CN113249333B - Hybridoma cell strain RSVN4C3 secreting anti-respiratory syncytial virus monoclonal antibody - Google Patents
Hybridoma cell strain RSVN4C3 secreting anti-respiratory syncytial virus monoclonal antibody Download PDFInfo
- Publication number
- CN113249333B CN113249333B CN202110281178.8A CN202110281178A CN113249333B CN 113249333 B CN113249333 B CN 113249333B CN 202110281178 A CN202110281178 A CN 202110281178A CN 113249333 B CN113249333 B CN 113249333B
- Authority
- CN
- China
- Prior art keywords
- respiratory syncytial
- syncytial virus
- monoclonal antibody
- rsvn4c3
- colloidal gold
- 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.)
- Active
Links
- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 35
- 241000700605 Viruses Species 0.000 title claims abstract description 12
- 230000003097 anti-respiratory effect Effects 0.000 title claims abstract description 12
- 230000003248 secreting effect Effects 0.000 title claims abstract description 10
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 27
- 206010061603 Respiratory syncytial virus infection Diseases 0.000 claims abstract description 20
- 241000725643 Respiratory syncytial virus Species 0.000 claims abstract description 18
- 208000030925 respiratory syncytial virus infectious disease Diseases 0.000 claims abstract description 18
- 238000012360 testing method Methods 0.000 claims abstract description 18
- 108090001074 Nucleocapsid Proteins Proteins 0.000 claims abstract description 13
- 238000008157 ELISA kit Methods 0.000 claims abstract description 5
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 238000001514 detection method Methods 0.000 claims description 23
- 239000012528 membrane Substances 0.000 claims description 21
- 239000000020 Nitrocellulose Substances 0.000 claims description 18
- 229920001220 nitrocellulos Polymers 0.000 claims description 18
- 101710141454 Nucleoprotein Proteins 0.000 claims description 15
- 238000003908 quality control method Methods 0.000 claims description 14
- 239000011248 coating agent Substances 0.000 claims description 10
- 238000000576 coating method Methods 0.000 claims description 10
- 241000283707 Capra Species 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000011521 glass Substances 0.000 claims description 4
- 229920002678 cellulose Polymers 0.000 claims description 3
- 239000001913 cellulose Substances 0.000 claims description 3
- 238000003745 diagnosis Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 208000015181 infectious disease Diseases 0.000 abstract description 3
- 230000009257 reactivity Effects 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 24
- 210000004027 cell Anatomy 0.000 description 20
- 238000000034 method Methods 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 239000000427 antigen Substances 0.000 description 10
- 102000036639 antigens Human genes 0.000 description 10
- 108091007433 antigens Proteins 0.000 description 10
- 238000002965 ELISA Methods 0.000 description 9
- 239000012634 fragment Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000010166 immunofluorescence Methods 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 238000012258 culturing Methods 0.000 description 7
- 230000003053 immunization Effects 0.000 description 7
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 6
- 241000711920 Human orthopneumovirus Species 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 238000002649 immunization Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 102000011931 Nucleoproteins Human genes 0.000 description 5
- 108010061100 Nucleoproteins Proteins 0.000 description 5
- 229920001213 Polysorbate 20 Polymers 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 229960005486 vaccine Drugs 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000001131 transforming effect Effects 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 241001529459 Enterovirus A71 Species 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 206010057190 Respiratory tract infections Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000001804 emulsifying effect Effects 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 241000712461 unidentified influenza virus Species 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- 241000709687 Coxsackievirus Species 0.000 description 1
- 241001429382 Coxsackievirus A16 Species 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000032754 Infant Death Diseases 0.000 description 1
- 206010024971 Lower respiratory tract infections Diseases 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 241000711504 Paramyxoviridae Species 0.000 description 1
- 241000711902 Pneumovirus Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 244000191761 Sida cordifolia Species 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 230000008076 immune mechanism Effects 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008518 non respiratory effect Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
- G01N2333/135—Respiratory syncytial virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a hybridoma cell strain RSVN4C3 secreting anti-respiratory syncytial virus monoclonal antibody, which is preserved in China Center for Type Culture Collection (CCTCC), wherein the preservation number is CCTCC NO: C2020254, the monoclonal antibody secreted by the hybridoma cell strain RSVN4C3 and resisting respiratory syncytial virus nucleocapsid protein is used for preparing a kit for detecting or diagnosing respiratory syncytial virus infection, the kit can be an immune colloidal gold test strip or ELISA kit, and the hybridoma cell strain RSVN4C3 provided by the invention can stably secrete the monoclonal antibody (MAb) resisting respiratory syncytial virus, and the reactivity and the specificity of the monoclonal antibody (MAb) are identified, thereby laying a foundation for diagnosing the respiratory syncytial virus infection and researching an infection mechanism in future.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a hybridoma cell strain RSVN4C3 secreting an anti-respiratory syncytial virus monoclonal antibody.
Background
Human respiratory syncytial virus (Human respiratory syncytial virus, RSV) is the leading cause of acute lower respiratory tract infections (ali) worldwide, leading to hospitalization in children under 5 years of age and immunocompromised adults. RSV infection is associated with high morbidity and mortality in children with acute lower respiratory infections (alis) in developed and developing countries. In developed countries, it is a major cause of infant hospitalization for one month of infant birth; while RSV is the leading cause of infant death in developing countries, children under 3400 ten thousand years old develop severe RSV disease each year, with 350 ten thousand children requiring hospitalization and 6.6 ten thousand to 19.9 ten thousand deaths, 99% of which occur in developing countries. Although RSV creates a tremendous disease burden, no licensed vaccine is currently marketed, and the world health organization has listed the development of RSV vaccines as one of the priority development programs for the global vaccine program.
RSV is a non-segmented, single-stranded negative-strand RNA virus belonging to the genus paramyxoviridae, pneumovirus. The RSV genome is approximately 15.2kb in length and encodes 11 proteins NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2 and L10. Among all proteins encoded by the RSV genome, nucleoprotein (N protein) is the most conserved protein, either between subtypes A, B or within the same subtype. Many studies at home and abroad report that the N protein gene is used as a target gene for molecular biological detection. N protein is an important component of the RNA-dependent RNA polymerase complex in RSV, and N protein is also one of the important antigens recognized by cytotoxic T cells. Therefore, the research of the N gene and the coded protein thereof has important significance for further researching the pathogenesis and immune mechanism of the RSV and developing rapid diagnostic reagent of the RSV. But currently, N protein is rarely studied in China.
Disclosure of Invention
Aiming at the technical problems, the invention provides a hybridoma cell strain RSVN4C3 secreting an anti-respiratory syncytial virus monoclonal antibody.
The technical scheme of the invention is as follows:
a hybridoma cell strain RSVN4C3 is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C2020254.
Further, the preparation method of the hybridoma cell strain RSVN4C3 comprises the following steps:
s1, taking a strain B133 strain Hep-2 cell culture supernatant of an isolated strain B133 in 2017 of Guizhou, RSV, and extracting total RNA of the RSV; reverse transcription is carried out to synthesize a first strand of cDNA, and specific primers are designed by taking the first strand as a template, and the sequence is as follows:
P1：5′-AATGGATCCATGGCTCTTAGCAAAGTC-3' (underlined is the BamHI cleavage site),
P2：5′-ATTGTCGACAAGCTCTACATCATTATC-3' (Sall cleavage site underlined),
s2, using P1 and P2 as primers, amplifying RSV N gene fragments by PCR, wherein the reaction conditions are as follows: 94 ℃ for 30s; extending at 52 ℃ for 30s at 72 ℃ for 1min for 35 cycles; obtaining a 908bp PCR amplification product, and respectively carrying out double enzyme digestion on the PCR product and a pQE30 expression vector by using BamHI and Sall to recover a target gene fragment and a vector fragment;
s3, connecting a target gene fragment and a carrier fragment by using T4 DNA ligase, transforming a connection product into competent escherichia coli XL1-blue, coating an LB ampicillin plate, standing and culturing at 37 ℃ overnight, respectively picking single colonies the next day, inoculating into 3ml of LB culture solution, culturing for 16 hours at 37 ℃ in a shaking way, extracting plasmids, carrying out double enzyme digestion identification, carrying out agarose gel electrophoresis analysis, and carrying out gene sequencing on positive plasmids;
s4, transforming escherichia coli XL1-blue bacteria by using a recombinant expression plasmid with correct sequence, inoculating the escherichia coli XL1-blue bacteria into an LB medium tube containing 100 mug/ml ampicillin (Amp), and carrying out shake culture at 37 ℃ for overnight; respectively inoculating into 5ml LB bacteria culture medium tube containing 100 μg/ml Amp and 200ml LB culture medium triangular flask according to 1% inoculum size, shake culturing at 37deg.C until bacterial liquid A600 value is about 0.6, adding IPTG with final concentration of 100 μg/ml, shake culturing at 37deg.C for 4 hr; collecting thallus, ultrasonic crushing, centrifuging, collecting supernatant, and adding Ni 2+ Purifying by an affinity chromatography column; purifying the expression product, and performing 12% SDS-PAGE and Western blot identification analysis to obtain purified recombinant RSV nucleoprotein;
s5, immunizing BALA/C mice with purified recombinant RSV nucleoprotein, performing immunization according to the dose of 100 micrograms of each mouse, emulsifying the mice with Freund' S complete adjuvant for the first immunization, emulsifying the mice with incomplete adjuvant for the second immunization and the third immunization, wherein the immunization interval time is two weeks, blood sampling is performed at the tail of the mice after 7d of the third immunization, serum antibody titer is measured, 3d of the mice are boosted for one time before fusion, spleen cells of the immunized mice are taken and SP2/0 are subjected to cell fusion according to a conventional method, and HAT selection culture medium is used for screening and culturing fusion hybridoma cells;
s6, detecting hybridoma cell supernatants of the grown hybridoma cells by an indirect ELISA method, an indirect immunofluorescence method and a western-blot method, and screening hybridoma cell strains secreting anti-respiratory syncytial virus nucleocapsid protein monoclonal antibodies.
The invention also provides application of the hybridoma cell strain RSVN4C3 in preparing a reagent for detecting or diagnosing respiratory syncytial virus infection.
Further, the monoclonal antibody secreted by the hybridoma cell strain RSVN4C3 and resisting the nucleocapsid protein of the respiratory syncytial virus reacts with the RSV N protein to generate antigen-antibody, belonging to IgG type antibody. The monoclonal antibody of the anti-respiratory syncytial virus nucleocapsid protein secreted by the hybridoma cell strain RSVN4C3 reacts with the RSV N protein to generate antigen-antibody, belonging to IgG type antibody.
The invention also provides a kit for detecting the respiratory syncytial virus antigen by an indirect immunofluorescence method and detecting the respiratory syncytial virus antigen by a colloidal gold method to diagnose respiratory syncytial virus infection, a kit for detecting the respiratory syncytial virus antigen by an ELISA method to diagnose respiratory syncytial virus infection and a method for detecting serum respiratory syncytial virus specific IgM antibody of a patient by an IgM capture method to diagnose respiratory syncytial virus infection. The 4 methods and kits comprise monoclonal antibodies secreted by the hybridoma cell line RSVN4C3 against respiratory syncytial virus nucleocapsid protein.
Optionally, the kit is an immune colloidal gold test strip.
Furthermore, the test strip sequentially comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane, water absorbing paper and a PVC bottom plate which is positioned below and is used as an assembly platform according to the connection sequence, wherein the nitrocellulose membrane is overlapped with the colloidal gold pad, the nitrocellulose membrane is overlapped with the water absorbing paper, the colloidal gold pad is composed of a glass cellulose membrane adsorbed with a colloidal gold marked anti-RSVN 4C3 monoclonal antibody, and the nitrocellulose membrane is provided with a quality control line coated with goat anti-mouse IgG polyclonal antibody and a detection line coated with an anti-RSVN protein monoclonal antibody.
Further, the quality control line is obtained by coating goat anti-mouse IgG polyclonal antibody on a nitrocellulose membrane at a concentration of 0.8 mg/ml; the detection line was obtained by coating an anti-RSV N protein monoclonal antibody on a nitrocellulose membrane at a concentration of 0.5 mg/ml.
Still further, the detection line and the quality control line are spaced apart by 0.5cm.
Furthermore, the detection method for detecting human respiratory syncytial virus by using the immune colloidal gold test strip comprises the following steps:
s1, adding 200 mu L of a test sample into sample application Kong Di, and reacting for 10-15min to observe a result;
s2, judging a result:
the quality control line and the detection line are both provided with red stripes, and the detection line is positive;
only a red strip appears on the quality control line, and the color is negative;
if no strip appears on the quality control line, the result is judged to be invalid whether the red strip appears on the detection line or not.
Alternatively, the kit for detecting or diagnosing respiratory syncytial virus is an ELISA kit.
The beneficial effects of the invention are as follows: the invention clones RSV N protein full-length gene, expresses recombinant N protein in colibacillus, purifies, uses purified recombinant nucleoprotein to immunize BALB/C mouse, fuses immune mouse spleen lymphocyte with mouse myeloma SP2/0 cell, screens hybridoma cell strain secreting anti-respiratory syncytial virus nucleocapsid protein monoclonal antibody. The hybridoma cell strain can stably secrete monoclonal antibodies (MAbs) resisting respiratory syncytial viruses, the reactivity and the specificity of the monoclonal antibodies (MAbs) are identified, and a foundation is laid for the diagnosis of RSV infection, the research of infection mechanism and vaccine development in the future.
Drawings
FIG. 1 is a SDS-PAGE analysis of recombinant RSV nucleocapsid proteins expressed and purified in E.coli.
FIG. 2 is a graph showing the result of Western blot of monoclonal antibodies secreted by the hybridoma cell line RSVN4C3 and purified recombinant RSV nucleocapsid protein expressed in E.coli.
FIG. 3 is a graph showing the immunofluorescence results of specific recognition of RSV-infected Hep-2 cells by monoclonal antibody secreted by hybridoma cell line RSV N4C3. Wherein A: normal uninfected Hep-2 cells; RSV infects Hep-2 cells.
FIG. 4 shows the results of a specific assay for antigen detection diagnostic reagents prepared from colloidal gold labeled RSVN4C3 monoclonal antibodies.
FIG. 5 shows the result of a sensitivity test for the colloidal gold antigen detection reagent of RSVN4C3 monoclonal antibody.
Detailed Description
Example 1
The hybridoma cell strain RSVN4C3 secreting the anti-respiratory syncytial virus monoclonal antibody is preserved in China Center for Type Culture Collection (CCTCC), the address is Wuhan in China, the preservation date is 12 months 1 in 2020, and the preservation number is CCTCC NO: C2020254.
The preparation method of the hybridoma cell strain RSVN4C3 comprises the following steps:
materials and methods
The culture supernatant of the strain B133 Hep-2 cell of the isolate B133 of Guizhou 2017 of RSV is taken, the total RNA of RSV is extracted according to the Trizol reagent specification, the first strand of cDNA is synthesized by reverse transcription, and a specific primer is designed by taking the first strand as a template, and the sequence is as follows:
P1：5′-AATGGATCCATGGCTCTTAGCAAAGTC-3' (underlined is the BamHI cleavage site),
P2：5′-ATTGTCGACAAGCTCTACATCATTATC-3' (Sall cleavage site underlined);
PCR amplification of RSV N gene segment with P1 and P2 as primer, and the reaction conditions are as follows: 94 ℃ for 30s; extending at 52 ℃ for 30s at 72 ℃ for 1min for 35 cycles; obtaining a 908bp PCR amplification product, and respectively carrying out double enzyme digestion on the PCR product and a pQE30 expression vector by using BamHI and SalI to recover a target gene fragment and a vector fragment;
ligating with T4 DNA ligase, transforming competent E.coli XL1-blue with the ligation product, coating an LB ampicillin plate, standing at 37 ℃ for overnight, picking single colonies each the next day, inoculating into 3ml LB culture solution, shaking at 37 ℃ for 16 hours, extracting plasmids, performing double enzyme digestion identification, performing agarose gel electrophoresis analysis, and performing gene sequencing on positive plasmids;
e.coli XL1-blue bacteria transformed by the recombinant expression plasmid with correct sequence are inoculated in LB medium tube containing 100 mug/ml ampicillin (Amp) and cultured overnight at 37 ℃ in an oscillating way; respectively inoculating into 5ml LB bacteria culture medium tube containing 100 μg/ml Amp and 200ml LB culture medium triangular flask according to 1% inoculum size, shake culturing at 37deg.C until bacterial liquid A600 value is about 0.6, adding IPTG with final concentration of 100 μg/ml, shake culturing at 37deg.C for 4 hr; the bacterial cells are collected, crushed by ultrasound, centrifuged, the supernatant is taken and purified by a Ni2+ affinity chromatography column, and the purified expression products are subjected to 12% SDS-PAGE and Western blot identification analysis.
Expression and identification of recombinant RSV-N proteins
The RSV-N gene is amplified and cloned to form island expression vector pQE-30, and the recombinant plasmid expresses RSV-N protein product in colibacillus and through analysis of 12% SDS-PAGE, specific protein band with relative molecular weight of about 30kD is detected, and the size is in accordance with the expected. Western blot analysis shows that the purified recombinant protein and the mouse anti-RSV-N monoclonal antibody react specifically, and the expressed protein is RSV-N protein. See fig. 1 and 2.
Establishment and identification of hybridoma cell lines
BAL A/C mice are immunized by purified recombinant RSV nucleocapsid protein, immunized spleen cells are fused with SP2/0 myeloma cells, and hybridoma cells are selected by HAT selection culture.
Positive hybridoma cells are screened by using indirect immunofluorescence and indirect ELISA, and after subcloning by a limiting dilution method for 3 times, 1 hybridoma cell strain which stably secretes an anti-RSV-N protein monoclonal antibody is obtained and is named as RSVN-4C3.
Monoclonal antibodies secreted by the hybridoma cells do not react with uninfected Hep-2 cells, react with RSV-infected Hep-2 cells, and exhibit specific immunofluorescence in cytoplasm (FIG. 3, wherein A is normal uninfected Hep-2 cells; B is RSV-infected Hep-2 cells).
The indirect immunofluorescence method specificity identification shows that the monoclonal antibodies only react with RSV infected cells, but do not react with influenza virus (H1N 1, PR8 strain), EV71 virus and coxsackievirus (CA 16) infected cells.
Injecting RSVN4C3 hybridoma cells into the abdominal cavity of a mouse to prepare the monoclonal antibody titer of ascites, and measuring by an indirect ELISA method of coating recombinant nucleoprotein, wherein the titer of the RSVN4C3 ascites antibody is 1:1,000,000; indirect immunofluorescence assay, rsvp 3 titer 1:100000; all are significantly higher than the titers of immune serum.
Through identification of the monoclonal antibody subclass, the RSVN4C3 monoclonal antibody heavy chains are all IgG1, and the light chains are all kappa chains.
The monoclonal antibody recognizes RSV nucleocapsid protein, and the recognition amino acid sequence is as follows:
MALSKVKLNDTLNKDQLLSSSKYTIQRSTGDSIDTPNYDVQKHINKLCGMLLI TEDANHKFTGLIGMLYAMSRLGREDTIKILKDAGYHVKANGVDVTTHRQDIN GKEMKFEVLTLASLTTEIQINIEIESRKSYKKMLKEMGEVAPEYRHDSPDCGM IILCIAALVITKLAAGDRSGLTAVIRRANNVLKNEMKRYKGLLPKDIANSFYE VFEKYPHFIDVFVHFGIAQSSTRGGSRVEGIFAGLFMNAYGAGQVMLRWGVL AKSVKNIMLGHASVQAEMEQVVEVYEYAQKLGGEAGFYHILNNPKASLLSL TQFPHFSSVVLGNAAGLGIMGEYRGTPRNQDLYDAAKAYAEQLKENGVINY SVLDLTAEELEAIKHQLNPKDNDVEL
example 2
Clinical respiratory syncytial virus infection was detected by indirect immunofluorescence using monoclonal antibodies directed against respiratory syncytial virus nucleocapsid protein secreted by hybridoma cell line RSVN4C3.
Taking a nasopharyngeal secretion or nasopharyngeal lotion sample of a patient with respiratory tract infection, diluting with normal saline, centrifuging at 2000 rpm for 5 minutes, collecting nasopharyngeal exfoliative cells, suspending exfoliative cells in 100 microliter of normal saline, dripping the cells onto a glass slide, drying, and fixing the cells with acetone. RSVN4C3 monoclonal antibody was added at 1:10000 dilution, the dilutions were reacted with Phosphate Buffer (PBS) containing 3% Bovine Serum Albumin (BSA) for 30 min at 37 ℃, the slides were washed with PBS for 10 min, 1: 50-dilution FITC fluorescent labeling goat anti-mouse IgG antibody, reacting for 30 minutes at 37 ℃, washing a slide with PBS for 10 minutes, drying the slide, adding a fluorescent protective agent, adding a cover slip, sealing the cover slip with nail polish, observing cells by a fluorescent microscope, finding positive green fluorescent particles in the cells, indicating respiratory syncytial virus infection, otherwise, detecting negative and non-respiratory syncytial virus infection without the green fluorescent particles in the cells.
Example 3
The embodiment provides a kit for detecting or diagnosing respiratory syncytial virus infection, which is an immune colloidal gold test strip and comprises a monoclonal antibody of anti-respiratory syncytial virus nucleocapsid protein secreted by the hybridoma cell strain RSVN4C3.
The test strip sequentially comprises a sample pad, a colloidal gold pad, a nitrocellulose membrane, water absorbing paper and a PVC bottom plate which is positioned below and used as an assembly platform according to a connection sequence, wherein an overlap joint is arranged between the nitrocellulose membrane and the colloidal gold pad, an overlap joint is arranged between the nitrocellulose membrane and the water absorbing paper, the colloidal gold pad is composed of a glass cellulose membrane adsorbed with a colloidal gold marked anti-RSVN 4C3 monoclonal antibody, and the nitrocellulose membrane is provided with a quality control line coated with goat anti-mouse IgG polyclonal antibody and a detection line coated with an anti-RSVN protein monoclonal antibody.
The quality control line is obtained by coating goat anti-mouse IgG polyclonal antibody on nitrocellulose membrane at concentration of 0.8 mg/ml; the detection line was obtained by coating an anti-RSV N protein monoclonal antibody on a nitrocellulose membrane at a concentration of 0.5 mg/ml. The distance between the detection line and the quality control line is 0.5cm.
Example 4
The embodiment provides a detection method for detecting human respiratory syncytial virus by using the immune colloidal gold test strip of the embodiment 3, which comprises the following steps:
s1, adding 200 mu L of a test sample into sample application Kong Di, and reacting for 10-15min to observe a result;
s2, judging a result:
the quality control line and the detection line are both provided with red stripes, and the detection line is positive;
only a red strip appears on the quality control line, and the color is negative;
if no strip appears on the quality control line, the result is judged to be invalid whether the red strip appears on the detection line or not.
Example 5
This example provides a specificity test of the immune colloidal gold test strip of example 3.
Antigen detection diagnostic reagent prepared by colloidal gold labeled RSVN4C3 monoclonal antibody specifically recognizes respiratory syncytial virus, presents positive reaction with both genotypes A and B of RSV, and has no cross reaction with influenza virus H1N1, enterovirus 71 (EV 71) and Coxsackie virus A16 (CA 16) (figure 4).
Example 6
This example provides a sensitivity test of the immune colloidal gold test strip of example 3.
Sensitivity test analysis of antigen detection diagnosis kit prepared by colloidal gold labeled RSVN4C3 monoclonal antibody shows that the antigen detection reagent can detect 10 5 Viral particles above PFU (fig. 5).
Example 7
The embodiment provides an ELISA kit for detecting respiratory syncytial virus antigen by a double-antibody sandwich method so as to diagnose respiratory syncytial virus infection.
Rsvp 4℃ 3 monoclonal antibody diluted with 1:10000 phosphate buffer (PBS ph 7.2), 96-well ELISA reaction plates were added 100 μl per well, coating was performed overnight at 4 ℃,100 μl of 3% bsa per well was added, room temperature was blocked for 1 hour, PBS-Tween20 was washed 3 times, 100 μl of patient nasopharyngeal wash was added, reaction was performed for 1 hour at 37 ℃, PBS-Tween20 was washed 3 times, 100 μl of diluted horseradish peroxidase (HRP) -labeled rsvp 4℃ 3 monoclonal antibody was added, reaction was performed for 1 hour at 37 ℃, PBS-Tween20 was washed 3 times, HRP substrate (TMB or OPD) was added, reaction was terminated after 30 minutes of development reaction at 37 ℃, absorbance OD value of each sample well was measured with a microplate reader, and OD value was higher than 2 times that of negative control, and positive was judged.
Example 8
The embodiment provides an ELISA kit for detecting specific IgM antibodies of patient serum respiratory syncytial virus by an IgM capture method so as to diagnose respiratory syncytial virus infection.
The goat anti-human IgM antibody diluted with 1:500 phosphate buffer (PBS pH 7.2), 100. Mu.L of each well was added to a 96-well ELISA reaction plate, coated overnight at 4 ℃, 100. Mu.L of 3% BSA was added to each well, blocked at room temperature for 1 hour, ELISA plates were washed 3 times with PBS-Tween20, 100. Mu.L of 1:100 diluted patient serum was added, reacted at 37℃for 1 hour, ELISA plates were washed 3 times with 100. Mu.L (50 ng) recombinant respiratory syncytial virus N protein, reacted at 37℃for 1 hour, diluted horseradish peroxidase (HRP) labeled N4℃ 3 monoclonal antibody, reacted at 37℃for 1 hour, ELISA plates were washed 3 times with PBS-Tween20, HRP substrates (TMB or OPD) were added, the reaction was terminated after 30 minutes of chromogenic reaction at 37℃and the absorbance OD value of each sample well was measured with a microplate reader and was judged positive by an OD value 2 times or higher than that of negative control. Respiratory syncytial virus-specific IgM antibody positivity can diagnose recent infection with respiratory syncytial virus.
Sequence listing
<110> Guizhou province people hospital
<120> hybridoma cell line RSVN4C3 secreting anti-respiratory syncytial virus monoclonal antibody
<130> none of
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 27
<212> DNA
<213> artificial sequence
<221> misc_feature
<222> (1)..(27)
<223> specific primers for HRSV N Gene fragment amplification were designed according to the experimental requirements
<400> 1
aatggatcca tggctcttag caaagtc
27
<210> 2
<211> 27
<212> RNA
<213> artificial sequence
<221> misc_feature
<222> (1)..(27)
<223> specific primers for HRSV N Gene fragment amplification were designed according to the experimental requirements
<400> 2
attgtcgacaagctctacatcattatc
27
Claims (8)
1. The hybridoma cell strain RSVN4C3 secreting the anti-respiratory syncytial virus monoclonal antibody is characterized by being preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C2020254.
2. Use of a hybridoma cell line RSVN4C3 according to claim 1 for the preparation of a reagent for the detection or diagnosis of respiratory syncytial virus infection.
3. A monoclonal antibody against respiratory syncytial virus nucleocapsid protein secreted by hybridoma cell line rsvp N4C3 according to claim 1, wherein said monoclonal antibody is antigen-antibody reactive with RSV N protein, belonging to IgG type antibody.
4. A kit for detecting or diagnosing a respiratory syncytial virus infection, comprising the monoclonal antibody of claim 3.
5. An immune colloidal gold test strip for detecting or diagnosing respiratory syncytial virus infection, which comprises the monoclonal antibody according to claim 3.
6. The immune colloidal gold test strip for detecting or diagnosing respiratory syncytial virus infection according to claim 5, which is characterized by comprising a sample pad, a colloidal gold pad, a nitrocellulose membrane, water absorbing paper and a PVC bottom plate which is positioned below and is used as an assembly platform in sequence according to the connection sequence, wherein the nitrocellulose membrane is lapped with the colloidal gold pad, the nitrocellulose membrane is lapped with the water absorbing paper, the colloidal gold pad consists of a glass cellulose membrane adsorbed with the monoclonal antibody of claim 3 marked by colloidal gold, and the nitrocellulose membrane is provided with a quality control line coated by goat anti-mouse IgG polyclonal antibody and a detection line coated by anti-RSV N protein monoclonal antibody.
7. The immune colloidal gold test strip for detecting or diagnosing respiratory syncytial virus infection according to claim 6, wherein the quality control line is obtained by coating goat anti-mouse IgG polyclonal antibody on nitrocellulose membrane at a concentration of 0.8 mg/ml; the detection line was obtained by coating an anti-RSV N protein monoclonal antibody on a nitrocellulose membrane at a concentration of 0.5 mg/ml.
8. A kit for detecting or diagnosing a respiratory syncytial virus infection according to claim 4, wherein the kit is an ELISA kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110281178.8A CN113249333B (en) | 2021-03-16 | 2021-03-16 | Hybridoma cell strain RSVN4C3 secreting anti-respiratory syncytial virus monoclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110281178.8A CN113249333B (en) | 2021-03-16 | 2021-03-16 | Hybridoma cell strain RSVN4C3 secreting anti-respiratory syncytial virus monoclonal antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113249333A CN113249333A (en) | 2021-08-13 |
| CN113249333B true CN113249333B (en) | 2023-06-02 |
Family
ID=77181047
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110281178.8A Active CN113249333B (en) | 2021-03-16 | 2021-03-16 | Hybridoma cell strain RSVN4C3 secreting anti-respiratory syncytial virus monoclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113249333B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114057867B (en) * | 2021-12-14 | 2023-05-12 | 河南联科物联网科技有限公司 | Monoclonal antibody for resisting egg drop syndrome and application thereof |
| CN115838419B (en) * | 2022-09-27 | 2023-06-30 | 深圳重链生物科技有限公司 | Anti-respiratory syncytial virus antibodies and related uses thereof |
| CN115960216B (en) * | 2022-09-27 | 2023-07-07 | 深圳重链生物科技有限公司 | Anti-respiratory syncytial virus antibodies and related uses thereof |
| CN115925909B (en) * | 2022-09-27 | 2023-07-21 | 深圳重链生物科技有限公司 | Anti-respiratory syncytial virus antibodies and related uses thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006117456A1 (en) * | 2005-04-29 | 2006-11-09 | Institut National De La Recherche Agronomique (Inra) | Preparation of soluble n-protein/truncated p-protein complexes or n-proteins soluble in a virus of the paramyxoviridae family and use thereof in vaccines |
| WO2007101441A1 (en) * | 2006-03-06 | 2007-09-13 | Symphogen A/S | Recombinant polyclonal antibody for treatment of respiratory syncytial virus infections |
| CN101130765A (en) * | 2006-08-21 | 2008-02-27 | 北京阿斯可来生物工程有限公司 | Reagent kit for detecting syncytial virus of respiratory passage |
| WO2008106980A2 (en) * | 2007-03-06 | 2008-09-12 | Symphogen A/S | Recombinant antibodies for treatment of respiratory syncytial virus infections |
| WO2009042794A2 (en) * | 2007-09-26 | 2009-04-02 | Vanderbilt University | Venezuelan equine encephalitis replicons expressing paramyxovirus glycoproteins as vaccine |
| WO2011020079A1 (en) * | 2009-08-13 | 2011-02-17 | Calmune Corporation | Antibodies against human respiratory syncytial virus (rsv) and methods of use |
| WO2017040387A2 (en) * | 2015-08-31 | 2017-03-09 | Technovax, Inc. | Human respiratory syncytial virus (hrsv) virus-like particles (vlps) based vaccine |
-
2021
- 2021-03-16 CN CN202110281178.8A patent/CN113249333B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006117456A1 (en) * | 2005-04-29 | 2006-11-09 | Institut National De La Recherche Agronomique (Inra) | Preparation of soluble n-protein/truncated p-protein complexes or n-proteins soluble in a virus of the paramyxoviridae family and use thereof in vaccines |
| WO2007101441A1 (en) * | 2006-03-06 | 2007-09-13 | Symphogen A/S | Recombinant polyclonal antibody for treatment of respiratory syncytial virus infections |
| CN101130765A (en) * | 2006-08-21 | 2008-02-27 | 北京阿斯可来生物工程有限公司 | Reagent kit for detecting syncytial virus of respiratory passage |
| WO2008106980A2 (en) * | 2007-03-06 | 2008-09-12 | Symphogen A/S | Recombinant antibodies for treatment of respiratory syncytial virus infections |
| WO2009042794A2 (en) * | 2007-09-26 | 2009-04-02 | Vanderbilt University | Venezuelan equine encephalitis replicons expressing paramyxovirus glycoproteins as vaccine |
| WO2011020079A1 (en) * | 2009-08-13 | 2011-02-17 | Calmune Corporation | Antibodies against human respiratory syncytial virus (rsv) and methods of use |
| WO2017040387A2 (en) * | 2015-08-31 | 2017-03-09 | Technovax, Inc. | Human respiratory syncytial virus (hrsv) virus-like particles (vlps) based vaccine |
Non-Patent Citations (6)
| Title |
|---|
| Monoclonal Antibody-Based Capture Enzyme Immunoassays for Specific Serum Immunoglobulin G (IgG), IgA, and IgM Antibodies to Respiratory Syncytial Virus;DEAN D. ERDMAN等;《JOURNAL OF CLINICAL MICROBIOLOGY》;19901231;第28卷(第12期);2744-2749 * |
| 北京地区人呼吸道合胞病毒核蛋白基因的序列分析及原核表达;孙宇等;《病毒学报》;20071115;第23卷(第06期);459-465 * |
| 呼吸道合胞病毒L蛋白单克隆抗体的制备;向江艳等;《中国免疫学杂志》;20180920;第34卷(第09期);1354-1359 * |
| 呼吸道合胞病毒单克隆抗体的制备及鉴定;陈晓琦等;《中国免疫学杂志》;20071120;第23卷(第11期);1015-1018 * |
| 抗呼吸道合胞病毒(RSV)单克隆抗体的制备及其对RSV病毒株抗原性的分析;田慕贞等;《中国病毒学》;19920630;第7卷(第02期);181-185 * |
| 用呼吸道合胞病毒单克隆抗体快速诊断合胞病毒感染;余福勋等;《中国卫生检验杂志》;19960630;第6卷(第1期);10-11 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113249333A (en) | 2021-08-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113249333B (en) | Hybridoma cell strain RSVN4C3 secreting anti-respiratory syncytial virus monoclonal antibody | |
| EP2716656B1 (en) | Monoclonal antibodies binding to avian influenza virus subtype H5 haemagglutinin and uses thereof | |
| JP6925336B2 (en) | Full human antibody of antirespiratory syncytial virus | |
| US10196438B2 (en) | Human antibodies binding to RSV G protein | |
| CN105713087B (en) | Human papilloma virus 58 monoclonal antibody and application thereof | |
| CN105585631B (en) | Human papilloma virus 16 type monoclonal antibody and application thereof | |
| CN113603769A (en) | Hybridoma cell strain capable of stably secreting anti-novel coronavirus nucleocapsid protein monoclonal antibody and establishment method and application thereof | |
| WO2016192652A1 (en) | Broad-spectrum monoclonal anti-flu b antibody and uses thereof | |
| CN113249334B (en) | Hybridoma cell strain SFTSN5G12 secreting anti-fever with thrombocytopenia syndrome virus monoclonal antibody | |
| CN113416245A (en) | Neutralizing antibody capable of combining SARS-CoV-2 virus RBD protein and application thereof | |
| WO2016173559A1 (en) | Preparation and use of murine monoclonal antibody against gi.1 norovirus | |
| CN110272488A (en) | Feline calicivirus monoclonal antibody and its application | |
| CN110078821B (en) | Sequence of enterovirus D group 68 type VP1 monoclonal antibody and application thereof | |
| CN113402604B (en) | Kit for rapidly detecting virus and preparation method thereof | |
| TWI487537B (en) | Monoclonal antibodies against enterovirus 71 and uses thereof | |
| CN113817687A (en) | Hybridoma cell strain, influenza A virus nucleoprotein monoclonal antibody and application thereof | |
| JP2011069800A (en) | Method of immunologically discriminating and detecting human influenza virus h1 subtype | |
| CA2198279A1 (en) | Antigenic peptides derived from the g protein of rsv for type- and subtype-specific diagnosis of respiratory syncytial virus (rsv) infection | |
| CN114989296B (en) | Monoclonal antibody 2F2 of rabies virus and general type rabies virus antibody rapid detection test paper | |
| WO2019165019A1 (en) | Antibodies to human respiratory syncytial virus protein f pre-fusion conformation and methods of use therefor | |
| CN115806611A (en) | Antibody against novel coronavirus N protein and application thereof | |
| CN113354734B (en) | Kit for rapidly detecting viruses and preparation method thereof | |
| CN116655779A (en) | Novel coronavirus antibodies and uses thereof | |
| CN112359020B (en) | Hybridoma cell strain, avian H1N 1-like swine influenza virus HA protein MAb, epitope and application | |
| KR101080071B1 (en) | Rift valley fever competition ELISA using monoclonal antibodies against recombinant N protein |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |