgG/IgM重组抗体按实施例3中表达MA-9G7TB2重组抗体的制备方式进行制备。
图4为21C5TB2杂合重组抗体的电泳结果。
3.CTNI 21C5TB2重组抗体荧光层析平台应用：
3.1CTNI 21C5TB2、CTNI 21C5TB3、全长抗体CTNI-TBQ、CTNI 21C5TB1和CTNI 21C5荧光层析检测试剂卡的制备
3.1.1硝酸纤维素膜的制备
硝酸纤维素膜的制备：用包被缓冲液将CTNI 21C5TB2重组抗体稀释至1～2mg/ml，划T线(检测线)，T线靠近金标垫端，距金标垫端约5mm；用包被缓冲液将羊抗鼠IgG抗体(菲鹏生物股份有限公司生产，货号BA-PAB-MU0001)稀释至1～2mg/ml，划C线(控制线)，C线靠近吸收垫，距吸收垫约3mm。37℃烘干，封装备用。
3.1.2荧光层析标记单克隆抗体的制备
取1ml 1％固含量的荧光微球，离心去上清后，加入和微球等体积的标记活化液超声混匀，再加入活化剂EDC(终浓度5mg/ml)和NHS(终浓度5mg/ml)，避光震荡混匀20min后离心去上清，加入和微球等体积的标记偶联液超声混匀后加入另一株CTNI标记抗体0.2～1mg，避光震荡混匀3h，最后加入标记封闭液进行封闭，避光震荡混合45min后终止标记，离心去上清，用微球标记保存液复溶微球，超声混匀，置4℃备用，有效期1个月。
3.1.3荧光标记垫的制备
用标记物稀释液将标记物稀释5～10倍后，使用喷垫仪将稀释后的标记物喷于玻璃纤维垫上。封装标记垫，置4℃备用。
3.1.4试纸条样品垫的制备
将玻璃纤维垫浸泡于封闭液(含BSA)中30min后，于37℃烘干，封装样品垫，置4℃备用。
3.1.5检测试剂卡的组装
将吸收垫(购自Millipore公司)、硝酸纤维素膜、标记垫、样品垫设置在不吸水的支撑薄片上，切成3mm宽的小条，装入卡壳中，每根卡一包，加入干燥剂，铝箔袋真空封装，得到CTNI 21C5TB2荧光层析检测试剂卡。
使用CTNI 21C5TB3、全长抗体CTNI-TBQ、CTNI 21C5TB1和CTNI 21C5荧光层析检测试剂卡按照针对制备CTNI 21C5TB2荧光层析检测试剂卡所描述的方法分别制备荧光层析检测试剂卡；CTNI 21C5TB1、全长抗体CTNI-TBQ、CTNI 21C5TB2、CTNI 21C5TB3 IgG/IgM杂合重组抗体T线杂合重组抗体的质量浓度与对照抗体相同。
3.2 CTNI 21C5TB2重组抗体荧光层析平台测试结果
以购买的CTNI抗原作为检测样本。撕开铝箔袋，取出测试卡。对于每个检测样本，吸取50μL的样本，将样本加入样本稀释液中，充分混匀后吸取50μL加入到测试卡的加样孔中；测试卡加样后的反应时间为15min；荧光检测仪将自动进行测试并读取结果。同时，以从医院收集的30份临床定值标本作为检测样本，检测21C5TB2或21C5抗体的标本相关性r。具体测试结果如表5所示，显示21C5TB2抗体的检测灵敏度大于21C5。同理，按照上述的测试方法，测得使用21C5TB1、21C5TB3及21C5TBQ抗体检测30份标本的相关性r均在0.9以上，且灵敏度21C5TB2＞21C5TB3＞21C5TBQ＞21C5TB1。
表5
临床赋值标本(ng/ml) 21C5 21C5TB2
50 2.168 6.209
18 0.746 3.022
10.5 0.450 1.700
3 0.180 0.460
0.7 0.045 0.110
0.15 0.026 0.040
0.08 0.020 0.027
0.04 0.017 0.023
0 0.015 0.015
检测30份标本的相关性r 0.96 0.98
实施例7 CKMB 10C5TB4IgG/IgA杂合重组抗体的构建、制备及荧光层析平台应用
本实施例中IgG抗体采用Anti-CKMB 10C5单克隆抗体(以下称之为CKMB 10C5)，该单克隆抗体的序列记载于中国专利公开号CN111349168A，其通过引用并入本文。抗体可以随机选择任意一种具有CN111349168A的【0096】～【0098】段突变组合1～突变组合56的任意一组突变的抗体，例如突变组合11，它们具有相似的性质。
1.CKMB 10C5TB4 IgG/IgA杂合重组抗体的表达质粒的构建
首先将编码CKMB 10C5的VH区+部分CH1区段的多核苷酸通过桥式PCR的方式和编码IgA CH区的铰链区起始至C末端的片段的多核苷酸进行连接。通过PCR获得1.5kb左右的DNA片段，并通过HindIII/EcoRI双酶切，连接至pcDNA TM 3.4
Figure PCTCN2022078975-appb-000012
vector中，简称pcDNA3.4A-10C5TB3VCH。
CKMB 10C5TB1 IgG/IgM重组抗体的表达质粒参照实施例2中MA-9G7TB1的表达质粒的构建方式进行构建。
所表达的CKMB 10C5TB1 IgG/IgM重组抗体是将CKMB 10C5IgG的VH段与上文的IgM的CH段衔接而成的抗体。
另外，构建了CTNI-TBQ IgG/IgM重组抗体的表达质粒。具体地，所表达的CTNI-TBQ IgG/IgM重组抗体将CKMB 10C5全长抗体(包含完整恒定区)和上述相同的IgA的片段连接而成的抗体。
本实施例中的抗体的轻链克隆使用中国专利公开号CN111349168A公开的轻链质粒的构建方法进行构建。
2.CKMB 10C5TB4、CKMB 10C5TB1和CTNI-TBQ IgG/IgM重组抗体按实施例3中表达MA-9G7TB2重组抗体的方式进行制备，利用captoL(cytiva)和CHT(Bio-Rad)两种填料，通过亲和层析分两步纯化获得抗体。
3.CKMB 10C5TB4重组抗体荧光层析平台应用：
3.1全长抗体CKMB-TBQ、CKMB 10C5和CKMB 10C5TB4的荧光层析检测试剂卡的制备同实施例6所述；区别在于，本实施例各组杂合重组抗体均是IgA的二聚体，全长抗体CKMB-TBQ、CKMB 10C5TB4 IgG/IgA杂合重组抗体T线杂合重组抗体的质量浓度与对照抗体相同。
3.2 CKMB 10C5TB4重组抗体荧光层析平台测试结果
以购买的CKMB抗原作为检测样本。撕开铝箔袋，取出测试卡。对于每个检测样本，吸取50μL的样本，将样本加入样本稀释液中，充分混匀后吸取50μL加入到测试卡的加样孔中；测试卡加样后的反应时间为15min；荧光检测仪将自动进行测试并读取结果。同时，以从医院收集的30份临床定值标本作为检测样本，检测10C5TB4或10C5抗体的标本相关性r。具体测试结果如表6所示，可知10C5TB4抗体的检测灵敏度大于10C5，检测30份标本的相关性r均在0.9以上。同理，按照上述的测试方法，测得使用10C5TB1、10C5TBQ抗体检测30份标本的相关性r均在0.9以上，且灵敏度10C5TB4＞10C5TBQ＞10C5TB1。
表6
Figure PCTCN2022078975-appb-000013
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。

Claims (18)

  1. 一种免疫球蛋白，其由第一多肽和第二多肽融合得到：
    所述第一多肽包括位于N端的IgG可变区；以及
    所述第二多肽包括位于C端的IgA CH2-CH3区，或IgM CH3-CH4区。
  2. 根据权利要求1所述的免疫球蛋白，其中，所述第一多肽中的IgG还具有CH1区；
    可选的，所述第二多肽中IgA或IgM还具有CH1区；
    可选的，所述第一多肽还包含IgG的铰链区；
    可选的，所述第二多肽还包含IgA或IgM的铰链区；
    可选的，所述第一多肽中IgG还具有CH1-CH2区；
    可选的，所述第一多肽中IgG还具有CH1-CH2-CH3区；
    可选的，所述第二多肽中IgM或IgA还具有CH1-CH2区；
    可选的，所述IgG为IgG1、IgG2、IgG3或IgG4；
    优选的，所述第一多肽和所述第二多肽中的至少一个包含CH1区；
    优选的，所述第一多肽和所述第二多肽中的至少一个包含铰链区。
  3. 多聚体，其以权利要求1或2所述的免疫球蛋白为单体聚合得到；
    可选的，所述的多聚体为2、3或4个具有IgA CH2-CH3区的免疫球蛋白单体聚合得到；或为5个具有IgM CH3-CH4区的免疫球蛋白单体聚合得到；
    可选的，所述多聚体还缀合有信号物质。
  4. 分离的核酸，其编码权利要求1或2所述的免疫球蛋白。
  5. 载体，其含有权利要求4所述的核酸。
  6. 宿主细胞，其含有权利要求4所述的核酸或被权利要求5所述的载体所转化。
  7. 一种免疫球蛋白的制备方法，包括表达权利要求6所述的宿主细胞。
  8. 一种免疫球蛋白的制备方法，包括将第一多肽和第二多肽进行融合表达：
    所述第一多肽包括位于N端的IgG可变区；以及
    所述第二多肽包括位于C端的IgA CH2-CH3区，或IgM CH3-CH4区。
  9. 根据权利要求8所述的制备方法，其中，所述第一多肽中的IgG还具有CH1区；
    可选的，所述第二多肽中IgA或IgM还具有CH1区；
    可选的，所述第一多肽还包含IgG的铰链区；
    可选的，所述第二多肽还包含IgA或IgM的铰链区；
    可选的，所述第一多肽中IgG还具有CH1-CH2区；
    可选的，所述第一多肽中IgG还具有CH1-CH2-CH3区；
    可选的，所述第二多肽中IgM或IgA还具有CH1-CH2区；
    可选的，所述IgG为IgG1、IgG2、IgG3或IgG4；
    优选的，所述第一多肽和所述第二多肽中的至少一个包含CH1区；
    优选的，所述第一多肽和所述第二多肽中的至少一个包含铰链区。
  10. 一种由权利要求7～9任一项所述制备方法制备得到的免疫球蛋白。
  11. 一种多聚体的制备方法，包括将以权利要求7～9任一项所述制备方法制备得到的免疫球蛋白为单体进行聚合；
    可选的，所述的多聚体为2、3或4个具有IgA CH2-CH3区的免疫球蛋白单体聚合；或为5个具有IgM CH3-CH4区的免疫球蛋白单体聚合。
  12. 一种由权利要求11所述制备方法制备得到的多聚体。
  13. 固相载体，其表面包被有权利要求3或12所述的多聚体；
    可选的，所述固相载体是塑料载体、微粒或膜载体。
  14. 含有权利要求3或12所述的多聚体，或权利要求13所述固相载体的试剂盒或试纸条；
    可选的，所述试纸条包括样品垫、结合垫、反应膜和吸收垫，所述反应膜上设置有检测区和质检区；
    所述多聚体包被于所述检测区和/或滴加在所述结合垫；
    可选的，所述试剂盒包括样品预处理试剂、清洗液、缓冲液以及显色试剂。
  15. 检测抗原的方法，包括：
    用权利要求3或12所述的多聚体，或权利要求13所述的固相载体与所述抗原接触形成缀合物，和
    对所述缀合物进行检测，
    其中，所述抗原为能够与所述IgG可变区发生特异性结合的抗原。
  16. 权利要求3或12所述的多聚体、权利要求13所述的固相载体或权利要求14所述的试剂盒或试纸条在免疫检测中的应用。
  17. 用于免疫检测的权利要求3或12所述的多聚体、权利要求13所述的固相载体或权利要求14所述的试剂盒或试纸条。
  18. 一种免疫检测的方法，包括使用权利要求3或12所述的多聚体、权利要求13所述的固相载体或权利要求14所述的试剂盒或试纸条检测抗原。
PCT/CN2022/078975 2021-06-17 2022-03-03 嵌合的免疫球蛋白 WO2022262321A1 (zh)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101980603A (zh) * 2007-10-11 2011-02-23 比奥根艾迪克Ma公司 LINGO-1和TrkB拮抗剂的用途
CN102643344A (zh) * 2003-07-26 2012-08-22 新兴产品开发西雅图有限公司 结合构建体及其使用方法
CN106029694A (zh) * 2013-12-06 2016-10-12 达纳-法伯癌症研究院公司 治疗性肽
CN111018983A (zh) 2018-10-10 2020-04-17 东莞市朋志生物科技有限公司 一种抗人心肌肌钙蛋白i的抗体及其应用
CN111349168A (zh) 2018-12-20 2020-06-30 东莞市朋志生物科技有限公司 一种抗人ckmb的抗体及其应用
CN111363044A (zh) 2018-12-25 2020-07-03 东莞市朋志生物科技有限公司 一种抗泛种特异性疟原虫乳酸脱氢酶的抗体
CN112239501A (zh) 2020-10-29 2021-01-19 东莞市朋志生物科技有限公司 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒
CN112239500A (zh) 2020-10-29 2021-01-19 东莞市朋志生物科技有限公司 抗新型冠状病毒的抗体和检测新型冠状病毒的试剂盒

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2114168A1 (en) * 1993-03-05 1994-09-06 Zlatko Dembic Chimeric human interferon-gamma-receptor/immunoglobulin polypeptides
EP2706116A1 (en) * 2001-01-17 2014-03-12 Emergent Product Development Seattle, LLC Binding domain-immunoglobulin fusion proteins
PL206701B1 (pl) * 2001-03-07 2010-09-30 Merck Patent Gmbh Immunoglobulinowe białko fuzyjne, kodujący je kwas nukleinowy, replikujący wektor ekspresji zawierający taki kwas nukleinowy, eukariotyczna komórka gospodarza zawierająca taki wektor ekspresji oraz sposób zwiększania ekspresji takiego białka fuzyjnego
HUE030208T2 (en) * 2009-12-10 2017-04-28 Regeneron Pharma Heavy-chain antibody-producing mice
GB201412821D0 (en) * 2014-07-18 2014-09-03 Liverpool School Tropical Medicine Polymeric proteins and uses thereof
CN111732664B (zh) * 2020-07-06 2020-12-01 北京金智准科技有限公司 一种新型冠状病毒重组蛋白、兔-人嵌合抗体、其制备方法及应用
CN112961237B (zh) * 2021-03-10 2022-03-22 中国计量科学研究院 人源化IgM单克隆抗体标准物质及制备方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102643344A (zh) * 2003-07-26 2012-08-22 新兴产品开发西雅图有限公司 结合构建体及其使用方法
CN101980603A (zh) * 2007-10-11 2011-02-23 比奥根艾迪克Ma公司 LINGO-1和TrkB拮抗剂的用途
CN106029694A (zh) * 2013-12-06 2016-10-12 达纳-法伯癌症研究院公司 治疗性肽
CN111018983A (zh) 2018-10-10 2020-04-17 东莞市朋志生物科技有限公司 一种抗人心肌肌钙蛋白i的抗体及其应用
CN111349168A (zh) 2018-12-20 2020-06-30 东莞市朋志生物科技有限公司 一种抗人ckmb的抗体及其应用
CN111363044A (zh) 2018-12-25 2020-07-03 东莞市朋志生物科技有限公司 一种抗泛种特异性疟原虫乳酸脱氢酶的抗体
CN112239501A (zh) 2020-10-29 2021-01-19 东莞市朋志生物科技有限公司 抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒
CN112239500A (zh) 2020-10-29 2021-01-19 东莞市朋志生物科技有限公司 抗新型冠状病毒的抗体和检测新型冠状病毒的试剂盒

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Food immunology", 31 December 2007, CHEMICAL INDUSTRY PRESS , CN, ISBN: 7-5025-9603-8, article FAN, YUANJING: "Chapter 2 Antigen-antibody reaction and immune chemicals: Functional Characteristics of Various Immunoglobulins", pages: 45 - 80, XP009541964 *
"Medical genetics", 31 December 2006, PEOPLE'S HEALTH PUBLISHING HOUSE, CN, ISBN: 7-117-08256-9, article LIANG, SUHUA: "Chapter 14 Developmental genetics: Secondary Genetic Recombination during B lymphocyte Differentiation", pages: 184 - 185, XP009542945 *
"Recombinant antibody", 31 May 2005, SCIENCE PRESS, CN, ISBN: 7-03-014638-7, article SHEN, BEIFEN ET AL.: "Class Switch of Antibodies and its Mechanism", pages: 9 - 11, XP009542388 *
HUANG JIAN, SHIBAGUCHI HIROTOMO, MASAHIDE KUROKI: "Replacement of the constant region gene of a novel anti-CEA antibody and characterization of its biological activity", CHINESE JOURNAL OF CELLULAR AND MOLECULAR IMMUNOLOGY, FOURTH MILITARY MEDICAL UNIVERSITY, XI'AN, CN, vol. 22, no. 5, 31 December 2006 (2006-12-31), CN , pages 650 - 653, XP093014871, ISSN: 1007-8738 *
MIRETE-BACHILLER, S. ET AL.: "Immunoglobulin T genes in Actinopterygii.", FISH AND SHELLFISH IMMUNOLOGY., vol. 108, 3 December 2020 (2020-12-03), XP086417929, DOI: 10.1016/j.fsi.2020.11.027 *

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