CN116425868A - Anti-coxsackievirus A10 monoclonal antibody, and preparation method and application thereof - Google Patents

Anti-coxsackievirus A10 monoclonal antibody, and preparation method and application thereof Download PDF

Info

Publication number
CN116425868A
CN116425868A CN202210348791.1A CN202210348791A CN116425868A CN 116425868 A CN116425868 A CN 116425868A CN 202210348791 A CN202210348791 A CN 202210348791A CN 116425868 A CN116425868 A CN 116425868A
Authority
CN
China
Prior art keywords
antibody
antigen
ser
binding fragment
coxsackievirus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210348791.1A
Other languages
Chinese (zh)
Inventor
张改梅
高帆
李国顺
陈磊
赵丽丽
谢学超
马廷涛
顾美荣
刘建凯
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Minhai Biotechnology Co ltd
Original Assignee
Beijing Minhai Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Minhai Biotechnology Co ltd filed Critical Beijing Minhai Biotechnology Co ltd
Priority to CN202210348791.1A priority Critical patent/CN116425868A/en
Publication of CN116425868A publication Critical patent/CN116425868A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • C07K16/1009—Picornaviridae, e.g. hepatitis A virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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]
    • 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
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5765—Hepatitis delta antigen
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Communicable Diseases (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)

Abstract

The invention relates to the technical field of antibodies, in particular to an anti-coxsackievirus A10 monoclonal antibody, and a preparation method and application thereof. The heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the antibody or antigen binding fragment thereof have amino acid sequences shown in SEQ ID NO.1-3, and the light chain complementarity determining regions CDR1, CDR2 and CDR3 have amino acid sequences shown in SEQ ID NO. 4-6. The antibody or antigen binding fragment thereof has CV-A10 specificity and in-type broad spectrum, can cross-bind and neutralize different subtype strains of CV-A10, has better protection effect on CV-A10 infected animals, and has important significance on CV-A10 detection, vaccine research and development, evaluation and clinical diagnosis, and treatment of CV-A10 infection and related diseases.

Description

Anti-coxsackievirus A10 monoclonal antibody, and preparation method and application thereof
Technical Field
The invention relates to the technical field of antibodies, in particular to an anti-coxsackievirus A10 monoclonal antibody, and a preparation method and application thereof.
Background
Coxsackie virus group 10 (CV-A10), one of the important members of enteroviruses, was first discovered in 1965 as an infectious hepatitis outbreak. Previous studies have shown that CV-A10 can cause hepatitis, hand-foot-and-mouth disease (Hand, foot and mouth disease, HFMD), and may be closely associated with serious neurological complications such as aseptic encephalitis, viral meningitis, etc. In recent years, CV-A10 has become one of the major pathogens causing hand-foot-and-mouth disease in addition to Enterovirus type 71 (Enterovirus 71, EV71), coxsackie virus type A16 (CV-A16). The CV-A10 strain that has been popular in recent years is predominantly of the C genotype (including both the C1 and C2 genotypes).
There is no antiviral drug against CV-A10, and researchers have found by analyzing CV-A10 structure that the entrance and intermediate regions at the VP1 pocket can bind to a highly conserved lead compound (ICA 135) that has an inhibitory effect on CV-A10 virus.
Monoclonal antibodies have the characteristics of high targeting, low toxicity and the like, are an important means for clinical diagnosis and treatment of virus infection, are also an important means for modern life science, and are widely applied to the aspects of clinical diagnosis, treatment, vaccine evaluation and the like. Therefore, the development of monoclonal antibodies against CV-A10 is of great importance for the diagnosis, treatment and vaccine production of the viral infection.
Disclosure of Invention
The invention aims to provide an anti-coxsackievirus A10 antibody and an antigen binding fragment thereof, and a preparation method and application of the antibody and the antigen binding fragment thereof.
Specifically, the invention provides the following technical scheme:
in a first aspect, the present invention provides an antibody or antigen binding fragment thereof having the heavy chain complementarity determining regions CDR1, CDR2, CDR3 of SEQ ID No.1-3 and/or the light chain complementarity determining regions CDR1, CDR2, CDR3 of the antibody or antigen binding fragment thereof having the amino acid sequences of SEQ ID No. 4-6.
In the present invention, an antigen-binding fragment refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, or that competes with the full-length antibody for specific binding to the antigen.
In some embodiments of the invention, the heavy chain complementarity determining regions CDR1, CDR2, CDR3 of the antibody or antigen binding fragment thereof have the amino acid sequences shown in SEQ ID NO.1-3 and the light chain complementarity determining regions CDR1, CDR2, CDR3 have the amino acid sequences shown in SEQ ID NO. 4-6.
In some embodiments of the invention, the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2, CDR3 of the antibody or antigen binding fragment thereof are shown in SEQ ID NO.1-3, and the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2, CDR3 are shown in SEQ ID NO. 4-6.
The antibody or the antigen binding fragment thereof provided by the invention has CV-A10 specificity and in-type broad spectrum, and can cross-bind and neutralize strains of different subtypes of CV-A10.
Preferably, the heavy chain variable region of the antibody or antigen binding fragment thereof has an amino acid sequence as shown in SEQ ID No.7 or has an amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID No. 7; and/or the light chain variable region of the antibody or antigen binding fragment thereof has an amino acid sequence as shown in SEQ ID No.8 or has an amino acid sequence having at least 80% homology with the amino acid sequence as shown in SEQ ID No. 8.
In some embodiments of the invention, the heavy chain variable region of the antibody or antigen binding fragment thereof has an amino acid sequence as shown in SEQ ID No.7 and the light chain variable region has an amino acid sequence as shown in SEQ ID No. 8.
In some embodiments of the invention, the heavy chain variable region of the antibody or antigen binding fragment thereof has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence homology to the amino acid sequence as shown in SEQ ID No.7, and the light chain variable region has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence homology to the amino acid sequence as shown in SEQ ID No.8, with the heavy chain and light chain complementarity determining regions CDR1, CDR2, CDR3 described above.
In some embodiments of the invention, the antibody or antigen binding fragment thereof further comprises a signal peptide at the N-terminus of the heavy chain variable region.
In some embodiments of the invention, the signal peptide at the N-terminus of the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 9.
In some embodiments of the invention, the antibody or antigen binding fragment thereof further comprises a signal peptide at the N-terminus of the light chain variable region.
In some embodiments of the invention, the signal peptide at the N-terminus of the light chain variable region has the amino acid sequence shown in SEQ ID No. 10.
In the present invention, the antibody may be a monoclonal antibody, a bispecific antibody or a multispecific antibody.
Wherein the monoclonal antibody may be a murine antibody, a chimeric antibody or a humanized antibody.
In the present invention, the antigen binding fragment may be Fab, fab ', F (ab') 2, fd, fv, dAb, a complementarity determining region fragment, or a single chain antibody.
In some embodiments of the invention, the antibody further comprises a heavy chain constant region.
In some embodiments of the invention, the heavy chain of the antibody is IgG2b.
In some embodiments of the invention, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO. 11.
In some embodiments of the invention, the antibody further comprises a light chain constant region.
In some embodiments of the invention, the light chain of the antibody is Kappa.
In some embodiments of the invention, the amino acid sequence of the light chain constant region is shown in SEQ ID NO. 12.
In a second aspect, the present invention provides a labelled antibody, which is an antibody or antigen binding fragment thereof as described above, labelled with an enzyme, biotin, fluorescent dye, chemiluminescent dye and/or radioisotope.
In a third aspect, the invention provides a nucleic acid molecule encoding the antibody or antigen binding fragment thereof.
Based on the amino acid sequences of the above antibodies or antigen binding fragments thereof, the skilled artisan can obtain nucleotide sequences of nucleic acid molecules encoding the above antibodies or antigen binding fragments thereof. Because of the degeneracy of the codons, the nucleotide sequences of the nucleic acid molecules encoding the antibodies or antigen binding fragments thereof are not unique, and all nucleic acid molecules capable of encoding the antibodies or antigen binding fragments thereof are within the scope of the invention.
In some embodiments of the invention, the nucleotide sequence of a nucleic acid molecule encoding the heavy chain of the above-described antibody is shown in SEQ ID NO.13 and the nucleotide sequence of a nucleic acid molecule encoding the light chain of the above-described antibody is shown in SEQ ID NO. 14.
In a fourth aspect, the present invention provides a biological material comprising a nucleic acid molecule as described above; the biological material is an expression cassette, a vector or a host cell.
The expression cassette may be a recombinant DNA obtained by operably linking the nucleic acid molecule with a promoter.
Such vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, artificial chromosome vectors, and the like.
Such host cells include microbial cells, animal cells or cell lines. Wherein the microbial cells include, but are not limited to, E.coli, yeast, etc., the animal cells include, but are not limited to, insect cells, and the cell lines include, but are not limited to, CHO cells, 293T cells, etc.
In a fifth aspect, the invention provides a method of preparing the antibody or antigen binding fragment thereof, the method comprising: culturing a host cell capable of expressing the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.
In a sixth aspect, the invention provides the use of any of the following antibodies or antigen binding fragments thereof or the labelled antibodies or the nucleic acid molecules or the biological material:
(1) Use in the preparation of a product for detecting the presence or level of coxsackievirus a10 in a sample;
(2) Use in the manufacture of a product for diagnosing coxsackievirus a10 infection or a disease caused by coxsackievirus a10 infection;
(3) Use in the preparation of a product for neutralising coxsackievirus a10 virulence in a sample;
(4) The application in preparing a medicine for preventing or treating coxsackievirus A10 infection or diseases caused by coxsackievirus A10 infection;
(5) The application in detecting the antigenicity and immunogenicity of the Coxsackie virus A10 vaccine;
(6) The application in the quality control of the production of coxsackievirus A10 vaccine;
(7) Use in the detection of the specificity of coxsackievirus a10 antigen.
In the application of (1) above, detecting the presence of coxsackievirus A10 in the sample means detecting whether the sample contains coxsackievirus A10, and detecting the level of coxsackievirus A10 means detecting the content of coxsackievirus A10 in the sample.
In the applications described in the above (1) and (3), the sample may be a sample derived from a living human or animal (including blood, excrement, oral-nasal secretion, etc.), or may be a cell sample cultured in vitro.
In the applications described in the above (1) and (2), the product may be a detection reagent or a kit.
In the applications described in (2) and (4), the diseases caused by Coxsackie virus A10 infection include hand-foot-and-mouth disease.
In the application of the above item (5), the immunogenicity test is specifically to test the performance of the Coxsackie virus A10 vaccine in inducing immune response in animal body, including the evaluation of humoral immune function (such as neutralizing antibody and its level, and affinity of antibody) of immunized animal, etc.
In the application of the above (6), the quality control of the coxsackievirus A10 vaccine specifically comprises detecting whether the quality, content, stability and the like of the antigen in the coxsackievirus A10 vaccine are acceptable.
In the above applications, the detection method may use enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, etc. The antibody or the antigen binding fragment thereof provided by the invention can be used as a binding antibody in the detection method and is used for detecting the Coxsackie virus A10 vaccine antigen.
In a seventh aspect, the invention provides a kit comprising said antibody or antigen-binding fragment thereof or said labeled antibody.
The kit has any one of the following purposes:
(1) Detecting the presence or level of coxsackievirus a10 in the sample;
(2) Diagnosing coxsackievirus a10 infection or a disease caused by coxsackievirus a10 infection;
(3) Detecting antigenicity and immunogenicity of the coxsackievirus A10 vaccine;
(4) Quality control of coxsackievirus a10 vaccine production;
(5) The specificity of coxsackievirus a10 antigen was detected.
The above kit may comprise, in addition to the antibody or antigen-binding fragment thereof or the labeled antibody, a secondary antibody carrying a detectable label to detect the antibody or antigen-binding fragment thereof of the present invention. Among them, detectable labels include, but are not limited to, enzymes, radioisotopes, fluorescent dyes, and the like.
In an eighth aspect, the invention provides a medicament comprising the antibody or antigen-binding fragment thereof.
The medicament has any one of the following purposes:
(1) Preventing coxsackievirus a10 infection or diseases caused by coxsackievirus a10 infection;
(2) Treating Coxsackie virus A10 infection or diseases caused by Coxsackie virus A10 infection.
The above-mentioned medicaments may contain, in addition to the antibody or antigen-binding fragment thereof, a pharmaceutically acceptable carrier, excipient or other active ingredient.
In a ninth aspect, the invention provides a method of detecting the presence or level of coxsackievirus a10 in a sample, the method comprising detecting the presence or level of coxsackievirus a10 in the sample using an antibody or antigen binding fragment thereof of the invention. The method may be used for diagnostic purposes (the sample is from a living human or animal) or for non-diagnostic purposes (the sample is a cell sample cultured in vitro, not a sample from a living human or animal).
In a tenth aspect, the invention provides a method of diagnosing coxsackievirus a10 infection or a disease caused by coxsackievirus a10 infection, the method comprising: the antibodies or antigen binding fragments thereof of the invention are used to diagnose coxsackievirus a10 infection or diseases caused by coxsackievirus a10 infection.
In an eleventh aspect, the invention provides a method for neutralising coxsackievirus type a10 virulence in a sample, the method comprising contacting a sample comprising coxsackievirus type a10 with an antibody or antigen binding fragment thereof of the invention. The method may be used for therapeutic or non-therapeutic purposes (the sample is a cell sample cultured in vitro, not a sample from a living human or animal).
In a twelfth aspect, the present invention provides a method for preventing or treating a disease (including hand-foot-and-mouth disease, etc.) caused by coxsackievirus a10 infection, the method comprising: administering to the subject a prophylactically or therapeutically effective amount of an antibody or antigen-binding fragment thereof of the invention or a medicament comprising said antibody or antigen-binding fragment thereof.
In a thirteenth aspect, the present invention provides a quality control method for the production of a coxsackievirus type a10 vaccine, the method comprising the step of detecting coxsackievirus type a10 using an antibody or antigen binding fragment thereof of the invention.
The beneficial effects of the invention at least comprise the following aspects:
(1) The antibody or the antigen binding fragment thereof provided by the invention has CV-A10 specificity and broad spectrum in, can specifically bind CV-A10, and has no cross reaction with other antigens and Vero host cell proteins; but also cross-binding strains of different subtypes of CV-A10.
(2) The antibody or the antigen binding fragment thereof provided by the invention has broad-spectrum neutralization activity and can cross-neutralize strains of different subtypes of CV-A10.
(3) The antibody or the antigen binding fragment thereof provided by the invention can competitively inhibit the serum of natural infection of a human body and the serum of recovery period of the patient.
(4) The antibody or the antigen binding fragment thereof provided by the invention has a better protection effect on CV-A10 infected animals, and the protection effect and the antibody dosage show an effect relationship.
The antibody or the antigen thereof provided by the invention can be applied to detection of the existence or content level of CV-A10, and evaluation of antigenicity and immunogenicity of CV-A10 vaccine, and has important significance to detection of CV-A10, research and development of CV-A10 vaccine, evaluation and clinical diagnosis; can also be applied to the development of CV-A10 preventive or therapeutic drugs, and has important significance for the treatment of CV-A10 infection and related diseases.
Drawings
FIG. 1 shows solid virus particles and hollow virus particles identified by electron microscopy after sucrose density gradient centrifugation in example 1 of the present invention, wherein the left graph is hollow particles, the right graph is solid particles, and the scale in the graph is 100nm.
FIG. 2 shows the SDS-PAGE detection of the monoclonal antibody of example 3 of the present invention.
FIG. 3 shows the results of detection of specific binding reactions of monoclonal antibodies in example 3 of the present invention.
FIG. 4 shows a competition inhibition test of monoclonal antibodies and human serum in example 3 of the present invention.
FIG. 5 is a reaction curve of the enzyme-labeled antibody of example 5 of the present invention.
FIG. 6 shows the results of three evaluations of the linear range of the antigen detection method of example 7 of the present invention.
FIG. 7 shows the results of the specificity evaluation of the antigen detection method in example 7 of the present invention.
FIG. 8 is a survival curve of each group in example 8 of the present invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Example 1 preparation of CV-A10 antigen
Virus culture: 1 CV-A10 strain was taken, diluted at MOI=0.0001, inoculated into 4 10-layer cell factories each of which was full of monolayer Vero cells, and cultured in an incubator at 35℃for 3 to 5 days in 2L each. And harvesting the virus liquid when the lesion reaches 100%. Subpackaging the virus harvest liquid into a centrifugal cup, centrifuging at 6000rpm for 40min, and harvesting supernatant to obtain CV-A10 supernatant.
Ultrafiltration concentration: the supernatant was concentrated by ultrafiltration using a 300KD membrane pack. The membrane is washed with 10mmol/L PBST solution for 2 times, and the membrane washing liquid and the unfiltered liquid are combined to be ultrafiltration products.
Sucrose density gradient centrifugation: the ultrafiltration product was diluted 3.5-fold with 10mmol/L PBS solution (0.15 mol/L NaCl) and subjected to gradient centrifugation. The concentration of sucrose is 40% -55%. Centrifuge at 30000rpm for 15-18 h, 35ml per tube, collect 20 tubes. Sampling for SDS-PAGE detection, combining the hollow particles and the solid particles according to the detection result, and identifying the hollow solid particles by using an electron microscope.
The electron microscope comparison chart of solid virus particles and hollow virus particles identified by the electron microscope after sucrose density gradient centrifugation is shown in figure 1.
EXAMPLE 2 preparation of monoclonal antibodies
Hybridoma cell line preparation: the hollow particles and the solid particles prepared in the example 1 are collected and then desugared, and the hollow particles and the solid particles are combined according to the volume of 1:1, so that CV-A10 virus purified liquid is obtained. Immunizing NIH mice by using the CV-A10 virus purified liquid, and immunizing 5 mice; subcutaneous multipoint immunization at 5 needle backs total at 0,2,4,6,8 weeks; immunization dose: needle 1 was 50 μg/Freund's complete adjuvant; needle 2, 3 25 μg/dose of Freund's incomplete adjuvant; needle 4 without adjuvant, 25 μg/patient, tail vein injection; and (3) blood collection detection: the indirect ELISA titers are detected by blood sampling 1 week after immunization of the 2 nd needle and the 3 rd needle respectively, and the antibody titers reach 10 4 The mice were given 4 th intraperitoneal injections for booster immunization.
Cell fusion: cell fusion was performed using electrofusion methods. The average fusion efficiency was about 2500 spleen cells capable of fusion to produce 1 hybridoma.
Screening and verification: the supernatant of the fused cells was initially screened by an indirect ELISA method, and the supernatant positive for the immunogen was selected. For all positive clones obtained in the preliminary screening stage, screening was confirmed by indirect ELISA. Neutralization activity verification is carried out on positive clones confirmed to be screened by adopting a method of neutralizing antibody measurement. Positive clones with high binding titers and neutralizing activity were screened.
Subcloning, expansion culture and cryopreservation: positive master clone cells were transferred to 24 well plates for expansion culture and cell supernatants were collected for re-validation. Finally, the positive parent clone is subjected to twice subcloning by adopting a limiting dilution method, and screening is carried out by adopting an indirect ELISA method and a neutralizing antibody measuring method. Positive clone strain confirmed after secondary subcloning is subjected to expansion culture, and ascites is prepared by immunized mice.
EXAMPLE 3 purification and identification of monoclonal antibodies
Antibody purification: ascites prepared by immunizing a mouse in example 2 is centrifuged at 4000-8000 r/min for 5-15 minutes at 2-8 deg.c, the supernatant is filtered with 0.45 μm filter membrane, the antibody is purified by Protein A/G affinity chromatography, and the purified antibody is stored in Phosphate Buffer (PBS) by dialysis. The Lowry method is adopted to detect the protein content of 4.15mg/ml. And (3) detecting the purity and determining the titer of the purified monoclonal antibody.
SDS-PAGE purity assay: analysis showed that the heavy and light chains of monoclonal antibody 2D2 (hereinafter referred to as mab 2D 2) were around 50KD and 25KD, respectively, as shown in fig. 2.
Binding antibody titer determination: EV-A71, CV-A16, CV-A6, CV-A10 virus purified solutions and Vero cell host proteins were diluted to 1.0. Mu.g/ml with carbonate buffer, respectively, and 96-well ELISA plates were coated overnight at 4 ℃. Washing 3-5 times by adding 0.01M Phosphate Buffer (PBST) with final concentration of 0.05% Tween 20, adding 150 μl PBST containing 3-6% BSA into each well, sealing at 37deg.C for 1-2 hr, discarding sealing solution, and drying. Serial gradient dilution is carried out on the monoclonal antibody to be detected and the negative control (sample diluent) to 10 according to a 10-fold gradient method 8 Doubling, sequentially adding 10 -2 To 10 -8 The diluted samples are added into the 96-well plate, 100 mu l of PBST is added into each well for incubation for 1 hour at 37 ℃, 300 mu l of PBST is added into each well, washing is carried out for 2-5 times, 100 mu l of goat anti-mouse IgG-HRP diluted by 1:5000 is added into each well, incubation is carried out for 45 minutes at 37 ℃, after the PBST is washed for 2-5 times, the plates are patted dry, and substrate color development liquid is added into the plates for developing color for 10-15 minutes at room temperature and in dark place, and 1M H is carried out 2 SO 4 The reaction was terminated and read at a wavelength of 450nm on a microplate reader. Negative-positive threshold judgment criterion: should be equal to or greater than the negative control mean x 2.1. The results show that the monoclonal antibody 2D2 specifically binds to CV-A10 antigen with a binding antibody titer of 10 7 But not with other types and Verso cell host protein binding (fig. 3) indicates that mab 2D2 has good specificity.
Neutralizing antibody titer determination: sample to be tested (1:8 dilution) is added into 96-well cell culture plate, serial dilution is carried out by 2 times, and each well is respectively added with 100CCID (complementary serial port) and 0.05 ml/well 50 (half cell infection amount) CV-A10 virus suspension at 37 ℃ and 2 hours; adding 1-2X 10 5 RD cell suspension at a concentration of 0.1 ml/well, 35.+ -. 0.5 ℃ and 5% CO 2 Culturing in an incubator for 7 days. The highest dilution that inhibited 50% of cytopathic effect was designated as neutralizing antibody titer and expressed as the reciprocal of the dilution fold. Each test is provided with a virus drip-back test, and the drip-back result is 32-320 CCID 50 The test was judged to be true at the time of the well. The neutralization antibody is positive when the neutralization titer is more than or equal to 8, the neutralization antibody is negative when the neutralization titer is less than 8, and the geometric average titer (geometric mean titer, GMT) of the negative antibody is calculated according to 4. The neutralizing antibody titer of the monoclonal antibody 2D2 is 1:32678, which shows that the monoclonal antibody has high in vitro neutralizing activity.
Subtype identification: purified mab 2D2 was diluted to 200ng/ml with PBST, 50. Mu.l/well was added to pre-coated ELISA plates (manufacturer: proteintech), and 1 Xsheep anti-mouse IgM+IgG-HRP was added to the sample wells, 50. Mu.l/well mixed gently and incubated for 1 hour at room temperature. The liquid in the wells was discarded, the plates were washed 3 times with PBST, and the plates were dried by patting. The color-developing solution was added at 100. Mu.l/well. Light is prevented from being used for 10-20 min at room temperature. The reaction was stopped by adding 100. Mu.l of stop solution to each well. And reading OD450nm through an enzyme-labeled instrument, wherein the corresponding subtype is the most deep hole of the OD value. The results of the mab subtype assay are shown in table 1. The heavy chain of mab 2D2 is IgG2b and the light chain is Kappa.
TABLE 1 subtype identification results
Subtype classification IgG1 IgG2a IgG2b IgG2c IgG3 IgM Kappa Lambda
Absorbance at 450nm 0.222 0.274 3.35 0.103 0.094 0.083 1.34 0.112
In vitro neutralization activity: the method for detecting the neutralization activity in vitro by adopting a micro cytopathic method comprises the following specific steps: taking 96-well plate, diluting 1mg/ml monoclonal antibody 2D2 with 1:8-1:16384 for 2 times, and respectively mixing with 100CCID 50 The strain C2 genotype strain (strain isolated from China 2014-2019) and the strain C10 strain (genotype A, genbank No. AY 421767) of (Coxsackie virus A10 strain R06030451 was deposited with China general microbiological culture Collection center (CGMCC) at day 13 of 7 months of 2021, address: north Chen West Lu No.1, beijing Korea, university of China, post code 100101), classification name Coxsackie virus A10 type, and accession number CGMCC No.19533, which strain has been disclosed in patent application CN113564130 a; the neutralization activity detection is entrusted to the Chinese food and drug verification institute, and other strains are all from the Chinese food and drug verification institute and used for testing) are neutralized for 2 hours at 37 ℃; the concentration of the mixture is (1-2) multiplied by 10 5 RD cell suspension at 100. Mu.L/well, 35 ℃, CO 2 Culturing in an incubator for 7d; cytopathic effects (cytopathic effect, CPE) were observed and the reciprocal of the highest dilution that inhibited 50% CPE was judged as neutralizing antibody titer. The test is provided with cell contrast and virus back drop, only cell contrast has no CPE, and the virus back drop is between 30 and 300CCID 50 In the case of the well, the test method can be established. The neutralization titers measured for 8 strains including the prototype strain were 768-3072, MAX/MIN was 4 (Table 2), and a relatively uniform broad-spectrum neutralization capacity was shown, indicating that monoclonal antibody 2D2 has broad-spectrum and high-titer binding capacity and neutralization capacity for CV-A10, and can be used as a detection monoclonal antibody.
Table 2 neutralization capacity test
Figure BDA0003578298610000061
Competition inhibition study with human serum (pre-infection serum, patient convalescent serum, and human natural infection serum): the antigen of CV-A10 is diluted to 0.01 mug/ml and coated on a 96-well ELISA plate, human serum is diluted from 1:4 by sample dilution, and is diluted by 2 times ratio continuously, 100 mug/well, and incubated for 1h at 37 ℃; HRP-labeled mab 2D2 was 1:800 diluted with antibody dilution, 100 μl/well, incubated for 1h at 37 ℃; taking the average value of the control Kong Xiguang value as B0, taking the average value of the absorbance values of the sample group to be detected as B1, and inhibiting the absorbance values by = (B0-B1)/B0 by 100. The inhibition ratio is taken as an ordinate, the dilution of serum to be detected (log 2 is taken as a base log) is taken as an abscissa, and a four-parameter method is adopted to fit and determine a reaction curve (FIG. 4, R) 2 0.9907, 0.9894, 1.000, 0.9992, 0.9951, 0.9949 in this order).
The results show that the serum before human infection and the monoclonal antibody 2D2 have no obvious competitive inhibition, while the serum in the convalescence of the patient and the serum after natural human infection have obvious competitive inhibition with the monoclonal antibody 2D 2. The epitope corresponding to the monoclonal antibody 2D2 is proved to play an important role in immune response caused by human infection.
Isolation and identification of the gene sequence of monoclonal antibody 2D 2: total RNA is extracted from well-grown hybridoma cell lines by using Trizol reagent, cDNA is synthesized by reverse transcription, and then light and heavy chain variable region genes are amplified by PCR, and amino acid sequence determination is carried out. Sequencing results show that the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 of the monoclonal antibody 2D2 are shown as SEQ ID NO.1-3, and the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2 and CDR3 are shown as SEQ ID NO. 4-6; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.8, the amino acid sequence of the heavy chain is shown as SEQ ID NO.15, and the amino acid sequence of the light chain is shown as SEQ ID NO. 16.
EXAMPLE 4 monoclonal antibody 2D2 specifically binds CV-A10 viral particle antigen
Sandwich ELISA experiments: diluting rabbit polyclonal antibody (1:1000, 1:5000, 1:10000, 1:20000) against CV-A10 with coating solution carbonate buffer (CBS pH 9.6), adding ELISA plate (detachable from Corning), coating 100 μl per well at 2-8deg.C overnight; washing with PBST for 3 times, and drying; blocking with 3% BSA PBST, 150 μl per well, incubation for 2h at 37deg.C; washing with PBST for 3 times, and drying; diluting the antigen reference to 100ng/ml with sample diluent (0.5 BSA PBS solution) as positive control, sample diluent as negative control, incubating for 2h at 37 ℃ in 100 μl per well; washing with PBST for 3 times, and drying; monoclonal antibody 2D2 was diluted to appropriate concentrations (1:1000, 1:5000, 1:10000, 1:20000) with antibody dilutions (10% fbs PBST), 100 μl per well, and allowed to act at 37 ℃ for 1h; washing with PBST for 3 times, and drying; diluting goat anti-mouse-HRP with antibody diluent to 1:8000, 100 μl per well, and allowing the mixture to act at 37deg.C for 30-60 min; washing with PBST for 4 times, and drying; adding 100 μl of TMB substrate color development liquid into each hole, and developing at room temperature in dark for 15min; 1mol/L H is added 2 SO 4 Mu.l of each well was added. Read at 450nm/630 nm. The results show that the P/N value is maximal when the dilution factor of the coated polyclonal antibody is 1:10000 and the monoclonal antibody 2D2 is diluted 1:5000 as the detection antibody. The above results indicate that monoclonal antibody 2D2 can be used as a detection antibody.
EXAMPLE 5 Horseradish peroxidase-labeled monoclonal antibody 2D2
HRP 5mg was dissolved in 0.2mol/L, pH 5.6 acetate buffer 0.5 ml; adding freshly prepared 0.1mol/L NaIO 4 0.25ml, at which time the solution turns from original brown to dark green, and standing at 4deg.C for 30min, at which time the solution turns from original brown to dark green; 0.5ml of 2.5% ethylene glycol was added thereto, and the mixture was stirred slightly at room temperature for 1 hour to terminate the reaction; 10mg of antibody to be labeled is added, and the pH value is adjusted to 9.0 by 1.0mol/L and pH value is 9.5 CBS; mixing, and standing at 4deg.C overnight; adding 0.1ml of sodium borohydride solution, uniformly mixing, and standing at 4 ℃ for 3 hours; dialyzing overnight with 0.15mol/L PBS solution at pH7.4 at 4deg.C, and changing the solution for 3 times; centrifuging at 3000r/min for 30min, and removing precipitate; collecting supernatant to obtain enzyme-labeled antibody. Adding 60% glycerol, packaging, and storing at below-20deg.C.
Titer determination of enzyme-labeled antibodies: CV-A10 antigen was diluted to about 10. Mu.g/ml with 0.05mol/L of coating buffer, 100. Mu.l was added to the wells of polystyrene plate, and washed 3 times with PBST overnight at 4 ℃. The plate was blocked with 3% BSA-PBST, 150. Mu.l/well, incubated for 2 hours at 37℃and washed 3 times with PBST. Enzyme-labeled antibodies were diluted from 1:12800 with 0.5% BSA-PBST, serially diluted 2-fold to 204800, added to each reaction well, and incubated at 37℃for 1 hour in two wells for each dilution, followed by washing. Then, 100. Mu.l of the color-developing solution was added to each well, and the wells were protected from light at room temperature for 10 minutes. In 1mol/L H 2 SO 4 The reaction was stopped, 50 μl per well.
The ELISA microplate reader was used to read the OD450nm/630nm values of each well, and the dilution of the microplate antibody was plotted on the ordinate with OD (450 nm-630 nm) on the abscissa, and a titration curve was drawn (FIG. 5). The dilution of the enzyme-labeled antibody when the OD value is about 1.0 and the slope of the curve is maximum is found from the curve, and the antibody titer of the marker is obtained. The results are shown in Table 3, and the titer of the enzyme-labeled antibody is 153600.
TABLE 3 absorbance values for enzyme-labeled antibody reactions
Dilution degree Absorbance 1 Absorbance 2 Mean value of
12800 3.533 3.732 3.633
25600 3.346 3.031 3.189
51200 2.428 2.243 2.336
102400 1.509 1.109 1.309
204800 0.88 0.687 0.784
409600 0.485 0.38 0.433
819200 0.262 0.203 0.233
1638400 0.144 0.113 0.129
EXAMPLE 6 establishment of antigen detection method
The rabbit polyclonal antibody is used as a coating antibody, and the monoclonal antibody 2D2 is used as a detection antibody to establish a double-antibody sandwich ELISA detection method for detecting CV-A10 antigen. The test is carried out by adopting a chessboard method, and the specific method is as follows: the rabbit polyclonal antibody was diluted to 1.6. Mu.g/ml, 0.32. Mu.g/ml, 0.16. Mu.g/ml, 0.08. Mu.g/ml with carbonate buffer (pH 9.6), coated with 96-well ELISA plates, 100. Mu.l/well, and coated for 2 hours at 37 ℃. The plates were washed 3 times with PBST and the non-specific binding sites were blocked with blocking solution (0.01M PBS containing 3% BSA, 0.5% Tween 20), 150. Mu.l/well, at 37℃for 2 hours. CV-A10 vaccine antigens were diluted to 100ng/ml with PBS (0.5% BSA), and ELISA plates were added at 100. Mu.l/well, and a blank control was set with sample dilutions as negative controls, each 2 wells, and incubated at 37℃for 2 hours. PBST plates were washed 4 times, HRP-labeled mab 2D2 (1:5000, 1:10000) was added and incubated at 37℃for 30-45 min. PBST plate was washed 5 times, 100. Mu.l of TMB developing solution was added to each well to develop the color at room temperature in the dark for 15min, and 50. Mu.l of stop solution (1M sulfuric acid solution) was added to each well to stop the reaction, and the value was read at 450/630nm in an ELISA reader. The results are shown in Table 4, and when the P/N value is highest, the coated antibody is coated with 0.08 mug/ml, the enzyme-labeled antibody is diluted with 1:5000, and the combination is optimal. The above results demonstrate that the double antibody sandwich method using monoclonal antibody 2D2 as the detection antibody can be used for CV-A10 antigen detection.
Table 4 chessboard method
Figure BDA0003578298610000081
Note that: p represents the mean value of the sample to be detected, N represents the mean value of negative control
EXAMPLE 7 evaluation of antigen detection method
The antigen detection method established in example 6 was evaluated as follows:
linear range: according to the established antigen detection method, national antigen standards were serially diluted 2-fold from 100ng/ml to 8 dilutions (100 ng/ml,50 ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.125ng/ml, 1.5625ng/ml, 0.78125 ng/ml), each dilution being multiplexed. The measurement was performed 3 times independently. Taking the logarithm of the content of the standard substance as an abscissa, taking the logarithm of the light absorption value as an ordinate, drawing a standard curve, and examining R of three results 2 Is a uniform property of (a). The negative mean value of 2.1 is used as a negative and positive judgment standard.
R of the results of the three tests 2 The method is good in linearity and parallelism, and the linearity is good with reference within 50-3.125 ng/ml, as shown in FIG. 6.
Specificity evaluation: referring to the method of example 6, CV-A10 was diluted to 1000ng/mL as a sample to be tested (calculated as protein content) together with EV-A71, CV-A16, CV-A6 antigen and host protein of production cell matrix Vero cells of the family Microriboviridae, and other components M199, DMEM, PEG6000, 58% sucrose, 10mmol/L PBST (1M NaCl, 0.1% Tween 80) possibly present in Poliovirus (PV) antigen and CV-A10 antigen sample were selected as a sample to be tested (abbreviated as S), CV-A10 vaccine antigen was used as a positive control, a sample dilution was used as a negative control, 2.1 times of the negative control was used as CUT-OFF value (abbreviated as CO), and the specificity of the antigen detection system for enterovirus detection was verified.
The results are shown in FIG. 7, and the antigen evaluation system has specific specificity for CV-A10 virus detection and does not respond to other enteroviruses.
Evaluation of binding ability of antigen detection kit to empty solid particles: according to the determined antigen detection method, the hollow virus particles and the solid virus particles are serially diluted according to a certain concentration, and under the condition of reaching the same OD value, the reciprocal of the concentration ratio of the hollow virus protein and the solid virus protein is added to obtain the ratio of the reaction capacity of the antigen detection kit to the hollow heart disease virus particles and the solid virus particles.
The results are shown in Table 5, with a 4:1 empty solid particle reaction capacity. The antigen detection kit can well detect hollow particles and solid particles.
TABLE 5 comparative reactivity to hollow and solid particles
Figure BDA0003578298610000082
EXAMPLE 8 experiments with monoclonal antibody 2D2 in vivo treatment of CV-A10 infection in mice
With lethal doses of R06010343/CV-A10 (424 CCID 50 Mice) were infected with 1-day-old BALB/c rats, 7 groups including a virus control group were provided, and after 1 day the experimental groups were intraperitoneally injected with 1 μg/mouse, 0.25 μg/mouse, 0.0625 μg/mouse, 0.015625 μg/mouse, 0.00390625 μg/mouse, respectively, with the same dose of MEM, and the survival status of the rats was recorded by continuous 21d observation.
As shown in fig. 8, MEM control groups survived all, and virus control group mice died 100% 10 days after challenge. The results show that: group 1 μg of mice survived all but did not show clinical symptoms of wasting and quadriplegia; the survival rate of the groups of 0.25 mug of suckling mice is 66.7%; the survival rate of the 0.0625 mug group of suckling mice is 50%; groups of mice below 0.015625 μg all died.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Beijing Minhai Biotech Co., ltd
<120> a monoclonal antibody against coxsackievirus A10, and preparation method and application thereof
<130> KHP221112873.7
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Ser Tyr Gly Ile His
1 5
<210> 2
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Val Ile Trp Ser Gly Gly Asn Arg Asn Tyr Asn Ala Gly Phe Thr Ser
1 5 10 15
<210> 3
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Asn Tyr Asp Gly Tyr Tyr Leu Tyr Tyr Phe Asp Tyr
1 5 10
<210> 4
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Ser Ser Ser Gln Ser Ile Val His Asn Asn Gly Asn Thr Tyr Leu Glu
1 5 10 15
<210> 5
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Arg Val Ser Asn Arg Phe Ser
1 5
<210> 6
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Phe Gln Gly Ser His Val Pro Trp Thr
1 5
<210> 7
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
Gln Val Gln Leu Lys Gln Ser Gly Pro Ala Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Ile His Trp Leu Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Arg Asn Tyr Asn Ala Gly Phe Thr
50 55 60
Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Val Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Asn Tyr Asp Gly Tyr Tyr Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Ser
115 120
<210> 8
<211> 112
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Arg Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Ser Ser Ser Gln Ser Ile Val His Asn
20 25 30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 9
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Met Ala Val Leu Gly Leu Leu Phe Cys Leu Val Thr Phe Pro Ser Cys
1 5 10 15
Val Leu Ser
<210> 10
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala
1 5 10 15
Ser Ser Ser
<210> 11
<211> 334
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly
1 5 10 15
Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Pro Ser
35 40 45
Gly Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met
50 55 60
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val
65 70 75 80
Thr Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys
85 90 95
Leu Glu Pro Arg Gly Pro Lys Ile Asp Pro Cys Pro Pro Cys Lys Glu
100 105 110
Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val Phe
115 120 125
Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro
130 135 140
Met Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val
145 150 155 160
Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr
165 170 175
Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala
180 185 190
Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys
195 200 205
Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser
210 215 220
Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu Pro Pro
225 230 235 240
Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys Leu Ala
245 250 255
Val Gly Phe Ser Pro Glu Asp Ile Ser Val Glu Trp Thr Ser Asn Gly
260 265 270
His Thr Glu Glu Asn Tyr Lys Asn Thr Ala Pro Val Leu Asp Ser Asp
275 280 285
Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asp Ile Lys Thr Ser Lys Trp
290 295 300
Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly Leu His
305 310 315 320
Ser Phe Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Lys
325 330
<210> 12
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
1 5 10 15
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
20 25 30
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
35 40 45
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
65 70 75 80
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
85 90 95
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
<210> 13
<211> 1422
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
atggctgtct tggggctgct cttctgcctg gtgacattcc caagctgtgt cctatcccag 60
gtgcagttga agcagtcagg acctgcccta gtgcagccct cacagagcct gtccatcacc 120
tgcacagtct ctggtttctc attaactagc tatggtatac actggcttcg ccagtctcca 180
ggaaagggtc tggagtggct gggagtgata tggagtggtg gaaacagaaa ctataatgca 240
ggtttcacat ccagactgag catcaccaag gacaattcca agagccaagt tttcttgaaa 300
gtgaacagtc tgcaagctga tgacacagcc atatattact gtgccagaaa ttatgatggt 360
tactaccttt attactttga ctattggggc caaggcacca ctctcacagt ctcctcagcc 420
aaaacaacac ccccatcggt ctatccactg gcccctgggt gtggagatac aactggttcc 480
tccgtgactc tgggatgcct ggtcaagggc tacttccctg agtcagtgac tgtgacttgg 540
aactctggat ccctgcccag cggtgtgcac accttcccag ctctcctgca gtctggactc 600
tacactatga gcagctcagt gactgtcccc tccagcacct ggccgagcca gaccgtcacc 660
tgcagcgttg ctcacccagc cagcagcacc acagtggaca aaaaacttga gcccagaggg 720
cccaaaatcg acccctgtcc tccatgcaag gagtgtcaca aatgcccagc tcctaacctc 780
gagggtggac cctccgtctt catctttcct ccaaatatca aggatgtact catgatctcc 840
ctgagcccca tggtcacatg tgtggtggtg gatgtgagcg aggatgaccc agatgtccag 900
atcagctggt ttgtgaacaa tgtggaagta cacacagctc agacacaaac ccatagagag 960
gattacaaca gtactctccg ggtggtcagt gccctcccca tccagcacca ggactggatg 1020
agtggcaagg agttcaaatg caaggtcaac aacaaagacc tcccagcgcc catcgagaga 1080
accatctcaa aaattaaagg gctagtcaga gctccacaag tatacatctt gccgccacca 1140
gcagagcagt tgtccaggaa ggatgtcagt ctgacttgcc tggccgtggg cttcagccct 1200
gaagacatca gtgtggagtg gaccagcaat gggcatacag aggagaacta caagaacacc 1260
gcaccagtcc tagactctga cggttcttac ttcatataca gcaagctcga tataaaaaca 1320
agcaagtggg agaaaacaga ttccttctca tgcaacgtga gacacgaggg tctgcacagt 1380
ttctacctga agaagaccat ctcccggtct ccgggtaaat ga 1422
<210> 14
<211> 717
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 60
gttgtgatga cccaaactcc actctccctg cctgtccgtc ttggagatca agcctccatc 120
tcttgcagtt ctagtcagag tattgtgcat aataatggaa acacctattt agaatggtac 180
ctgcagaagc caggccagtc tccaaaactc ctgatctaca gagtttccaa ccgattttct 240
ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300
agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcaca tgttccgtgg 360
acgttcggtg gaggcaccaa gttggaaatc aagcgggctg atgctgcacc aactgtatcc 420
atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 480
aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 540
aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 600
agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 660
actcacaaga catcaacttc acccatcgtc aagagcttca acaggaatga gtgttag 717
<210> 15
<211> 454
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
Gln Val Gln Leu Lys Gln Ser Gly Pro Ala Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Gly Ile His Trp Leu Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Ser Gly Gly Asn Arg Asn Tyr Asn Ala Gly Phe Thr
50 55 60
Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Val Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Arg Asn Tyr Asp Gly Tyr Tyr Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val
115 120 125
Tyr Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Ser Val Thr Val Thr
145 150 155 160
Trp Asn Ser Gly Ser Leu Pro Ser Gly Val His Thr Phe Pro Ala Leu
165 170 175
Leu Gln Ser Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser
180 185 190
Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Ser Val Ala His Pro Ala
195 200 205
Ser Ser Thr Thr Val Asp Lys Lys Leu Glu Pro Arg Gly Pro Lys Ile
210 215 220
Asp Pro Cys Pro Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro Asn
225 230 235 240
Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Asn Ile Lys Asp
245 250 255
Val Leu Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp
260 265 270
Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn
275 280 285
Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn
290 295 300
Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp
305 310 315 320
Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro
325 330 335
Ala Pro Ile Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu Val Arg Ala
340 345 350
Pro Gln Val Tyr Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys
355 360 365
Asp Val Ser Leu Thr Cys Leu Ala Val Gly Phe Ser Pro Glu Asp Ile
370 375 380
Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys Asn
385 390 395 400
Thr Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys
405 410 415
Leu Asp Ile Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys
420 425 430
Asn Val Arg His Glu Gly Leu His Ser Phe Tyr Leu Lys Lys Thr Ile
435 440 445
Ser Arg Ser Pro Gly Lys
450
<210> 16
<211> 219
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 16
Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Arg Leu Gly
1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Ser Ser Ser Gln Ser Ile Val His Asn
20 25 30
Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
85 90 95
Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
115 120 125
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
130 135 140
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
145 150 155 160
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
180 185 190
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
195 200 205
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215

Claims (10)

1. An antibody or antigen-binding fragment thereof, characterized in that the heavy chain complementarity determining regions CDR1, CDR2, CDR3 of the antibody or antigen-binding fragment thereof have the amino acid sequences shown in SEQ ID NO.1-3,
and/or the number of the groups of groups,
the light chain complementarity determining regions CDR1, CDR2, CDR3 of the antibody or antigen binding fragment thereof have amino acid sequences as shown in SEQ ID NO. 4-6.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region of the antibody or antigen-binding fragment thereof has an amino acid sequence as shown in SEQ ID No.7 or has an amino acid sequence having at least 80% homology with the amino acid sequence as shown in SEQ ID No. 7;
and/or the light chain variable region of the antibody or antigen binding fragment thereof has an amino acid sequence as shown in SEQ ID No.8 or has an amino acid sequence having at least 80% homology with the amino acid sequence as shown in SEQ ID No. 8.
3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody is a monoclonal antibody, a bispecific antibody or a multispecific antibody; the antigen binding fragment is a Fab, fab ', F (ab') 2, fd, fv, dAb, complementarity determining region fragment, or single chain antibody.
4. A labeled antibody, characterized in that it is obtained by labeling the antibody or the antigen binding fragment thereof according to any one of claims 1 to 3 with an enzyme, biotin, a fluorescent dye, a chemiluminescent dye and/or a radioisotope.
5. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-3.
6. A biological material comprising the nucleic acid molecule of claim 5, wherein the biological material is an expression cassette, a vector or a host cell.
7. A method of producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising: culturing a host cell capable of expressing the antibody or antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment thereof.
8. Use of the antibody or antigen binding fragment thereof of any one of claims 1-3 or the labeled antibody of claim 4 or the nucleic acid molecule of claim 5 or the biological material of claim 6 for any one of the following:
(1) Use in the preparation of a product for detecting the presence or level of coxsackievirus a10 in a sample;
(2) Use in the manufacture of a product for diagnosing coxsackievirus a10 infection or a disease caused by coxsackievirus a10 infection;
(3) Use in the preparation of a product for neutralising coxsackievirus a10 virulence in a sample;
(4) The application in preparing a medicine for preventing or treating coxsackievirus A10 infection or diseases caused by coxsackievirus A10 infection;
(5) The application in detecting the antigenicity and immunogenicity of the Coxsackie virus A10 vaccine;
(6) The application in the quality control of the production of coxsackievirus A10 vaccine;
(7) Use in the detection of the specificity of coxsackievirus a10 antigen.
9. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3 or the labeled antibody of claim 4.
10. A medicament comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3.
CN202210348791.1A 2022-04-01 2022-04-01 Anti-coxsackievirus A10 monoclonal antibody, and preparation method and application thereof Pending CN116425868A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210348791.1A CN116425868A (en) 2022-04-01 2022-04-01 Anti-coxsackievirus A10 monoclonal antibody, and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210348791.1A CN116425868A (en) 2022-04-01 2022-04-01 Anti-coxsackievirus A10 monoclonal antibody, and preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN116425868A true CN116425868A (en) 2023-07-14

Family

ID=87082041

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210348791.1A Pending CN116425868A (en) 2022-04-01 2022-04-01 Anti-coxsackievirus A10 monoclonal antibody, and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN116425868A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117783524A (en) * 2024-02-26 2024-03-29 中国医学科学院医学生物学研究所 Establishment and application of double-antibody sandwich method for indirect quantitative detection of coxsackie A10 type virus antigen

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117783524A (en) * 2024-02-26 2024-03-29 中国医学科学院医学生物学研究所 Establishment and application of double-antibody sandwich method for indirect quantitative detection of coxsackie A10 type virus antigen
CN117783524B (en) * 2024-02-26 2024-05-03 中国医学科学院医学生物学研究所 Establishment and application of double-antibody sandwich method for indirect quantitative detection of coxsackie A10 type virus antigen

Similar Documents

Publication Publication Date Title
WO2016173558A1 (en) Preparation and use of anti-norovirus gii.4 type murine monoclonal antibody
CN111606993B (en) Fully human broad-spectrum neutralizing antibody 4F1 for resisting respiratory syncytial virus and application thereof
CN109265542B (en) Antibody specifically binding norovirus GII.4 genotype VP1 protein or VLP, and preparation method and application thereof
WO2016173559A1 (en) Preparation and use of murine monoclonal antibody against gi.1 norovirus
CN115724958A (en) Monoclonal antibody of anti-norovirus GII genome capsid protein VP1 and application thereof
CN116425868A (en) Anti-coxsackievirus A10 monoclonal antibody, and preparation method and application thereof
CN104745534A (en) Procalcitonin monoclonal antibody hybrid tumor 2H4 and monoclonal antibody
CN116199774B (en) Monoclonal antibody for hepatitis B virus surface antigen mutant strain
CN115806611B (en) Antibodies against novel coronavirus N proteins and uses thereof
CN116462754A (en) Monoclonal antibody for identifying N protein of canine distemper virus, detection reagent and application
CN116217716A (en) Monoclonal antibody for identifying Coxsackie viruses A2, A4 and A5 and application thereof
CN110702913B (en) Monoclonal antibody composition for quantitatively detecting coxiella burnetii I strain
CN111205364B (en) Monoclonal neutralizing antibody against HPV31L1 and application thereof
CN116425869A (en) Anti-coxsackievirus A6 monoclonal antibody, and preparation method and application thereof
CN112225804A (en) Nanobodies and uses thereof
CN115850454A (en) Anti-coxsackievirus A16 antibody and preparation method and application thereof
KR102241520B1 (en) An antibody having immune reactivity to Foot-and-Mouth Disease Virus type O and composition for detecting FMDV type O comprising the same
KR102241521B1 (en) Method of detecting antibody produced by foot-and-mouth disease vaccination using an antibody having immune reactivity to FMDV type O
CN112225805B (en) Nanobodies and uses thereof
CN112225803B (en) Nanobodies and uses thereof
CN110760483B (en) Preparation and application of anti-TNF-alpha monoclonal antibody with cattle and sheep cross reaction
CN117659172A (en) anti-PRV gD protein monoclonal antibody, preparation method and application thereof
CN115975016A (en) anti-GII.2 type norovirus monoclonal antibody and application thereof
CN116239682A (en) Monoclonal antibody for identifying Coxsackie viruses A2 and A4 and application thereof
CN115975017A (en) anti-GII.17 type norovirus monoclonal antibody and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination