WO2022027703A1 - Monoclonal antibody of n antigen of sars-cov-2, detection method therefor and use thereof - Google Patents
Monoclonal antibody of n antigen of sars-cov-2, detection method therefor and use thereof Download PDFInfo
- Publication number
- WO2022027703A1 WO2022027703A1 PCT/CN2020/108323 CN2020108323W WO2022027703A1 WO 2022027703 A1 WO2022027703 A1 WO 2022027703A1 CN 2020108323 W CN2020108323 W CN 2020108323W WO 2022027703 A1 WO2022027703 A1 WO 2022027703A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antigen
- cov
- sars
- antibody
- gdmcc
- Prior art date
Links
- 108091007433 antigens Proteins 0.000 title claims abstract description 147
- 239000000427 antigen Substances 0.000 title claims abstract description 143
- 102000036639 antigens Human genes 0.000 title claims abstract description 142
- 238000001514 detection method Methods 0.000 title claims abstract description 124
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 24
- 208000025721 COVID-19 Diseases 0.000 claims abstract description 21
- 210000002700 urine Anatomy 0.000 claims abstract description 17
- 238000003745 diagnosis Methods 0.000 claims abstract description 12
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims abstract description 12
- 210000002966 serum Anatomy 0.000 claims abstract description 10
- 241001678559 COVID-19 virus Species 0.000 claims abstract 15
- 238000012360 testing method Methods 0.000 claims description 28
- 210000004408 hybridoma Anatomy 0.000 claims description 25
- 150000001413 amino acids Chemical class 0.000 claims description 22
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 19
- 102000039446 nucleic acids Human genes 0.000 claims description 18
- 108020004707 nucleic acids Proteins 0.000 claims description 18
- 150000007523 nucleic acids Chemical class 0.000 claims description 18
- 238000003317 immunochromatography Methods 0.000 claims description 17
- 229940126619 mouse monoclonal antibody Drugs 0.000 claims description 17
- 238000004587 chromatography analysis Methods 0.000 claims description 10
- 229910052693 Europium Inorganic materials 0.000 claims description 3
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 claims description 3
- 239000004005 microsphere Substances 0.000 claims description 3
- 239000000084 colloidal system Substances 0.000 claims 1
- 239000010931 gold Substances 0.000 claims 1
- 229910052737 gold Inorganic materials 0.000 claims 1
- 238000003018 immunoassay Methods 0.000 claims 1
- 230000028327 secretion Effects 0.000 claims 1
- 238000012216 screening Methods 0.000 abstract description 6
- 241001529936 Murinae Species 0.000 abstract 2
- 238000005516 engineering process Methods 0.000 description 15
- 241000699670 Mus sp. Species 0.000 description 14
- 230000035945 sensitivity Effects 0.000 description 11
- 101710141454 Nucleoprotein Proteins 0.000 description 10
- 241000711573 Coronaviridae Species 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 206010003445 Ascites Diseases 0.000 description 6
- 239000002250 absorbent Substances 0.000 description 6
- 230000002745 absorbent Effects 0.000 description 6
- 206010036790 Productive cough Diseases 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 239000002671 adjuvant Substances 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 210000003802 sputum Anatomy 0.000 description 5
- 208000024794 sputum Diseases 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 4
- 101001024637 Severe acute respiratory syndrome coronavirus 2 Nucleoprotein Proteins 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 230000002163 immunogen Effects 0.000 description 4
- 238000009629 microbiological culture Methods 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- 206010037660 Pyrexia Diseases 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000002818 protein evolution Methods 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 244000309467 Human Coronavirus Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 241000315672 SARS coronavirus Species 0.000 description 2
- 208000037847 SARS-CoV-2-infection Diseases 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000010923 batch production Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000037029 cross reaction Effects 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 210000003200 peritoneal cavity Anatomy 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 208000031504 Asymptomatic Infections Diseases 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000494545 Cordyline virus 2 Species 0.000 description 1
- 102100031673 Corneodesmosin Human genes 0.000 description 1
- 101710139375 Corneodesmosin Proteins 0.000 description 1
- 108700002099 Coronavirus Nucleocapsid Proteins Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 108091005774 SARS-CoV-2 proteins Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- MUUGCDGGIVCSDT-UHFFFAOYSA-N [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O Chemical compound [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O MUUGCDGGIVCSDT-UHFFFAOYSA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000003771 laboratory diagnosis Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000011330 nucleic acid test Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000004926 tubular epithelial cell Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1002—Coronaviridae
- C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/08—RNA viruses
- C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
-
- 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
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
-
- G—PHYSICS
- 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
Definitions
- the invention relates to the technical field of immunodiagnosis, in particular to the detection of viral infection by viral antigens, and in particular to a specific diagnostic method for novel coronavirus pneumonia (COVID-19) caused by novel coronavirus (SARS-CoV-2) infection.
- COVID-19 novel coronavirus pneumonia
- SARS-CoV-2 novel coronavirus
- Acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the pathogen that causes coronavirus disease-2019 (Corona Virus Disease 2019, COVID-19), and has been identified by the World Health Organization (world health organization) , WHO) officially named. As of July 2020, there have been more than 80,000 confirmed cases of COVID-19 in mainland China, and the epidemic has spread to more than 100 countries and regions around the world. The cumulative number of cases worldwide has exceeded 15 million. The clinical manifestations of COVID-19 within 7 days of onset are not specific, so the disease cannot be diagnosed by clinical symptoms, signs and general examinations. A specific, early, fast, simple, and suitable diagnosis method for grassroots use is the basis for "early detection, early isolation, and early treatment", and is also the key and necessary technology for epidemic prevention and control.
- nucleic acid detection In clinical practice, the laboratory diagnosis of COVID-19 relies on nucleic acid detection and antibody detection.
- the overall detection rate of nucleic acid detection is high, but gene amplification is required, the false negative and false positive rates are high, and sometimes repeated detection is required, the repeatability is poor, and the sample is easily contaminated, requiring professional instruments such as PCR amplifier and gel electrophoresis.
- the equipment requires high technical conditions for sample processing and detection, and the detection takes a long time. Professional technicians are required to operate and judge the test results.
- Antibody detection because of the late emergence of antibodies, cannot be diagnosed early, and the detection rate of antibodies is low, resulting in a large number of missed diagnoses.
- the technical problem to be solved by the present invention is to establish a COVID-19 diagnostic method for detecting SARS-CoV-2 virus based on antibodies against the N antigen of SARS-CoV-2.
- the present invention provides a method for detecting the N antigen (nucleocapsid protein, also known as nucleocapsid protein, N protein) of SARS-CoV-2: N antigen mouse monoclonal antibody, find the epitope-antibody pair with diagnostic value, based on the above epitope-antibody pair, through colloidal gold, fluorescein and other labels, to achieve high sensitivity, strong specificity, fast, simple, It is suitable for screening and diagnosis of COVID-19 in hospitals, grassroots, community, family and other places, and efficiently diagnoses COVID-19.
- the invention can promote the early detection, isolation and treatment of the source of infection, and effectively prevent the spread of the epidemic.
- the first step to manufacture the N-antigen series monoclonal antibody of SARS-CoV-2 The first step to manufacture the N-antigen series monoclonal antibody of SARS-CoV-2
- the N antigen gene was cloned and expressed in E. coli.
- the Escherichia coli engineering bacteria expressing the N antigen of SARS-CoV-2 are subjected to fermentation technology to obtain a stock solution containing the N antigen.
- the stock solution of N antigen was separated and purified by chromatography technology to obtain 10 mg of N antigen with a purity of more than 95%, which was stored at -20 degrees Celsius for future use.
- mice Take 20 ⁇ g of the above N antigen as an immunogen mixed with complete Freund's adjuvant subcutaneously to immunize Balb/c mice for the first time, a total of 20 mice; take the same amount of the above N antigen and incomplete Freund's adjuvant on 7 days and 14 days respectively.
- Mixed boost Balb/c mice On the 28th day after the primary immunization, retro-orbital blood was collected from the mice to detect the anti-N antigen antibody titer. Mice with a titer greater than 1:160 were sacrificed by cervical dislocation and the spleen was taken to obtain a single-cell suspension of spleen cells to prepare hybridoma cells. Fifteen monoclonal antibody cell lines were obtained by infinite dilution method and identification, and were frozen for future use.
- the above 15 monoclonal antibody cell lines were taken and injected into the peritoneal cavity of mice to prepare ascites, and the ascites was purified to obtain 15 monoclonal antibody lines.
- Step 2 Discover epitope-antibody pairs with diagnostic value
- colloidal gold technology and fluorescent technology were used to label antibodies, and a method for N antigen detection of SARS-CoV-2 was established.
- Fluorescein-labeled SARS-CoV-2 N antigen monoclonal antibodies M1, M2, and M3 were made into fluorescein test strips to detect the spare SARS-CoV-2 N antigen in the first step. Any N antigen above 10ng can be detected as positive .
- the method of the present invention was used to detect the N antigen of SARS-CoV-2 in nasopharyngeal swabs and urine samples.
- the N antigen immunodetection method of SARS-CoV-2 in the present invention includes antibodies, including capture antibodies M1, M2 and M3 that are labeled with detection signal molecules and can specifically bind to the N antigen of SARS-CoV-2, and can specifically bind to SARS-CoV-2.
- the detection antibody M4 of the N antigen of CoV-2, the detection signal molecule can be colloidal gold or fluorescein or other markers commonly used in the art.
- the capture antibody combination is mouse monoclonal antibodies M1, M2, and M3 in a ratio of 1:1:1, which specifically bind to amino acids 1-69, 119-213, and 212-341 of the N antigen of SARS-CoV-2, respectively.
- Amino acid sequence; the detection antibody is M4, which is a mouse monoclonal antibody that specifically binds to the amino acid sequence of amino acids 337-422 of the N antigen of SARS-CoV-2.
- the antibody combination M1, M2, M3 and the monoclonal antibody M4 are all immunoglobulin IgG, which are respectively secreted by the hybridoma cell strains whose deposit numbers are GDMCC 61007, GDMCC 61008, GDMCC 61009 and GDMCC 61010.
- the detection signal molecule may be colloidal gold, fluorescein, or the like.
- the detection device used in the detection method is a colloidal gold test strip, and the colloidal gold test strip includes a sample pad, a binding pad, a reaction pad and a water-absorbing pad connected in sequence; it is characterized in that , the binding pad is coated with a combination of colloidal gold-labeled M1, M2, and M3 antibodies.
- the detection device used in the detection method is a fluorescence immunochromatography detection test strip
- the fluorescence immunochromatography detection test strip includes a sample pad, a binding pad, a reaction pad and A water-absorbing pad; characterized in that the binding pad is coated with a combination of M1, M2, and M3 antibodies labeled with europium microspheres.
- the capture antibody and detection antibody used in the present invention are selected from 15 monoclonal antibodies that specifically bind to the N antigen of SARS-CoV-2.
- the combination of capture antibody M1, M2, M3 and detection antibody M4 binds to different epitopes of N antigen respectively, which not only significantly improves the sensitivity, specificity and stability of detection with a single antibody, reduces clinical missed detection, but is also suitable for nasopharyngeal Swab, urine, blood, sputum and other different samples are tested.
- the use of monoclonal antibodies overcomes the cross-reaction of polyclonal antiserum with other common viruses and coronaviruses, with good repeatability and easy standardization.
- the present invention provides an immunodetection method for detecting the N antigen of SARS-CoV-2, which is characterized in that an antibody against the N antigen is used to detect the N antigen.
- the N antigen capture antibodies M1, M2, M3, the detection antibody M4, optionally, the detection signal molecule is selected from colloidal gold or fluorescein.
- the present invention also provides a capture antibody, which is characterized by mouse monoclonal antibodies M1, M2, and M3, which specifically bind to amino acids 1-69, 119-213, and 212-341 of the N antigen of SARS-CoV-2, respectively. sequence.
- the present invention also provides a detection antibody M4, which is characterized by a mouse monoclonal antibody that specifically binds to the amino acid sequence of amino acids 337-422 of the N antigen of SARS-CoV-2.
- the invention also provides an N antigen colloidal gold immunochromatography device for detecting SARS-CoV-2, which uses M1, M2 and M3 monoclonal antibodies labeled with colloidal gold.
- the present invention also provides an N antigen fluorescence immunochromatography device for detecting SARS-CoV-2, which uses fluorescein-labeled M1, M2, and M3 monoclonal antibodies.
- the invention also provides an immune detection method, which is characterized in that the detection method can specifically detect the N antigen of SARS-CoV-2 in human samples such as nasopharyngeal swabs, serum, and urine.
- the present invention also provides an immune detection method, characterized in that the N antigen of SARS-CoV-2 detected by the detection method can be used to diagnose COVID-19, and the coincidence rate with the nucleic acid detection and diagnosis method is more than 99%.
- the specific detection of the N antigen of SARS-CoV-2 is achieved, and the specific diagnosis of COVID-19 is realized, and the specificity reaches 99%.
- the epitope-antibody pair by selecting the epitope-antibody pair, high-sensitivity detection of the N antigen of SARS-CoV-2 is achieved, and the detection sensitivity of the N antigen of SARS-CoV-2 reaches the ng level.
- the N antigen of SARS-CoV-2 can be detected in the nasopharyngeal swab sample on the 3rd day of fever through the early detection of the N antigen of SARS-CoV-2 by selecting the epitope-antibody pair to realize the early detection of the N antigen of SARS-CoV-2. diagnosis.
- the general and simple technology of colloidal gold and fluorescently labeled test paper is adopted, and the result is obtained within 10 minutes, so as to achieve the rapid detection of the N antigen of SARS-CoV-2, and realize rapid diagnosis.
- the detection method is reliable, simple and suitable for medical institutions, grassroots, families and individuals. Provide better means for the screening and diagnosis of suspected patients and asymptomatic infections and for early and rapid prevention of the spread of the epidemic.
- a hybridoma cell M1 its taxonomic name is Mus musculus hybridoma 1-10: M1
- the strain was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on April 30, 2020, and was preserved No. GDMCC 61007, deposit address: Guangdong Institute of Microbiology, Building 59, Yard No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong province, China, Postal Code: 510070.
- a hybridoma cell M2 its taxonomic name is Mus musculus hybridoma 11-20: M2, the strain was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on April 30, 2020, and was preserved No. GDMCC 61008, deposit address: Guangdong Institute of Microbiology, Building 59, Yard No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong province, China, Postal Code: 510070.
- a hybridoma cell M3 its taxonomic name is Mus musculus hybridoma 21-30: M3, the strain was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on April 30, 2020, and was preserved No. GDMCC 61009, deposit address: Guangdong Institute of Microbiology, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong province, China, zip code: 510070.
- GDMCC Guangdong Microbial Culture Collection Center
- a hybridoma cell M4 its taxonomic name is Mus musculus hybridoma 31-40: M4, the strain was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on April 30, 2020, and deposited No. GDMCC 61010, deposit address: Guangdong Institute of Microbiology, Building 59, Yard No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong province, China, Postal Code: 510070.
- GDMCC Guangdong Microbial Culture Collection Center
- the present invention can be carried out by the following embodiments, but the present invention is not limited thereto.
- the immunogen used for preparing the monoclonal antibody of the present invention is the N antigen of genetically recombined SARS-CoV-2.
- the genetically recombined N antigen is prepared by an engineered strain carrying the N antigen gene.
- the preparation method is carried out according to the conventional method, and the N antigen is obtained by purifying by the method of molecular sieve chromatography.
- the purified N antigen was identified by western blot, which was consistent with the theoretical molecular weight.
- mice 4-6 week old female BALB/c mice were immunized for the first time with complete Freund's adjuvant and an equal volume of N antigen for a total of 3 times. Before fusion, each mouse was injected intraperitoneally with 100 ⁇ g of N antigen to boost the immunization .
- the detection method of the present invention is realized by a colloidal gold detection test strip, including a sample pad, a binding pad, a reaction pad and a water-absorbing pad which are connected in sequence, wherein the sample pad, the binding pad, the reaction pad and the water-absorbing pad are connected in sequence.
- the sample pads, the binding pads, the reaction pads and the water absorption pads are arranged on the bottom plate.
- a diluent for diluting the sample to be tested is provided.
- the detection method of the present invention is realized by a fluorescent immunochromatography test strip, including a sample pad, a binding pad, a reaction pad and a water-absorbing pad connected in sequence, and the sample pad, the binding pad, the reaction pad and the water-absorbing pad are connected in sequence.
- the sample pads, the binding pads, the reaction pads and the water absorption pads are arranged on the bottom plate.
- a diluent for diluting the sample to be tested is provided.
- the detection method of the present invention in clinical suspected cases of COVID-19, takes the nucleic acid detection of nasopharyngeal swabs as the gold standard, and uses the method of the present invention to detect the N antigen of SARS-CoV-2 in nasopharyngeal swabs, urine and serum samples , to evaluate the clinical diagnostic significance of the detection method.
- the raw materials and equipment used in the present invention are the common raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are the conventional methods in the art.
- compositions and methods/processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein and any additional or optional ingredients, components, steps or limitations described herein.
- the sample includes at least one of nasopharyngeal swab, sputum, bronchoalveolar lavage fluid, blood, and urine, more preferably, the sample includes nasopharyngeal swab, sputum, bronchoalveolar lavage fluid, blood, For at least two kinds of urine, the samples are tested separately.
- the N antigen gene was cloned and expressed in E. coli.
- the Escherichia coli engineering bacteria expressing the N antigen of SARS-CoV-2 are subjected to fermentation technology to obtain a stock solution containing the N antigen.
- the stock solution of N antigen was separated and purified by chromatography technology to obtain 10 mg of N antigen with a purity of more than 95%, which was stored at -20°C for later use.
- mice Take 20 ⁇ g of the above N antigen as an immunogen mixed with complete Freund's adjuvant subcutaneously to immunize Balb/c mice for the first time, a total of 20 mice; take the same amount of the above N antigen and incomplete Freund's adjuvant on 7 days and 14 days respectively.
- Mixed boost Balb/c mice On the 28th day after the primary immunization, retro-orbital blood was collected from the mice to detect the anti-N antigen antibody titer. Mice with a titer greater than 1:160 were sacrificed by cervical dislocation and the spleen was taken to obtain a single-cell suspension of spleen cells to prepare hybridoma cells. Fifteen monoclonal antibody cell lines were obtained by infinite dilution method and identification, and were frozen for future use.
- Example 2 Anti-SARS-CoV-2 N protein monoclonal antibody M1-4 specifically binds to the N of SARS-CoV-2 protein
- the amino acid sequence of the N protein of common coronaviruses has more than 80% homology, only the monoclonal antibody that specifically recognizes the N protein of SARS-CoV-2 has diagnostic value for SARS-CoV-2 infection, and the above monoclonal antibody Manufacturing methods cannot avoid obtaining antibodies that bind to other coronaviruses.
- the 15 monoclonal antibodies obtained in Example 1 were further identified to search for monoclonal antibodies that only specifically bind to the N protein of SARS-CoV-2.
- Example 1 a 15 strains (M1 to M15) antibodies were produced in Example 1.
- yeast display technology to display different amino acid sequences of the N antigen of SARS-CoV-2 on the surface of yeast: amino acids 1-422, 1-213, 1-120, 68-213, 1-69, 68-120, 119-213, 214-422, 212-341, 337-422, using the flow cytometry technique based on the above-mentioned preparation of monoclonal antibodies, to determine the epitope sequences bound by each strain of monoclonal antibodies.
- a Flow cytometry detects the binding of different amino acid fragments of the N antigen of SARS-CoV-2 displayed by yeast to different monoclonal antibodies.
- the results showed the ratio of the fluorescence value of the binding between the mAb and the expressed antigen fragment detected by flow cytometry and the fluorescence value of the binding between the mAb and the empty expression vector (EBY100/pYD1), and a ratio greater than 2 was positive.
- N antigen amino acid sequence On the surface of EBY100 yeast cells, 10 different amino acid sequence fragments covering the full length of N antigen were displayed to investigate which amino acid sequences of different mAbs bind to N antigen.
- M1 to M4 have specific diagnostic value for SARS-CoV-2 infection, so M1-[amino acids 1-69 of N antigen], M2-[amino acids 119-213 of N antigen], M2-[amino acids 119-213 of N antigen], M3-[amino acids 212-341 of N antigen] 3 epitope-antibody pairs as capture epitope-antibody pairs; M4-[amino acids 337-422 of N antigens] epitope-antibody pair as detection epitopes- antibody pair.
- the effect of different antibody-epitope combinations on the detection sensitivity of the N antigen of SARS-CoV-2 was detected by chemiluminescence and fluorescence chromatography.
- the detection sensitivity was based on the lowest content ( ⁇ g/ml) of N antigen that could be detected. The results are shown in Table 4.
- the optimal combination of capture antibodies thus obtained is M1, M2, M3.
- This embodiment provides a colloidal gold immunochromatography device for detecting the N antigen of SARS-CoV-2, which can detect positive if the N antigen is more than 30 ng. It is characterized in that the binding pad is coated with colloidal gold-labeled M1, M2, M3 antibody combination.
- the colloidal gold immunochromatography device for detecting the N antigen of SARS-CoV-2 is realized by a colloidal gold detection test strip, and the colloidal gold detection test strip includes a sample pad, a binding pad, and a reaction pad connected in sequence. Pad and absorbent pad, the sample pad, binding pad, reaction pad and absorbent pad are overlapped and connected to each other, and the sample pad, binding pad, reaction pad and absorbent pad are arranged on the bottom plate.
- a detection line and a quality control line are sequentially arranged on the reaction pad along the flow direction of the sample, the detection line is coated with a detection antibody M4, and the quality control line is coated with a goat anti-mouse IgG secondary antibody.
- the capture antibody combination is mouse monoclonal antibodies M1, M2, and M3 in a ratio of 1:1:1, which specifically bind to amino acids 1-69, 119-213, and 212-341 of the N antigen of SARS-CoV-2, respectively.
- Amino acid sequence; the detection antibody is M4, which is a mouse monoclonal antibody that specifically binds to the amino acid sequence of amino acids 337-422 of the N antigen of SARS-CoV-2.
- Capture antibody combination M1, M2, M3 and detection antibody M4 bind to different epitopes of N antigen respectively, which not only significantly improves the sensitivity, specificity and stability of detection with a single antibody, reduces clinical missed detection, but also is suitable for nasopharyngeal Swab, urine, blood, sputum and other different samples are tested.
- nasopharyngeal swabs Twenty-two clinically suspected patients were selected, and nasopharyngeal swabs and urine samples were taken simultaneously.
- the nasopharyngeal swabs gold standard
- the nasopharyngeal swabs were detected in parallel with the nationally approved nucleic acid detection reagent, and the urine was detected with the colloidal gold test paper of the detection technology of the present invention.
- 15 cases of N antigen were detected in urine at the same time, the detection rate was 71.4%; N antigen was detected in the urine of 1 patient (4.5%) with negative nasopharyngeal swabs. See Table 5 for the results.
- Urine N antigen test results 1 +(24.1) + 2 +(19.7) + 3 +(27.6) + 4 +(24.3) + 5 +(35.5) - 6 +(24.5) + 7 +(22.2) + 8 +(26.2) + 9 +(33.1) + 10 +(33.9) + 11 +(32.5) + 12 +(30.4) + 13 +(32.1) + 14 +(34.5) - 15 +(35.4) - 16 +(33.6) - 17 +(32.3) - 18 +(34.6) + 19 - + 20 +(31.8) + twenty one +(29.5) + twenty two +(17.1) +
- Embodiment 6 fluorescence chromatography test paper throat swab sample clinical detection verification
- This embodiment provides an N antigen fluorescence immunochromatography device for detecting SARS-CoV-2, which can detect positive when N antigen is more than 10 ng. It is characterized in that the binding pad is coated with M1 and M2 labeled with europium microspheres. , M3 antibody combination.
- the N antigen fluorescent immunochromatography device for detecting SARS-CoV-2 is realized by a fluorescent immunochromatography test strip, and the fluorescent immunochromatography test strip includes a sample pad, a binding pad, a reaction pad and a reaction pad connected in sequence. Pad and absorbent pad, the sample pad, binding pad, reaction pad and absorbent pad are overlapped and connected to each other, and the sample pad, binding pad, reaction pad and absorbent pad are arranged on the bottom plate.
- a detection line and a quality control line are sequentially arranged on the reaction pad along the flow direction of the sample, the detection line is coated with a detection antibody M4, and the quality control line is coated with a rabbit anti-mouse secondary antibody.
- Described capture antibody combination is mouse monoclonal antibody M1, M2, M3, the ratio is 1:1:1, respectively specifically binds to amino acid 1-69, 119-213, 212-341 of N antigen of SARS-CoV-2 Amino acid sequence;
- the detection antibody is M4, which is a mouse monoclonal antibody that specifically binds to the amino acid sequence of amino acids 337-422 of the N antigen of SARS-CoV-2.
- Capture antibody combination M1, M2, M3 and detection antibody M4 bind to different epitopes of N antigen respectively, which not only significantly improves the sensitivity, specificity and stability of detection with a single antibody, reduces clinical missed detection, but also is suitable for nasopharyngeal Swab, urine, blood, sputum and other different samples are tested.
- nasopharyngeal swab samples were taken, and the nucleic acid of SARS-CoV-2 was detected in parallel with the nucleic acid detection reagent approved by the state and the N antigen of SARS-CoV-2 was detected by the fluorescence chromatography test strip of the detection technology of the present invention.
- the specificity of the present invention is 100%, and the positive pre-test value is 100%; the nucleic acid detection results in 90 minutes, while the N antigen detection of the present invention results in 10 minutes. Therefore, the N antigen detection of the present invention is a more rapid detection method.
- nasopharyngeal swab samples were taken, and the nucleic acid of SARS-CoV-2 was detected in parallel with the nucleic acid detection reagent approved by the state and the N antigen of SARS-CoV-2 was detected by the fluorescence chromatography test strip of the detection technology of the present invention.
- Antibodies were detected in serum samples taken from each patient on the same day the nasopharyngeal swab was taken.
- N antigen-positive samples 100% of N antigen-positive samples are from confirmed patients, and the specificity is 100%; for suspected patients with fever and pneumonia, 100% of patients who are excluded after repeated testing are negative for N antigen; serum samples
- the sensitivity of N antigen detection is higher than that of nasopharyngeal swab samples: serum samples, nucleic acid Ct value of 32.3 (low viral load), and antigen detection are also positive; in this batch of samples, fever for 3 days, nasopharyngeal swabs are N antigen can be detected.
- This example illustrates that the serum and nasopharyngeal swab samples of the present invention can detect N antigen by the method of the present invention, and the method of the present invention is an early diagnosis method.
- Antigen results refer to the results detected by fluorescence chromatography test strips. The patient's nasopharyngeal swab sample was detected at the same time as the patient's serum sample, and the patient's nasopharyngeal swab sample was detected at the same time as the patient's serum sample.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Virology (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nanotechnology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Communicable Diseases (AREA)
- Pulmonology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
An immune detection method on the basis of an antibody that specifically binds to an N antigen of SARS-CoV-2, which is used for the diagnosis of COVID-19. The method comprises marking a capture antibody combination that specifically binds to an N antigen having a detection signal molecule, and detecting the antibody. By means of the method, the presence of an antigen is detected by specifically binding an antibody to an antigen; and the capture antibody is a murine monoclonal antibody combination, which is composed of M1, M2 and M3, the detection antibody is a murine monoclonal antibody M4, and the detection signal molecule can be a colloidal gold or fluorescein. The detection method can be used to specifically detect the N antigen of SARS-CoV-2 in human samples such as from nasopharyngeal swabs, urine and serum, so as to diagnose COVID-19 early, which can be applied to rapid screening and diagnosis in hospitals and on-site.
Description
本发明涉及免疫诊断技术领域，具体涉及通过病毒抗原检测病毒感染，特别是涉及用于新型冠状病毒(SARS-CoV-2)感染引起的新冠肺炎(COVID-19)的特异性诊断方法。The invention relates to the technical field of immunodiagnosis, in particular to the detection of viral infection by viral antigens, and in particular to a specific diagnostic method for novel coronavirus pneumonia (COVID-19) caused by novel coronavirus (SARS-CoV-2) infection.
[根据细则9.2改正19.08.2020]
急性呼吸综合征冠状病毒-2(Acute respiratory syndrome coronavirus 2，SARS-CoV-2)是引起冠状病毒病-2019(Corona Virus Disease 2019，COVID-19)的病原体，并被世界卫生组织(world health organization，WHO)正式命名。截至2020年7月，COVID-19在中国大陆确诊病例8万余人，疫情波及全世界100多个国家和地区，全球累计病例已超过1500万例。COVID-19发病7天内临床表现没有特异性，因此不能通过临床症状、体征和一般检查诊断该病。而特异、早期、快速、简便、适合基层使用的诊断方法，是实现“早发现、早隔离、早治疗”的基础，也是疫情防控的关键、必须技术。[Correction 19.08.2020 in accordance with Rule 9.2]
Acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the pathogen that causes coronavirus disease-2019 (Corona Virus Disease 2019, COVID-19), and has been identified by the World Health Organization (world health organization) , WHO) officially named. As of July 2020, there have been more than 80,000 confirmed cases of COVID-19 in mainland China, and the epidemic has spread to more than 100 countries and regions around the world. The cumulative number of cases worldwide has exceeded 15 million. The clinical manifestations of COVID-19 within 7 days of onset are not specific, so the disease cannot be diagnosed by clinical symptoms, signs and general examinations. A specific, early, fast, simple, and suitable diagnosis method for grassroots use is the basis for "early detection, early isolation, and early treatment", and is also the key and necessary technology for epidemic prevention and control.
急性呼吸综合征冠状病毒-2(Acute respiratory syndrome coronavirus 2，SARS-CoV-2)是引起冠状病毒病-2019(Corona Virus Disease 2019，COVID-19)的病原体，并被世界卫生组织(world health organization，WHO)正式命名。截至2020年7月，COVID-19在中国大陆确诊病例8万余人，疫情波及全世界100多个国家和地区，全球累计病例已超过1500万例。COVID-19发病7天内临床表现没有特异性，因此不能通过临床症状、体征和一般检查诊断该病。而特异、早期、快速、简便、适合基层使用的诊断方法，是实现“早发现、早隔离、早治疗”的基础，也是疫情防控的关键、必须技术。[Correction 19.08.2020 in accordance with Rule 9.2]
Acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the pathogen that causes coronavirus disease-2019 (Corona Virus Disease 2019, COVID-19), and has been identified by the World Health Organization (world health organization) , WHO) officially named. As of July 2020, there have been more than 80,000 confirmed cases of COVID-19 in mainland China, and the epidemic has spread to more than 100 countries and regions around the world. The cumulative number of cases worldwide has exceeded 15 million. The clinical manifestations of COVID-19 within 7 days of onset are not specific, so the disease cannot be diagnosed by clinical symptoms, signs and general examinations. A specific, early, fast, simple, and suitable diagnosis method for grassroots use is the basis for "early detection, early isolation, and early treatment", and is also the key and necessary technology for epidemic prevention and control.
临床实践中，COVID-19的实验室诊断依赖于核酸检测、抗体检测。核酸检测总体检出率高，但需要基因扩增，假阴性、假阳性率较高，有时需要反复检测，可重复性差，样本易被污染，需要PCR扩增仪及凝胶电泳等专业的仪器设备，对样本处理和检测技术条件要求高，检测耗时长，需专业技术人员操作和判断检测结果。而抗体检测因为抗体出现晚，不能早期诊断，抗体检出率低，造成大量漏诊。In clinical practice, the laboratory diagnosis of COVID-19 relies on nucleic acid detection and antibody detection. The overall detection rate of nucleic acid detection is high, but gene amplification is required, the false negative and false positive rates are high, and sometimes repeated detection is required, the repeatability is poor, and the sample is easily contaminated, requiring professional instruments such as PCR amplifier and gel electrophoresis. The equipment requires high technical conditions for sample processing and detection, and the detection takes a long time. Professional technicians are required to operate and judge the test results. Antibody detection, because of the late emergence of antibodies, cannot be diagnosed early, and the detection rate of antibodies is low, resulting in a large number of missed diagnoses.
因此，临床亟需一种更早期、更灵敏、更快捷、更有效的检测新型冠状病毒COVID-19的诊断试剂，进行早期鉴别诊断。而病毒抗原检测，由于抗原为病毒特有，可实现高特异性诊断；病毒抗原出现早于抗体、早于临床症状，可实现早期诊断。因此，通过抗原检测诊断COVID-19是理想的方法。Therefore, there is an urgent need for an earlier, more sensitive, faster and more effective diagnostic reagent for the detection of the new coronavirus COVID-19 for early differential diagnosis. For virus antigen detection, because the antigen is unique to the virus, it can achieve high-specificity diagnosis; the virus antigen appears earlier than the antibody and earlier than the clinical symptoms, and the early diagnosis can be achieved. Therefore, the diagnosis of COVID-19 by antigen testing is ideal.
发明内容SUMMARY OF THE INVENTION
本发明要解决的技术问题是建立基于针对SARS-CoV-2的N抗原的抗体检测SARS-CoV-2病毒的COVID-19诊断方法。为了克服已有技术的缺陷，本发明提供一种SARS-CoV-2的N抗原(nucleocapsid protein，也称为核衣壳蛋白、N蛋白)检测方法：通过制造、筛选针对SARS-CoV-2的N抗原的小鼠单克隆抗体，找到具有诊断价值的表 位-抗体对，基于上述表位-抗体对，通过胶体金、荧光素等标记，实现了灵敏度高、特异性强，快速、简便，适合医院、基层、社区、家庭等多种场所的COVID-19筛查和诊断，高效确诊COVID-19。本发明能够促进传染源的早期发现、隔离、治疗，高效预防疫情扩散。The technical problem to be solved by the present invention is to establish a COVID-19 diagnostic method for detecting SARS-CoV-2 virus based on antibodies against the N antigen of SARS-CoV-2. In order to overcome the defects of the prior art, the present invention provides a method for detecting the N antigen (nucleocapsid protein, also known as nucleocapsid protein, N protein) of SARS-CoV-2: N antigen mouse monoclonal antibody, find the epitope-antibody pair with diagnostic value, based on the above epitope-antibody pair, through colloidal gold, fluorescein and other labels, to achieve high sensitivity, strong specificity, fast, simple, It is suitable for screening and diagnosis of COVID-19 in hospitals, grassroots, community, family and other places, and efficiently diagnoses COVID-19. The invention can promote the early detection, isolation and treatment of the source of infection, and effectively prevent the spread of the epidemic.
本发明通过以下技术方案解决上述技术问题：The present invention solves the above-mentioned technical problems through the following technical solutions:
第一步 制造SARS-CoV-2的N抗原系列单克隆抗体抗体The first step to manufacture the N-antigen series monoclonal antibody of SARS-CoV-2
基于发布的SARS-CoV-2的N抗原基因序列，克隆N抗原基因，在大肠杆菌中表达N抗原。将表达SARS-CoV-2的N抗原的大肠杆菌工程菌通过发酵技术，获得含有N抗原的原液。将N抗原的原液经过层析技术分离纯化N抗原，获得纯度95％以上的N抗原10mg，-20摄氏度保存备用。Based on the published N antigen gene sequence of SARS-CoV-2, the N antigen gene was cloned and expressed in E. coli. The Escherichia coli engineering bacteria expressing the N antigen of SARS-CoV-2 are subjected to fermentation technology to obtain a stock solution containing the N antigen. The stock solution of N antigen was separated and purified by chromatography technology to obtain 10 mg of N antigen with a purity of more than 95%, which was stored at -20 degrees Celsius for future use.
取用上述N抗原20μg作为免疫原与完全弗氏佐剂混合皮下注射初次免疫Balb/c小鼠，共20只；分别于7天、14天取上述等量N抗原与非完全弗氏佐剂混合加强免疫Balb/c小鼠。于初次免疫后第28天小鼠眼眶后取血检测抗N抗原抗体滴度，滴度大于1:160的小鼠脱颈处死、取脾脏，获得脾细胞单细胞悬液，制备杂交瘤细胞，通过无限稀释法和鉴定获得15个单克隆抗体细胞株，冻存备用。Take 20 μg of the above N antigen as an immunogen mixed with complete Freund's adjuvant subcutaneously to immunize Balb/c mice for the first time, a total of 20 mice; take the same amount of the above N antigen and incomplete Freund's adjuvant on 7 days and 14 days respectively. Mixed boost Balb/c mice. On the 28th day after the primary immunization, retro-orbital blood was collected from the mice to detect the anti-N antigen antibody titer. Mice with a titer greater than 1:160 were sacrificed by cervical dislocation and the spleen was taken to obtain a single-cell suspension of spleen cells to prepare hybridoma cells. Fifteen monoclonal antibody cell lines were obtained by infinite dilution method and identification, and were frozen for future use.
取上述15个单克隆抗体细胞株，分别注射入小鼠腹腔制备腹水，取腹水纯化获得单克隆抗体15株。The above 15 monoclonal antibody cell lines were taken and injected into the peritoneal cavity of mice to prepare ascites, and the ascites was purified to obtain 15 monoclonal antibody lines.
第二步 发现具有诊断价值的表位-抗体对Step 2 Discover epitope-antibody pairs with diagnostic value
基于上述15株单克隆抗体，检测与其它常见冠状病毒N抗原的交叉反应性，获得SARS-CoV-2的N抗原特异性(只与SARS-CoV-2的N抗原结合而与其它冠状病毒不结合)的单克隆抗体4株分别命名为M1、M2、M3和M4。Based on the above 15 monoclonal antibodies, the cross-reactivity with other common coronavirus N antigens was detected, and the N antigen specificity of SARS-CoV-2 was obtained (it only binds to the N antigen of SARS-CoV-2 and does not interact with other coronaviruses). The four strains of monoclonal antibodies that bind to ) were named M1, M2, M3 and M4, respectively.
采用酵母展示技术，明确上述4株单克隆抗体所结合的表位，获得4个诊断用表位-抗体对。Using yeast display technology, the epitopes bound by the above four monoclonal antibodies were identified, and four diagnostic epitope-antibody pairs were obtained.
第三步 标记与检测方法建立The third step is the establishment of labeling and detection methods
基于上述发现的4个表位-抗体对，采用胶体金技术、荧光技术标记抗体，建立SARS-CoV-2的N抗原检测方法。Based on the four epitope-antibody pairs found above, colloidal gold technology and fluorescent technology were used to label antibodies, and a method for N antigen detection of SARS-CoV-2 was established.
胶体金标记SARS-CoV-2的N抗原单克隆抗体M1、M2、M3，制成胶体金试纸，检测第一步中备用SARS-CoV-2的N抗原，N抗原30ng以上均可检出阳性。Colloidal gold labeled SARS-CoV-2 N antigen monoclonal antibodies M1, M2, M3, made into colloidal gold test paper, to detect the N antigen of SARS-CoV-2 in the first step, N antigen above 30ng can be detected as positive .
荧光素标记SARS-CoV-2的N抗原单克隆抗体M1、M2、M3，制成荧光素试纸，检测第一步中备用SARS-CoV-2的N抗原，N抗原10ng以上均可检出阳性。Fluorescein-labeled SARS-CoV-2 N antigen monoclonal antibodies M1, M2, and M3 were made into fluorescein test strips to detect the spare SARS-CoV-2 N antigen in the first step. Any N antigen above 10ng can be detected as positive .
第四步 临床验证Step 4 Clinical Validation
以临床疑似COVID-19患者为基础，以鼻咽拭子核酸检测为金标准，用本发明的方法检测鼻咽拭子和尿液样本SARS-CoV-2的N抗原。On the basis of clinically suspected COVID-19 patients and the nucleic acid detection of nasopharyngeal swabs as the gold standard, the method of the present invention was used to detect the N antigen of SARS-CoV-2 in nasopharyngeal swabs and urine samples.
本发明中SARS-CoV-2的N抗原免疫检测方法包括抗体，其中包括标记有检测信号分子的能特异结合SARS-CoV-2的N抗原的捕获抗体M1、M2、M3，能特异结合SARS-CoV-2的N抗原的检测抗体M4，检测信号分子可以是胶体金或荧光素或其他本领域常用的标记物。所述捕获抗体组合是小鼠单克隆抗体M1、M2、M3，比例1:1:1，分别特异性结合SARS-CoV-2的N抗原的氨基酸1-69、119-213、212-341的氨基酸序列；所述检测抗体是M4，其为特异性结合SARS-CoV-2的N抗原的氨基酸337-422的氨基酸序列的小鼠单克隆抗体。The N antigen immunodetection method of SARS-CoV-2 in the present invention includes antibodies, including capture antibodies M1, M2 and M3 that are labeled with detection signal molecules and can specifically bind to the N antigen of SARS-CoV-2, and can specifically bind to SARS-CoV-2. The detection antibody M4 of the N antigen of CoV-2, the detection signal molecule can be colloidal gold or fluorescein or other markers commonly used in the art. The capture antibody combination is mouse monoclonal antibodies M1, M2, and M3 in a ratio of 1:1:1, which specifically bind to amino acids 1-69, 119-213, and 212-341 of the N antigen of SARS-CoV-2, respectively. Amino acid sequence; the detection antibody is M4, which is a mouse monoclonal antibody that specifically binds to the amino acid sequence of amino acids 337-422 of the N antigen of SARS-CoV-2.
本发明检测方法中，所述抗体组合M1、M2、M3和单抗M4均是免疫球蛋白IgG，分别由保藏号为GDMCC 61007、GDMCC 61008、GDMCC 61009、GDMCC 61010的杂交瘤细胞株所分泌。In the detection method of the present invention, the antibody combination M1, M2, M3 and the monoclonal antibody M4 are all immunoglobulin IgG, which are respectively secreted by the hybridoma cell strains whose deposit numbers are GDMCC 61007, GDMCC 61008, GDMCC 61009 and GDMCC 61010.
本发明检测方法中，所述检测信号分子可以是胶体金、荧光素等。当所述检测信号分子为胶体金时，本检测方法所用检测装置为胶体金试纸条，所述胶体金试纸条包括依次相连的样品垫、结合垫、反应垫和吸水垫；其特征在于，所述结合垫上包被有胶体金标记的M1、M2、M3抗体组合。当所述检测信号分子为荧光素时，本检测方法所用检测装置为荧光免疫层析检测试纸条，所述荧光免疫层析检测试纸条包括依次相连的样品垫、结合垫、反应垫和吸水垫；其特征在于，所述结合垫上包被有铕微球标记的M1、M2、M3抗体组合。In the detection method of the present invention, the detection signal molecule may be colloidal gold, fluorescein, or the like. When the detection signal molecule is colloidal gold, the detection device used in the detection method is a colloidal gold test strip, and the colloidal gold test strip includes a sample pad, a binding pad, a reaction pad and a water-absorbing pad connected in sequence; it is characterized in that , the binding pad is coated with a combination of colloidal gold-labeled M1, M2, and M3 antibodies. When the detection signal molecule is fluorescein, the detection device used in the detection method is a fluorescence immunochromatography detection test strip, and the fluorescence immunochromatography detection test strip includes a sample pad, a binding pad, a reaction pad and A water-absorbing pad; characterized in that the binding pad is coated with a combination of M1, M2, and M3 antibodies labeled with europium microspheres.
本发明所用的捕获抗体和检测抗体是从15种与SARS-CoV-2的N抗原特异性结合的单克隆抗体中筛选出来的。其中，捕获抗体组合M1、M2、M3和检测抗体M4分别结合N抗原不同表位，不仅显著提高了用单个抗体检测的敏感性、特异性、稳定性，减少临床漏检，而且适用于鼻咽拭子、尿液、血液、痰等不同样本检测。同时，单克隆抗体的使用克服了多克隆抗血清与其它常见病毒冠状病毒的交叉反应，重复性好、易于标准化。The capture antibody and detection antibody used in the present invention are selected from 15 monoclonal antibodies that specifically bind to the N antigen of SARS-CoV-2. Among them, the combination of capture antibody M1, M2, M3 and detection antibody M4 binds to different epitopes of N antigen respectively, which not only significantly improves the sensitivity, specificity and stability of detection with a single antibody, reduces clinical missed detection, but is also suitable for nasopharyngeal Swab, urine, blood, sputum and other different samples are tested. At the same time, the use of monoclonal antibodies overcomes the cross-reaction of polyclonal antiserum with other common viruses and coronaviruses, with good repeatability and easy standardization.
本发明提供一种检测SARS-CoV-2的N抗原的免疫检测方法，其特征是用针对N抗原的抗体检测N抗原，所述抗体包括标记有检测信号分子的能特异结合SARS-CoV-2的N抗原的捕获抗体M1、M2、M3，检测抗体M4，任选地，检测信号分子选自胶体金或荧光素。The present invention provides an immunodetection method for detecting the N antigen of SARS-CoV-2, which is characterized in that an antibody against the N antigen is used to detect the N antigen. The N antigen capture antibodies M1, M2, M3, the detection antibody M4, optionally, the detection signal molecule is selected from colloidal gold or fluorescein.
本发明还提供一种捕获抗体，其特征是小鼠单克隆抗体M1、M2、M3，分别特异性 结合SARS-CoV-2的N抗原的氨基酸1-69、119-213、212-341的氨基酸序列。The present invention also provides a capture antibody, which is characterized by mouse monoclonal antibodies M1, M2, and M3, which specifically bind to amino acids 1-69, 119-213, and 212-341 of the N antigen of SARS-CoV-2, respectively. sequence.
本发明还提供一种检测抗体M4，其特征是特异性结合SARS-CoV-2的N抗原的氨基酸337-422的氨基酸序列的小鼠单克隆抗体。The present invention also provides a detection antibody M4, which is characterized by a mouse monoclonal antibody that specifically binds to the amino acid sequence of amino acids 337-422 of the N antigen of SARS-CoV-2.
本发明还提供一种检测SARS-CoV-2的N抗原胶体金免疫层析装置，使用胶体金标记的M1、M2、M3单克隆抗体。The invention also provides an N antigen colloidal gold immunochromatography device for detecting SARS-CoV-2, which uses M1, M2 and M3 monoclonal antibodies labeled with colloidal gold.
本发明还提供一种检测SARS-CoV-2的N抗原荧光免疫层析装置，使用荧光素标记的M1、M2、M3单克隆抗体。The present invention also provides an N antigen fluorescence immunochromatography device for detecting SARS-CoV-2, which uses fluorescein-labeled M1, M2, and M3 monoclonal antibodies.
本发明还提供一种免疫检测方法，其特征是该检测方法可以特异性检测鼻咽拭子、血清、尿液等人体样本中的SARS-CoV-2的N抗原。The invention also provides an immune detection method, which is characterized in that the detection method can specifically detect the N antigen of SARS-CoV-2 in human samples such as nasopharyngeal swabs, serum, and urine.
本发明还提供一种免疫检测方法，其特征是该检测方法所检测到的SARS-CoV-2的N抗原可用于诊断COVID-19，与核酸检测诊断方法符合率99％以上。The present invention also provides an immune detection method, characterized in that the N antigen of SARS-CoV-2 detected by the detection method can be used to diagnose COVID-19, and the coincidence rate with the nucleic acid detection and diagnosis method is more than 99%.
本发明具有如下有益效果：The present invention has the following beneficial effects:
本发明中，通过精选表位-抗体对，达到对SARS-CoV-2的N抗原的特异性检测，实现对COVID-19特异性诊断，特异性达99％。In the present invention, by selecting epitope-antibody pairs, the specific detection of the N antigen of SARS-CoV-2 is achieved, and the specific diagnosis of COVID-19 is realized, and the specificity reaches 99%.
本发明中，通过精选表位-抗体对，达到对SARS-CoV-2的N抗原的高灵敏度检测，SARS-CoV-2的N抗原检测灵敏度达ng水平。In the present invention, by selecting the epitope-antibody pair, high-sensitivity detection of the N antigen of SARS-CoV-2 is achieved, and the detection sensitivity of the N antigen of SARS-CoV-2 reaches the ng level.
本发明中，通过精选表位-抗体对对SARS-CoV-2的N抗原的早期检测，发热第3天即可在鼻咽拭子样本检出SARS-CoV-2的N抗原，实现早期诊断。In the present invention, the N antigen of SARS-CoV-2 can be detected in the nasopharyngeal swab sample on the 3rd day of fever through the early detection of the N antigen of SARS-CoV-2 by selecting the epitope-antibody pair to realize the early detection of the N antigen of SARS-CoV-2. diagnosis.
本发明中，采用胶体金和荧光标记试纸的通用、简便技术，10分钟内出结果，达到SARS-CoV-2的N抗原快速检测，实现快速诊断。In the present invention, the general and simple technology of colloidal gold and fluorescently labeled test paper is adopted, and the result is obtained within 10 minutes, so as to achieve the rapid detection of the N antigen of SARS-CoV-2, and realize rapid diagnosis.
本发明中，检测方法可靠、简便、适合医疗机构、基层、家庭和个人使用。为疑似患者、无症状感染者排查和诊断及为早期、快速预防疫情扩散提供更优手段。In the present invention, the detection method is reliable, simple and suitable for medical institutions, grassroots, families and individuals. Provide better means for the screening and diagnosis of suspected patients and asymptomatic infections and for early and rapid prevention of the spread of the epidemic.
生物保藏信息biological deposit information
一株杂交瘤细胞M1，其分类学名称为Mus musculus hybridoma 1-10：M1，该菌株于2020年4月30日保藏于广东省微生物菌种保藏中心(Guangdong Microbial Culture Collection Center，GDMCC)，保藏号为GDMCC 61007，保藏地址：中国广东省广州市越秀区先烈中路100号大院59号楼广东省微生物研究所，邮政编码：510070。A hybridoma cell M1, its taxonomic name is Mus musculus hybridoma 1-10: M1, the strain was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on April 30, 2020, and was preserved No. GDMCC 61007, deposit address: Guangdong Institute of Microbiology, Building 59, Yard No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, China, Postal Code: 510070.
一株杂交瘤细胞M2，其分类学名称为Mus musculus hybridoma 11-20：M2，该菌株于2020年4月30日保藏于广东省微生物菌种保藏中心(Guangdong Microbial Culture Collection Center，GDMCC)，保藏号为GDMCC 61008，保藏地址：中国广东省广州市越秀区先烈中路100号大院59号楼广东省微生物研究所，邮政编码：510070。A hybridoma cell M2, its taxonomic name is Mus musculus hybridoma 11-20: M2, the strain was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on April 30, 2020, and was preserved No. GDMCC 61008, deposit address: Guangdong Institute of Microbiology, Building 59, Yard No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, China, Postal Code: 510070.
一株杂交瘤细胞M3，其分类学名称为Mus musculus hybridoma 21-30：M3，该菌株于2020年4月30日保藏于广东省微生物菌种保藏中心(Guangdong Microbial Culture Collection Center，GDMCC)，保藏号为GDMCC 61009，保藏地址：中国广东省广州市越秀区先烈中路100号大院59号楼广东省微生物研究所，邮政编码：510070。A hybridoma cell M3, its taxonomic name is Mus musculus hybridoma 21-30: M3, the strain was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on April 30, 2020, and was preserved No. GDMCC 61009, deposit address: Guangdong Institute of Microbiology, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, China, zip code: 510070.
一株杂交瘤细胞M4，其分类学名称为Mus musculus hybridoma 31-40：M4，该菌株于2020年4月30日保藏于广东省微生物菌种保藏中心(Guangdong Microbial Culture Collection Center，GDMCC)，保藏号为GDMCC 61010，保藏地址：中国广东省广州市越秀区先烈中路100号大院59号楼广东省微生物研究所，邮政编码：510070。A hybridoma cell M4, its taxonomic name is Mus musculus hybridoma 31-40: M4, the strain was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on April 30, 2020, and deposited No. GDMCC 61010, deposit address: Guangdong Institute of Microbiology, Building 59, Yard No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, China, Postal Code: 510070.
本发明可通过以下实施方案进行实施，但本发明并不限于此。The present invention can be carried out by the following embodiments, but the present invention is not limited thereto.
免疫原制备Immunogen preparation
本发明用于制备单克隆抗体的免疫原为基因重组SARS-CoV-2的N抗原。基因重组N抗原是用一种携带N抗原基因的工程菌株制备的，其制备方法按照常规方法进行，经用分子筛层析的方法进行纯化获得N抗原，详细制备方法可参照制备单克隆抗体的使用手册。经蛋白质印迹对纯化N抗原进行鉴定，与理论分子量相符。The immunogen used for preparing the monoclonal antibody of the present invention is the N antigen of genetically recombined SARS-CoV-2. The genetically recombined N antigen is prepared by an engineered strain carrying the N antigen gene. The preparation method is carried out according to the conventional method, and the N antigen is obtained by purifying by the method of molecular sieve chromatography. For the detailed preparation method, please refer to the preparation of monoclonal antibodies. manual. The purified N antigen was identified by western blot, which was consistent with the theoretical molecular weight.
免疫小鼠：取4-6周龄雌性BALB/c小鼠，第一次采用完全弗氏佐剂与等体积N抗原免疫共3次，于融合前每只小鼠腹腔注射N抗原100μg加强免疫。Immunized mice: 4-6 week old female BALB/c mice were immunized for the first time with complete Freund's adjuvant and an equal volume of N antigen for a total of 3 times. Before fusion, each mouse was injected intraperitoneally with 100 μg of N antigen to boost the immunization .
杂交瘤细胞的制备和鉴定：加强免疫3天后，无菌条件下取小鼠脾脏制备单细胞悬液，与NS-1细胞按10:1融合。经96孔板培养后的上清用SARS-CoV的N抗原、SARS-CoV-2的N抗原双筛选。上清只结合SARS-CoV-2的N抗原的克隆被标记上克隆号保存。按常规鉴定杂交瘤细胞培养上清所含抗体的亚型。其中，确定M1、M2、M3、M4均为IgG，分别为IgG2a、IgG1、IgG3、IgG1。Preparation and identification of hybridoma cells: 3 days after boosting immunization, the mouse spleen was taken under sterile conditions to prepare a single cell suspension, which was fused with NS-1 cells at a ratio of 10:1. The supernatant after cultured in 96-well plate was double screened with the N antigen of SARS-CoV and the N antigen of SARS-CoV-2. The clones whose supernatant only binds the N antigen of SARS-CoV-2 are marked with the clone number and saved. The subtypes of antibodies contained in the culture supernatant of hybridoma cells were routinely identified. Among them, it was determined that M1, M2, M3, and M4 were all IgG, which were IgG2a, IgG1, IgG3, and IgG1, respectively.
单克隆抗体的制备和纯化：通过腹腔注射向小鼠腹腔上述杂交瘤细胞，不同克隆制备不同克隆的腹水，通过7号注射针头收集腹水。采用辛酸-硫酸铵沉淀法获得单克隆抗体粗品，进一步用分子筛层析技术获得单克隆抗体纯品，冻存、备用。Preparation and purification of monoclonal antibody: the above hybridoma cells were injected into the abdominal cavity of mice by intraperitoneal injection, and ascites of different clones were prepared from different clones, and the ascites was collected through a 7-gauge injection needle. The crude monoclonal antibody was obtained by the octanoic acid-ammonium sulfate precipitation method, and the pure monoclonal antibody was further obtained by molecular sieve chromatography, which was frozen and used for later use.
单克隆抗体-表位抗体对的优选：采用酵母展示技术在酵母表面展示SARS-CoV-2的N抗原不同的氨基酸序列，基于上述制备单克隆抗体，采用流式细胞术，确定各株单克隆抗 体结合的表位序列，其中，结合良好的M1-[N抗原的氨基酸1-69]、M2-[N抗原的氨基酸119-213]、M3-[N抗原的氨基酸212-341]的3个表位-抗体对可作为捕获表位-抗体对；结合良好的M4-[N抗原的氨基酸337-422]表位-抗体对可作为标记表位-抗体对。Optimization of monoclonal antibody-epitope antibody pair: using yeast display technology to display different amino acid sequences of the N antigen of SARS-CoV-2 on the surface of yeast, prepare monoclonal antibodies based on the above, and use flow cytometry to determine the monoclonal The epitope sequence to which the antibody binds, among which, three of M1-[amino acids 1-69 of N antigen], M2-[amino acids 119-213 of N antigen], and M3-[amino acids 212-341 of N antigen] that bind well Epitope-antibody pairs can be used as capture epitope-antibody pairs; well-binding M4-[amino acids 337-422 of N antigen] epitope-antibody pairs can be used as label epitope-antibody pairs.
N抗原检测方法的建立Establishment of N antigen detection method
本发明的检测方法，实施方式之一，通过胶体金检测试纸条实现，包括依次相连的样品垫、结合垫、反应垫和吸水垫，所述样品垫、结合垫、反应垫和吸水垫之间相互交叠连接，所述样品垫、结合垫、反应垫和吸水垫设于底板上。另提供一种用于稀释待检样品的稀释液。The detection method of the present invention, one of the embodiments, is realized by a colloidal gold detection test strip, including a sample pad, a binding pad, a reaction pad and a water-absorbing pad which are connected in sequence, wherein the sample pad, the binding pad, the reaction pad and the water-absorbing pad are connected in sequence. The sample pads, the binding pads, the reaction pads and the water absorption pads are arranged on the bottom plate. In addition, a diluent for diluting the sample to be tested is provided.
本发明的检测方法，实施方式之二，通过荧光免疫层析试纸条实现，包括依次相连的样品垫、结合垫、反应垫和吸水垫，所述样品垫、结合垫、反应垫和吸水垫之间相互交叠连接，所述样品垫、结合垫、反应垫和吸水垫设于底板上。另提供一种用于稀释待检样品的稀释液。The detection method of the present invention, the second embodiment, is realized by a fluorescent immunochromatography test strip, including a sample pad, a binding pad, a reaction pad and a water-absorbing pad connected in sequence, and the sample pad, the binding pad, the reaction pad and the water-absorbing pad are connected in sequence. The sample pads, the binding pads, the reaction pads and the water absorption pads are arranged on the bottom plate. In addition, a diluent for diluting the sample to be tested is provided.
检测方法的临床考核Clinical assessment of detection methods
将本发明的检测方法，在临床疑似COVID-19病例，以鼻咽拭子核酸检测为金标准，用本发明的方法检测鼻咽拭子、尿液和血清样本SARS-CoV-2的N抗原，评价该检测方法临床诊断意义。The detection method of the present invention, in clinical suspected cases of COVID-19, takes the nucleic acid detection of nasopharyngeal swabs as the gold standard, and uses the method of the present invention to detect the N antigen of SARS-CoV-2 in nasopharyngeal swabs, urine and serum samples , to evaluate the clinical diagnostic significance of the detection method.
本发明中所用原料、设备，若无特别说明，均为本领域的常用原料、设备；本发明中所用方法，若无特别说明，均为本领域的常规方法。The raw materials and equipment used in the present invention, unless otherwise specified, are the common raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are the conventional methods in the art.
如无特殊说明，本说明书中的术语的含义与本领域技术人员一般理解的含义相同，但如有冲突，则以本说明书中的定义为准。Unless otherwise specified, the meanings of the terms in this specification are the same as those generally understood by those skilled in the art, but in case of conflict, the definitions in this specification shall prevail.
本文中“包括”、“包含”、“含”、“含有”、“具有”或其它变体意在涵盖非封闭式包括，这些术语之间不作区分。术语“包含”是指可加入不影响最终结果的其它步骤和成分。术语“包含”还包括术语“由……组成”和“基本上由……组成”。本发明的组合物和方法/工艺包含、由其组成和基本上由本文描述的必要元素和限制项以及本文描述的任一的附加的或任选的成分、组分、步骤或限制项组成。"Comprising," "comprising," "including," "containing," "having," or other variations herein are intended to encompass non-closed includes, and no distinction is made between these terms. The term "comprising" means that other steps and ingredients may be added that do not affect the final result. The term "comprising" also includes the terms "consisting of" and "consisting essentially of." The compositions and methods/processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein and any additional or optional ingredients, components, steps or limitations described herein.
在说明书和权利要求书中使用的涉及组分量、工艺条件等的所有数值或表述在所有情形中均应理解被“约”修饰。涉及相同组分或性质的所有范围均包括端点，该端点可独立地组合。由于这些范围是连续的，因此它们包括在最小值与最大值之间的每一数值。还应理解的是，本申请引用的任何数值范围预期包括该范围内的所有子范围。All numerical values or expressions used in the specification and claims referring to component amounts, process conditions, etc., should be understood to be modified by "about" in all instances. All ranges referring to the same component or property are inclusive of the endpoints, which endpoints are independently combinable. Since these ranges are continuous, they include every value between the minimum and maximum values. It is also to be understood that any numerical range recited herein is intended to include all subranges within that range.
所述样品包括鼻咽拭子、痰液、肺泡灌洗液、血液、尿液中的至少一种，更优选地， 所述样品包括鼻咽拭子、痰液、肺泡灌洗液、血液、尿液中的至少两种，将所述样品分别单独进行检测。The sample includes at least one of nasopharyngeal swab, sputum, bronchoalveolar lavage fluid, blood, and urine, more preferably, the sample includes nasopharyngeal swab, sputum, bronchoalveolar lavage fluid, blood, For at least two kinds of urine, the samples are tested separately.
为了更好的理解上述技术方案，下面将结合具体的实施例对上述技术方案进行详细的说明。以下具体的实施例仅是本发明的优选实施方式，不是对本发明的限制。In order to better understand the above technical solutions, the above technical solutions will be described in detail below with reference to specific embodiments. The following specific examples are only preferred embodiments of the present invention, and are not intended to limit the present invention.
实施例Example
实施例1—SARS-CoV-2的N抗原系列单克隆抗体抗体的制造Example 1—Manufacture of N Antigen Series Monoclonal Antibody Antibodies of SARS-CoV-2
基于发布的SARS-CoV-2的N抗原基因序列，克隆N抗原基因，在大肠杆菌中表达N抗原。将表达SARS-CoV-2的N抗原的大肠杆菌工程菌通过发酵技术，获得含有N抗原的原液。将N抗原的原液经过层析技术分离纯化N抗原，获得纯度95％以上的N抗原10mg，-20℃保存备用。Based on the published N antigen gene sequence of SARS-CoV-2, the N antigen gene was cloned and expressed in E. coli. The Escherichia coli engineering bacteria expressing the N antigen of SARS-CoV-2 are subjected to fermentation technology to obtain a stock solution containing the N antigen. The stock solution of N antigen was separated and purified by chromatography technology to obtain 10 mg of N antigen with a purity of more than 95%, which was stored at -20°C for later use.
取用上述N抗原20μg作为免疫原与完全弗氏佐剂混合皮下注射初次免疫Balb/c小鼠，共20只；分别于7天、14天取上述等量N抗原与非完全弗氏佐剂混合加强免疫Balb/c小鼠。于初次免疫后第28天小鼠眼眶后取血检测抗N抗原抗体滴度，滴度大于1:160的小鼠脱颈处死、取脾脏，获得脾细胞单细胞悬液，制备杂交瘤细胞，通过无限稀释法和鉴定获得15个单克隆抗体细胞株，冻存备用。Take 20 μg of the above N antigen as an immunogen mixed with complete Freund's adjuvant subcutaneously to immunize Balb/c mice for the first time, a total of 20 mice; take the same amount of the above N antigen and incomplete Freund's adjuvant on 7 days and 14 days respectively. Mixed boost Balb/c mice. On the 28th day after the primary immunization, retro-orbital blood was collected from the mice to detect the anti-N antigen antibody titer. Mice with a titer greater than 1:160 were sacrificed by cervical dislocation and the spleen was taken to obtain a single-cell suspension of spleen cells to prepare hybridoma cells. Fifteen monoclonal antibody cell lines were obtained by infinite dilution method and identification, and were frozen for future use.
取上述15个单克隆抗体细胞株，分别注射入小鼠腹腔制备腹水，取腹水纯化获得单克隆抗体15株。表1所列为15种抗体单批次生产、鉴定结果。The above 15 monoclonal antibody cell lines were taken and injected into the peritoneal cavity of mice to prepare ascites, and the ascites was purified to obtain 15 monoclonal antibody lines. Table 1 lists the single batch production and identification results of 15 antibodies.
表1.抗SARS-CoV-2的N蛋白单克隆抗体的单批次生产、鉴定结果Table 1. Single batch production and identification results of anti-SARS-CoV-2 N protein monoclonal antibody
实施例2—抗SARS-CoV-2的N蛋白单克隆抗体M1-4特异性结合SARS-CoV-2的NExample 2 - Anti-SARS-CoV-2 N protein monoclonal antibody M1-4 specifically binds to the N of SARS-CoV-2
蛋白protein
因为常见冠状病毒N蛋白的氨基酸序列具有80％以上同源性，只有特异性识别SARS-CoV-2的N蛋白的单克隆抗体才对SARS-CoV-2感染具有诊断价值，而上述单克隆抗体制造方法不能避免获得与其它冠状病毒结合的抗体。为此，将实施例1获得的15株单克隆抗体进行进一步鉴定，寻找只特异性结合SARS-CoV-2的N蛋白的单克隆抗体。Because the amino acid sequence of the N protein of common coronaviruses has more than 80% homology, only the monoclonal antibody that specifically recognizes the N protein of SARS-CoV-2 has diagnostic value for SARS-CoV-2 infection, and the above monoclonal antibody Manufacturing methods cannot avoid obtaining antibodies that bind to other coronaviruses. To this end, the 15 monoclonal antibodies obtained in Example 1 were further identified to search for monoclonal antibodies that only specifically bind to the N protein of SARS-CoV-2.
以3株无关单克隆抗体(A50、B30和C31，特异结合SARS-CoV的S蛋白N末端氨基酸249-667的氨基酸序列)为阴性对照抗体，通过酶联免疫结合试验(ELISA)证明15株单抗均能够结合SARS-CoV-2的N蛋白；通过蛋白质印迹证明15株单抗只有11株结合能够结合SARS-CoV-2的N蛋白；通过与其它常见冠状病毒的免疫荧光检测，发现只有M1、M2、M3、M4单抗可特异结合SARS-CoV-2的N蛋白，其它单抗与其它常见冠状病毒有交叉反应。结果如表2。Using 3 unrelated monoclonal antibodies (A50, B30 and C31, which specifically bind to the amino acid sequence of the N-terminal amino acids 249-667 of the S protein of SARS-CoV) as negative control antibodies, 15 monoclonal antibodies were confirmed by enzyme-linked immunosorbent assay (ELISA). All the antibodies can bind to the N protein of SARS-CoV-2; it was proved by western blot that only 11 of the 15 monoclonal antibodies bind to the N protein of SARS-CoV-2; by immunofluorescence detection with other common coronaviruses, it was found that only M1 , M2, M3, and M4 mAbs can specifically bind to the N protein of SARS-CoV-2, and other mAbs have cross-reactions with other common coronaviruses. The results are shown in Table 2.
表2.单抗筛选结果Table 2. mAb screening results
a15株(M1～M15)抗体为实施例1所制造。
a 15 strains (M1 to M15) antibodies were produced in Example 1.
b纯化的重组表达SARS-CoV-2的N抗原，5μg/ml，每孔100μl，作为包被抗原，与纯化的抗体反应(稀释1:1000)。读取450nm光密度值(OD)。-表示阴性结果；+表示OD在0.2至1；++表示OD为1至2；+++表示OD>2。
b Purified recombinant expression of SARS-CoV-2 N antigen, 5 μg/ml, 100 μl per well, as coating antigen, reacted with purified antibody (diluted 1:1000). Read the optical density (OD) at 450 nm. - indicates a negative result; + indicates an OD of 0.2 to 1; ++ indicates an OD of 1 to 2; +++ indicates an OD>2.
c在PVDF膜上进行重组N抗原与纯化抗体(稀释1:1000)的蛋白质印迹反应。-表示无结合；+
W表示弱结合；+
M表示中度结合；+
S表示强结合。
c Western blotting of recombinant N antigen with purified antibody (diluted 1:1000) on PVDF membrane. - means no binding; + W means weak binding; + M means moderate binding; + S means strong binding.
d对SARS-CoV感染的Vero E6细胞、人冠状病毒229E感染的MRC-5细胞、人冠状病毒OC43感染的BS-C-1细胞、SARS-CoV-2感染的人肾小管上皮细胞进行间接免疫荧光检测。-表示阴性结果；+表示阳性结果；/表示未检测。
d Indirect immunization against SARS-CoV-infected Vero E6 cells, human coronavirus 229E-infected MRC-5 cells, human coronavirus OC43-infected BS-C-1 cells, and SARS-CoV-2-infected human renal tubular epithelial cells Fluorescence detection. - means negative result; + means positive result; / means not tested.
实施例3—筛选具有诊断价值的表位抗体对Example 3—Screening of diagnostically valuable epitope antibody pairs
采用酵母展示技术在酵母表面展示SARS-CoV-2的N抗原不同的氨基酸序列：氨基酸1-422、1-213、1-120、68-213、1-69、68-120、119-213、214-422、212-341、337-422，采用基于上述制备单克隆抗体的流式细胞技术，确定各株单克隆抗体结合的表位序列。Using yeast display technology to display different amino acid sequences of the N antigen of SARS-CoV-2 on the surface of yeast: amino acids 1-422, 1-213, 1-120, 68-213, 1-69, 68-120, 119-213, 214-422, 212-341, 337-422, using the flow cytometry technique based on the above-mentioned preparation of monoclonal antibodies, to determine the epitope sequences bound by each strain of monoclonal antibodies.
结果见下表3。The results are shown in Table 3 below.
表3. 15株单抗结合的N蛋白表位描图
a
Table 3. Epitope mapping of N protein bound by 15 mAbs a
a流式细胞仪检测酵母展示的SARS-CoV-2的N抗原不同氨基酸片段与不同单克隆抗体结合。结果显示的是流式细胞仪检测到的单抗与表达抗原片段结合的荧光值与单抗与空表达空载(EBY100/pYD1)结合荧光值比值大小，比值大于2为阳性。-：阴性(即比值小于2)；+：弱阳性(比值为2-5)；++：中度阳性(比值5-10)；+++：强阳性(比值大于10)。
a Flow cytometry detects the binding of different amino acid fragments of the N antigen of SARS-CoV-2 displayed by yeast to different monoclonal antibodies. The results showed the ratio of the fluorescence value of the binding between the mAb and the expressed antigen fragment detected by flow cytometry and the fluorescence value of the binding between the mAb and the empty expression vector (EBY100/pYD1), and a ratio greater than 2 was positive. -: negative (ie, the ratio is less than 2); +: weakly positive (the ratio is 2-5); ++: moderately positive (the ratio is 5-10); +++: strong positive (the ratio is greater than 10).
bN抗原氨基酸序列。在EBY100酵母细胞表面展示10个覆盖N抗原全长的不同氨基酸序列片段，以考察不同单抗结合N抗原的哪些氨基酸序列。
b N antigen amino acid sequence. On the surface of EBY100 yeast cells, 10 different amino acid sequence fragments covering the full length of N antigen were displayed to investigate which amino acid sequences of different mAbs bind to N antigen.
c实施例1所列15株单抗纳入检测。
c The 15 monoclonal antibodies listed in Example 1 were included in the detection.
结合实施例2，只有M1～M4具有特异诊断SARS-CoV-2感染价值，故选定结合良好的M1-[N抗原的氨基酸1-69]、M2-[N抗原的氨基酸119-213]、M3-[N抗原的氨基酸212-341]3个表位-抗体对作为捕获表位-抗体对；结合良好的M4-[N抗原的氨基酸337-422]表位-抗体对作为检测表位-抗体对。In combination with Example 2, only M1 to M4 have specific diagnostic value for SARS-CoV-2 infection, so M1-[amino acids 1-69 of N antigen], M2-[amino acids 119-213 of N antigen], M2-[amino acids 119-213 of N antigen], M3-[amino acids 212-341 of N antigen] 3 epitope-antibody pairs as capture epitope-antibody pairs; M4-[amino acids 337-422 of N antigens] epitope-antibody pair as detection epitopes- antibody pair.
实施例4—最佳抗体组合筛选Example 4 - Screening of optimal antibody combinations
为提高对SARS-CoV-2的N抗原的检测敏感性，通过化学发光法和荧光层析法，检测不同抗体-表位对组合对SARS-CoV-2的N抗原检测敏感性的影响。检测敏感性以所能检测到N抗原的最低含量(μg/ml)为标准，结果见表4。In order to improve the detection sensitivity of the N antigen of SARS-CoV-2, the effect of different antibody-epitope combinations on the detection sensitivity of the N antigen of SARS-CoV-2 was detected by chemiluminescence and fluorescence chromatography. The detection sensitivity was based on the lowest content (μg/ml) of N antigen that could be detected. The results are shown in Table 4.
表4.不同抗体组合检测敏感性比较Table 4. Comparison of detection sensitivity of different antibody combinations
由此获得最佳捕获抗体组合是M1、M2、M3。The optimal combination of capture antibodies thus obtained is M1, M2, M3.
实施例5—胶体金试纸在疑似患者尿液检测SARS-CoV-2的N抗原Example 5—Detection of N antigen of SARS-CoV-2 by colloidal gold test paper in the urine of suspected patients
本实施例提供一种检测SARS-CoV-2的N抗原的胶体金免疫层析装置，N抗原30ng以上均可检出阳性，其特征在于，所述结合垫上包被有胶体金标记的M1、M2、M3抗体组合。具体而言，所述检测SARS-CoV-2的N抗原的胶体金免疫层析装置通过胶体金检测试纸条实现，所述胶体金检测试纸条包括依次相连的样品垫、结合垫、反应垫和吸水垫，所述样品垫、结合垫、反应垫和吸水垫之间相互交叠连接，所述样品垫、结合垫、反应垫和吸水垫设于底板上。所述反应垫上沿着样品流动方向依次设有检测线和质控线，所述检测线上包被有检测抗体M4，所述质控线上包被有羊抗鼠IgG二抗。所述捕获抗体组合是小鼠单克隆抗体M1、M2、M3，比例1:1:1，分别特异性结合SARS-CoV-2的N抗原的氨基酸1-69、119-213、212-341的氨基酸序列；所述检测抗体是M4，其为特异性结合SARS-CoV-2的N抗原的氨基酸337-422的氨基酸序列的小鼠单克隆抗体。捕获抗体组合M1、M2、M3和检测抗体M4分别结合N抗原不同表位，不仅显著提高了相对于用单个抗体检测的敏感性、特异性、稳定性，减少临床漏检，而且适用于鼻咽拭子、尿液、血液、痰等不同样本检测。This embodiment provides a colloidal gold immunochromatography device for detecting the N antigen of SARS-CoV-2, which can detect positive if the N antigen is more than 30 ng. It is characterized in that the binding pad is coated with colloidal gold-labeled M1, M2, M3 antibody combination. Specifically, the colloidal gold immunochromatography device for detecting the N antigen of SARS-CoV-2 is realized by a colloidal gold detection test strip, and the colloidal gold detection test strip includes a sample pad, a binding pad, and a reaction pad connected in sequence. Pad and absorbent pad, the sample pad, binding pad, reaction pad and absorbent pad are overlapped and connected to each other, and the sample pad, binding pad, reaction pad and absorbent pad are arranged on the bottom plate. A detection line and a quality control line are sequentially arranged on the reaction pad along the flow direction of the sample, the detection line is coated with a detection antibody M4, and the quality control line is coated with a goat anti-mouse IgG secondary antibody. The capture antibody combination is mouse monoclonal antibodies M1, M2, and M3 in a ratio of 1:1:1, which specifically bind to amino acids 1-69, 119-213, and 212-341 of the N antigen of SARS-CoV-2, respectively. Amino acid sequence; the detection antibody is M4, which is a mouse monoclonal antibody that specifically binds to the amino acid sequence of amino acids 337-422 of the N antigen of SARS-CoV-2. Capture antibody combination M1, M2, M3 and detection antibody M4 bind to different epitopes of N antigen respectively, which not only significantly improves the sensitivity, specificity and stability of detection with a single antibody, reduces clinical missed detection, but also is suitable for nasopharyngeal Swab, urine, blood, sputum and other different samples are tested.
选择临床疑似患者22例，同时取鼻咽拭子和尿液样本，平行用国家批准核酸检测试剂检测鼻咽拭子(金标准)，用本发明检测技术的胶体金试纸检测尿液。在21例鼻咽拭子检测阳性患者中，同时尿液检出N抗原15例，检出率71.4％；1例(4.5％)鼻咽拭子检测阴性患者尿液中检出了N抗原。结果参见表5。Twenty-two clinically suspected patients were selected, and nasopharyngeal swabs and urine samples were taken simultaneously. The nasopharyngeal swabs (gold standard) were detected in parallel with the nationally approved nucleic acid detection reagent, and the urine was detected with the colloidal gold test paper of the detection technology of the present invention. Among the 21 patients with positive nasopharyngeal swabs, 15 cases of N antigen were detected in urine at the same time, the detection rate was 71.4%; N antigen was detected in the urine of 1 patient (4.5%) with negative nasopharyngeal swabs. See Table 5 for the results.
表5.咽拭子核酸检测结果和尿液检测N抗原检测结果Table 5. Nucleic acid detection results of throat swabs and N antigen detection results of urine detection
| 病人编号patient number | 咽拭子核酸检测结果Throat swab nucleic acid test results | 尿液N抗原检测结果Urine N antigen test results |
| 11 | +(24.1)+(24.1) | ++ |
| 22 | +(19.7)+(19.7) | ++ |
| 33 | +(27.6)+(27.6) | ++ |
| 44 | +(24.3)+(24.3) | ++ |
| 55 | +(35.5)+(35.5) | -- |
| 66 | +(24.5)+(24.5) | ++ |
| 77 | +(22.2)+(22.2) | ++ |
| 88 | +(26.2)+(26.2) | ++ |
| 99 | +(33.1)+(33.1) | ++ |
| 1010 | +(33.9)+(33.9) | ++ |
| 1111 | +(32.5)+(32.5) | ++ |
| 1212 | +(30.4)+(30.4) | ++ |
| 1313 | +(32.1)+(32.1) | ++ |
| 1414 | +(34.5)+(34.5) | -- |
| 1515 | +(35.4)+(35.4) | -- |
| 1616 | +(33.6)+(33.6) | -- |
| 1717 | +(32.3)+(32.3) | -- |
| 1818 | +(34.6)+(34.6) | ++ |
| 1919 | -- | ++ |
| 2020 | +(31.8)+(31.8) | ++ |
| 21twenty one | +(29.5)+(29.5) | ++ |
| 22twenty two | +(17.1)+(17.1) | ++ |
以上结果表明，核酸检测30分钟出结果，而本发明的N抗原检测10分钟即可出结果。因此，本发明的N抗原检测是更快速的检测方法。The above results show that the nucleic acid detection results in 30 minutes, while the N antigen detection of the present invention can provide results in 10 minutes. Therefore, the N antigen detection of the present invention is a more rapid detection method.
实施例6—荧光层析试纸咽拭子样本临床检测验证Embodiment 6—fluorescence chromatography test paper throat swab sample clinical detection verification
本实施例提供一种检测SARS-CoV-2的N抗原荧光免疫层析装置，N抗原10ng以上均可检出阳性，其特征在于，所述结合垫上包被有铕微球标记的M1、M2、M3抗体组合。具体而言，所述检测SARS-CoV-2的N抗原荧光免疫层析装置通过荧光免疫层析试纸条实现，所述荧光免疫层析试纸条包括依次相连的样品垫、结合垫、反应垫和吸水垫，所述样品垫、结合垫、反应垫和吸水垫之间相互交叠连接，所述样品垫、结合垫、反应垫和吸水垫设于底板上。所述反应垫上沿着样品流动方向依次设有检测线和质控线，所述检测线上包被有检测抗体M4，所述质控线上包被有兔抗鼠二抗。所述捕获抗体组合是小鼠单克隆抗体M1、M2、M3，比例1:1:1，分别特异性结合SARS-CoV-2的N抗原的氨基酸1-69、 119-213、212-341的氨基酸序列；所述检测抗体是M4，其为特异性结合SARS-CoV-2的N抗原的氨基酸337-422的氨基酸序列的小鼠单克隆抗体。捕获抗体组合M1、M2、M3和检测抗体M4分别结合N抗原不同表位，不仅显著提高了相对于用单个抗体检测的敏感性、特异性、稳定性，减少临床漏检，而且适用于鼻咽拭子、尿液、血液、痰等不同样本检测。This embodiment provides an N antigen fluorescence immunochromatography device for detecting SARS-CoV-2, which can detect positive when N antigen is more than 10 ng. It is characterized in that the binding pad is coated with M1 and M2 labeled with europium microspheres. , M3 antibody combination. Specifically, the N antigen fluorescent immunochromatography device for detecting SARS-CoV-2 is realized by a fluorescent immunochromatography test strip, and the fluorescent immunochromatography test strip includes a sample pad, a binding pad, a reaction pad and a reaction pad connected in sequence. Pad and absorbent pad, the sample pad, binding pad, reaction pad and absorbent pad are overlapped and connected to each other, and the sample pad, binding pad, reaction pad and absorbent pad are arranged on the bottom plate. A detection line and a quality control line are sequentially arranged on the reaction pad along the flow direction of the sample, the detection line is coated with a detection antibody M4, and the quality control line is coated with a rabbit anti-mouse secondary antibody. Described capture antibody combination is mouse monoclonal antibody M1, M2, M3, the ratio is 1:1:1, respectively specifically binds to amino acid 1-69, 119-213, 212-341 of N antigen of SARS-CoV-2 Amino acid sequence; the detection antibody is M4, which is a mouse monoclonal antibody that specifically binds to the amino acid sequence of amino acids 337-422 of the N antigen of SARS-CoV-2. Capture antibody combination M1, M2, M3 and detection antibody M4 bind to different epitopes of N antigen respectively, which not only significantly improves the sensitivity, specificity and stability of detection with a single antibody, reduces clinical missed detection, but also is suitable for nasopharyngeal Swab, urine, blood, sputum and other different samples are tested.
针对临床疑似患者113例，取鼻咽拭子样本，平行用国家批准核酸检测试剂检测SARS-CoV-2核酸和本发明检测技术的荧光层析试纸检测SARS-CoV-2的N抗原。For 113 clinically suspected patients, nasopharyngeal swab samples were taken, and the nucleic acid of SARS-CoV-2 was detected in parallel with the nucleic acid detection reagent approved by the state and the N antigen of SARS-CoV-2 was detected by the fluorescence chromatography test strip of the detection technology of the present invention.
表6.大量本临床验证结果*Table 6. Numerous results of this clinical validation*
*敏感性98％；特异性100％；准确性99％；*Sensitivity 98%; Specificity 100%; Accuracy 99%;
阳性预检值100％；阴性预检值49.5％Positive pre-test value 100%; negative pre-test value 49.5%
以核酸检测为金标准，则本发明的特异性为100％，阳性预检值100％；核酸检测90分钟出结果，而本发明的N抗原检测10分钟出结果。因此，本发明的N抗原检测是更快速的检测方法。Taking nucleic acid detection as the gold standard, the specificity of the present invention is 100%, and the positive pre-test value is 100%; the nucleic acid detection results in 90 minutes, while the N antigen detection of the present invention results in 10 minutes. Therefore, the N antigen detection of the present invention is a more rapid detection method.
实施例7—与抗体和核酸检测对比Example 7—Comparison with antibody and nucleic acid detection
针对临床疑似患者19例，取鼻咽拭子样本，平行用国家批准核酸检测试剂检测SARS-CoV-2核酸和本发明检测技术的荧光层析试纸检测SARS-CoV-2的N抗原，对每个患者在取样鼻咽拭子的同一天取样的血清样本检测抗体。与核酸结果相比：N抗原阳性的样本100％来自于确诊患者，特异性100％；有发热、肺炎表现的疑似患者，经反复检测，予以排除的患者，N抗原100％为阴性；血清样本的N抗原检测灵敏度高于鼻咽拭子样本：血清样本，核酸Ct值32.3(低病毒载量血症)，抗原检测也同样阳性；本批次样本中，发烧3天，鼻咽拭子即可测出N抗原。本实施例说明本发明的血清和鼻咽拭子样本能够通过本发明的方法检测到N抗原，本发明的方法是早期诊断方法。For 19 clinical suspected patients, nasopharyngeal swab samples were taken, and the nucleic acid of SARS-CoV-2 was detected in parallel with the nucleic acid detection reagent approved by the state and the N antigen of SARS-CoV-2 was detected by the fluorescence chromatography test strip of the detection technology of the present invention. Antibodies were detected in serum samples taken from each patient on the same day the nasopharyngeal swab was taken. Compared with nucleic acid results: 100% of N antigen-positive samples are from confirmed patients, and the specificity is 100%; for suspected patients with fever and pneumonia, 100% of patients who are excluded after repeated testing are negative for N antigen; serum samples The sensitivity of N antigen detection is higher than that of nasopharyngeal swab samples: serum samples, nucleic acid Ct value of 32.3 (low viral load), and antigen detection are also positive; in this batch of samples, fever for 3 days, nasopharyngeal swabs are N antigen can be detected. This example illustrates that the serum and nasopharyngeal swab samples of the present invention can detect N antigen by the method of the present invention, and the method of the present invention is an early diagnosis method.
表7.荧光标记检测N抗原的样本适用性及与现有方法对比Table 7. Sample applicability of fluorescent labeling to detect N antigen and comparison with existing methods
+表示阳性，-表示阴性，NA表示未检测到。IgM抗体阳性表示近期感染，IgG抗体阳性表示感染时间较长或既往感染。抗原结果指的是通过荧光层析试纸检测的结果。检测患者的鼻咽拭子样本的同时检测了该患者的血清样本，检测患者的血清样本的同时检测了该患者的鼻咽拭子样本。+ means positive, - means negative, NA means not detected. A positive IgM antibody indicates a recent infection, and a positive IgG antibody indicates a longer infection time or previous infection. Antigen results refer to the results detected by fluorescence chromatography test strips. The patient's nasopharyngeal swab sample was detected at the same time as the patient's serum sample, and the patient's nasopharyngeal swab sample was detected at the same time as the patient's serum sample.
以上所述实施例仅表达了本发明的一些实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制，但凡采用等同替换或等效变换的形式所获得的技术方案，均应落在本发明的保护范围之内。The above-mentioned embodiments only express some embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the patent of the present invention. Any technology obtained in the form of equivalent replacement or equivalent transformation All solutions should fall within the protection scope of the present invention.
Claims (10)
- 一种杂交瘤细胞，其保藏号分别为GDMCC 61007、GDMCC 61008、GDMCC 61009或GDMCC 61010。A hybridoma cell, whose deposit number is GDMCC 61007, GDMCC 61008, GDMCC 61009 or GDMCC 61010, respectively.
- 一种单克隆抗体，其由保藏号分别为GDMCC 61007、GDMCC 61008、GDMCC 61009或GDMCC 61010的杂交瘤细胞株分泌。A monoclonal antibody, which is secreted by the hybridoma cell lines whose deposit numbers are GDMCC 61007, GDMCC 61008, GDMCC 61009 or GDMCC 61010 respectively.
- 一种捕获抗体，其能够特异性结合SARS-CoV-2的N抗原，优选地，所述捕获抗体能够特异性结合选自SARS-CoV-2的N抗原的氨基酸1-69、119-213、212-341或其组合的氨基酸序列，任选地，所述捕获抗体分别由选自保藏号为GDMCC 61007、GDMCC 61008或GDMCC 61009的杂交瘤细胞株或其组合的杂交瘤细胞株分泌，任选地，所述捕获抗体包括比例为1:1:1的上述杂交瘤细胞株分泌的单克隆抗体，优选地，所述捕获抗体标记有检测信号分子，任选地，检测信号分子选自胶体金或荧光素。A capture antibody that can specifically bind to the N antigen of SARS-CoV-2, preferably, the capture antibody can specifically bind to amino acids 1-69, 119-213, 119-213, The amino acid sequence of 212-341 or a combination thereof, optionally, the capture antibody is secreted by a hybridoma cell line selected from the hybridoma cell line whose deposit number is GDMCC 61007, GDMCC 61008 or GDMCC 61009 or a combination thereof, optionally Preferably, the capture antibody comprises a monoclonal antibody secreted by the above-mentioned hybridoma cell strain in a ratio of 1:1:1, preferably, the capture antibody is labeled with a detection signal molecule, optionally, the detection signal molecule is selected from colloidal gold or fluorescein.
- 一种检测抗体，其能够特异性结合SARS-CoV-2的N抗原，优选地，所述检测抗体能够特异性结合SARS-CoV-2的N抗原的氨基酸337-422的氨基酸序列，任选地，所述捕获抗体由保藏号为GDMCC 61010的杂交瘤细胞株分泌。A detection antibody that can specifically bind to the N antigen of SARS-CoV-2, preferably, the detection antibody can specifically bind to the amino acid sequence of amino acids 337-422 of the N antigen of SARS-CoV-2, optionally , the capture antibody is secreted by the hybridoma cell line with the deposit number GDMCC 61010.
- 一种检测SARS-CoV-2的N抗原的免疫检测方法，其特征是用针对N抗原的抗体检测N抗原，所述抗体包括能特异性结合SARS-CoV-2的N抗原的标记有检测信号分子的选自小鼠单克隆抗体M1、M2、M3的捕获抗体和能特异性结合SARS-CoV-2的N抗原的检测抗体小鼠单克隆抗体M4，任选地，检测信号分子选自胶体金或荧光素，任选地，其中所述小鼠单克隆抗体M1、M2、M3的比例为1:1:1，任选地，所述小鼠单克隆抗体M1、M2、M3分别特异性结合SARS-CoV-2的N抗原的氨基酸1-69、119-213、212-341的氨基酸序列。An immunodetection method for detecting the N antigen of SARS-CoV-2, which is characterized in that an antibody against the N antigen is used to detect the N antigen, and the antibody comprises a detection signal labeled with a detection signal that can specifically bind to the N antigen of SARS-CoV-2 The molecule is selected from the capture antibody of mouse monoclonal antibodies M1, M2, M3 and the detection antibody that can specifically bind to the N antigen of SARS-CoV-2 Mouse monoclonal antibody M4, optionally, the detection signal molecule is selected from colloid Gold or fluorescein, optionally, wherein the ratio of the mouse monoclonal antibodies M1, M2, M3 is 1:1:1, optionally, the mouse monoclonal antibodies M1, M2, M3 are respectively specific Amino acid sequences of amino acids 1-69, 119-213, 212-341 that bind to the N antigen of SARS-CoV-2.
- 根据权利要求5所述的免疫检测方法，其特征是小鼠单克隆抗体M4是特异性结合SARS-CoV-2的N抗原的氨基酸337-422的氨基酸序列的小鼠单克隆抗体，任选地，所述捕获抗体M1、M2、M3分别由选自保藏号为GDMCC 61007、GDMCC 61008、GDMCC 61009的杂交瘤细胞株的杂交瘤细胞株分泌，任选地，所述检测抗体M4由保藏号为GDMCC 61010的杂交瘤细胞株分泌，。The immunoassay method according to claim 5, wherein the mouse monoclonal antibody M4 is a mouse monoclonal antibody that specifically binds to the amino acid sequence of amino acids 337-422 of the N antigen of SARS-CoV-2, optionally , the capture antibodies M1, M2, and M3 are respectively secreted by hybridoma cell lines selected from the hybridoma cell lines whose deposit numbers are GDMCC 61007, GDMCC 61008, and GDMCC 61009. Optionally, the detection antibody M4 is secreted by the deposit number of GDMCC 61010 hybridoma cell line secretion,.
- 根据权利要求5所述的免疫检测方法，其特征是该检测方法能够特异性检测鼻咽拭子、血清、尿液等人体样本中的SARS-CoV-2的N抗原，任选地，该检测方法所检测到的SARS-CoV-2的N抗原能够用于诊断COVID-19，与核酸检测诊断方法符合率99％以上。The immune detection method according to claim 5, wherein the detection method can specifically detect the N antigen of SARS-CoV-2 in human samples such as nasopharyngeal swabs, serum, urine, etc. The N antigen of SARS-CoV-2 detected by the method can be used to diagnose COVID-19, and the coincidence rate with the nucleic acid detection and diagnosis method is more than 99%.
- 一种检测SARS-CoV-2的N抗原的胶体金免疫层析装置，所述胶体金免疫层析装置使用胶体金标记的M1、M2、M3单克隆抗体，任选地，所述胶体金免疫层析装置为胶体金试纸条，包括依次相连的样品垫、结合垫、反应垫和吸水垫，其中所述结合垫上包被有胶体金标记的M1、M2、M3抗体组合，任选地，其中所述小鼠单克隆抗体M1、M2、M3的比例为1:1:1。A colloidal gold immunochromatography device for detecting the N antigen of SARS-CoV-2, the colloidal gold immunochromatography device uses colloidal gold-labeled M1, M2, M3 monoclonal antibodies, optionally, the colloidal gold immunochromatography The chromatography device is a colloidal gold test strip, comprising a sample pad, a binding pad, a reaction pad and a water-absorbing pad connected in sequence, wherein the binding pad is coated with a combination of colloidal gold-labeled M1, M2, and M3 antibodies, optionally, The ratio of the mouse monoclonal antibodies M1, M2, and M3 is 1:1:1.
- 一种检测SARS-CoV-2的N抗原的荧光免疫层析装置，所述荧光免疫层析装置使用荧光素标记的M1、M2、M3单克隆抗体，任选地，所述荧光免疫层析装置为荧光免疫层析检测试纸条，包括依次相连的样品垫、结合垫、反应垫和吸水垫，其中所述结合垫上包被有铕微球标记的M1、M2、M3抗体组合，任选地，其中所述小鼠单克隆抗体M1、M2、M3的比例为1:1:1。A fluorescence immunochromatography device for detecting the N antigen of SARS-CoV-2, the fluorescence immunochromatography device uses fluorescein-labeled M1, M2, M3 monoclonal antibodies, optionally, the fluorescence immunochromatography device It is a fluorescent immunochromatographic detection test strip, including a sample pad, a binding pad, a reaction pad and a water-absorbing pad connected in sequence, wherein the binding pad is coated with a combination of M1, M2, and M3 antibodies labeled with europium microspheres, optionally , wherein the ratio of the mouse monoclonal antibodies M1, M2, and M3 is 1:1:1.
- 一种检测SARS-CoV-2的试剂盒，其包括根据权利要求2所述的单克隆抗体，任选地，所述试剂盒包括保藏号分别为GDMCC 61007、GDMCC 61008、GDMCC 61009的杂交瘤细胞株分泌的单克隆抗体，任选地，所述试剂盒还包括保藏号为GDMCC 61010的杂交瘤细胞株分泌的单克隆抗体。A kit for detecting SARS-CoV-2, comprising the monoclonal antibody according to claim 2, optionally, the kit comprises hybridomas with deposit numbers GDMCC 61007, GDMCC 61008, GDMCC 61009 respectively The monoclonal antibody secreted by the strain, optionally, the kit further includes the monoclonal antibody secreted by the hybridoma cell strain whose deposit number is GDMCC 61010.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010783411.8A CN114058593A (en) | 2020-08-06 | 2020-08-06 | Monoclonal antibody of N antigen of SARS-CoV-2, detection method and use thereof |
| CN202010783411.8 | 2020-08-06 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2022027703A1 true WO2022027703A1 (en) | 2022-02-10 |
Family
ID=80118608
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2020/108323 WO2022027703A1 (en) | 2020-08-06 | 2020-08-11 | Monoclonal antibody of n antigen of sars-cov-2, detection method therefor and use thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN114058593A (en) |
| WO (1) | WO2022027703A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114544960A (en) * | 2022-04-22 | 2022-05-27 | 北京康乐卫士生物技术股份有限公司 | SARS-CoV-2 antigen detecting test paper strip |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114685628B (en) * | 2022-03-24 | 2023-06-06 | 中国人民解放军陆军军医大学 | Antigen epitope peptide of RBD of SARS-CoV-2 and its application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111153991A (en) * | 2020-02-26 | 2020-05-15 | 北京博奥森生物技术有限公司 | Human SARS-CoV-2 monoclonal antibody and its preparation method and use |
| CN111217917A (en) * | 2020-02-26 | 2020-06-02 | 康希诺生物股份公司 | Novel coronavirus SARS-CoV-2 vaccine and preparation method thereof |
| CN111269313A (en) * | 2020-03-07 | 2020-06-12 | 北京岳昊科技发展有限公司 | Monoclonal antibody for detecting novel coronavirus and application of monoclonal antibody in preparation of kit |
| CN111337668A (en) * | 2020-02-27 | 2020-06-26 | 四川省人民医院 | Novel coronavirus antigen colloidal gold rapid diagnosis kit and preparation method thereof |
| CN111440229A (en) * | 2020-04-13 | 2020-07-24 | 中国人民解放军军事科学院军事医学研究院 | Novel coronavirus T cell epitope and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111398597A (en) * | 2020-03-12 | 2020-07-10 | 中科欧蒙未一(北京)医学技术有限公司 | Kit for detecting IgM antibody against novel coronavirus SARS-CoV-2 in sample |
| CN111426840A (en) * | 2020-06-11 | 2020-07-17 | 北京中检安泰诊断科技有限公司 | Novel coronavirus detection test strip and preparation method and application thereof |
-
2020
- 2020-08-06 CN CN202010783411.8A patent/CN114058593A/en active Pending
- 2020-08-11 WO PCT/CN2020/108323 patent/WO2022027703A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111153991A (en) * | 2020-02-26 | 2020-05-15 | 北京博奥森生物技术有限公司 | Human SARS-CoV-2 monoclonal antibody and its preparation method and use |
| CN111217917A (en) * | 2020-02-26 | 2020-06-02 | 康希诺生物股份公司 | Novel coronavirus SARS-CoV-2 vaccine and preparation method thereof |
| CN111337668A (en) * | 2020-02-27 | 2020-06-26 | 四川省人民医院 | Novel coronavirus antigen colloidal gold rapid diagnosis kit and preparation method thereof |
| CN111269313A (en) * | 2020-03-07 | 2020-06-12 | 北京岳昊科技发展有限公司 | Monoclonal antibody for detecting novel coronavirus and application of monoclonal antibody in preparation of kit |
| CN111440229A (en) * | 2020-04-13 | 2020-07-24 | 中国人民解放军军事科学院军事医学研究院 | Novel coronavirus T cell epitope and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| BURBELO PETER D, RIEDO FRANCIS X, MORISHIMA CHIHIRO, RAWLINGS STEPHEN, SMITH DAVEY, DAS SANCHITA, STRICH JEFFREY R, CHERTOW DANIEL: "Sensitivity in Detection of Antibodies to Nucleocapsid and Spike Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 in Patients With Coronavirus Disease 2019", JOURNAL OF INFECTIOUS DISEASES, UNIVERSITY OF CHICAGO PRESS, US, vol. 222, no. 2, 29 June 2020 (2020-06-29), US , pages 206 - 213, XP055818914, ISSN: 0022-1899, DOI: 10.1093/infdis/jiaa273 * |
| LI MAOHUA, JIN RONGHUA, PENG YA, WANG CUIYAN, REN WENLIN, LV FUDONG, GONG SITAO, FANG FENG, WANG QIANYUN, LI JIANLI, SHEN TONG, SU: "Generation of antibodies against COVID-19 virus for development of diagnostic tools", MEDRXIV, 27 February 2020 (2020-02-27), pages 1 - 17, XP009531584, DOI: 10.1101/2020.02.20.20025999 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114544960A (en) * | 2022-04-22 | 2022-05-27 | 北京康乐卫士生物技术股份有限公司 | SARS-CoV-2 antigen detecting test paper strip |
| CN114544960B (en) * | 2022-04-22 | 2022-07-12 | 北京康乐卫士生物技术股份有限公司 | SARS-CoV-2 antigen detecting test paper strip |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114058593A (en) | 2022-02-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7260192B2 (en) | Immunological detection method and kit for Mycoplasma pneumoniae | |
| US8399207B2 (en) | Monoclonal antibodies against osteopontin | |
| JP7175147B2 (en) | Immunological detection method and kit for Mycoplasma pneumoniae | |
| JP7178224B2 (en) | Immunological detection method and kit for Mycoplasma pneumoniae | |
| WO2022193980A1 (en) | Antibody or antigen-binding fragment thereof for novel coronavirus nucleocapsid protein, and application thereof | |
| WO2022004622A1 (en) | Antibody against sars-cov-2, method for detecting sars-cov-2 using antibody and kit containing antibody | |
| WO2021129247A1 (en) | Monoclonal antibody of coxsackievirus a6 true virus and use thereof | |
| WO2022027703A1 (en) | Monoclonal antibody of n antigen of sars-cov-2, detection method therefor and use thereof | |
| WO2023036152A1 (en) | Detection method and kit for sars-cov-2 | |
| JP2024012473A (en) | Detection reagent or kit for detecting rs virus, and method for detecting rs virus | |
| KR100832870B1 (en) | Monoclonal antibody against nucleocapsid protein of SARS coronavirus and the use thereof | |
| CN113045646B (en) | Antibodies against novel coronavirus SARS-CoV-2 | |
| US10634676B2 (en) | Method and kit for simultaneously detecting human parvovirus B19 antigen and antibody | |
| JP2007145775A (en) | Method for detecting norovirus gi in high sensitivity | |
| WO2023277143A1 (en) | Antibody against nucleocapsid protein of sars-cov-2 | |
| JP5204036B2 (en) | Method for detecting pneumococci | |
| KR100832867B1 (en) | Monoclonal antibody against nucleocapsid protein of SARS coronavirus and the use thereof | |
| WO2021184390A1 (en) | Method for screening 2019 novel coronavirus and pre-diagnosing severe pneumonia based on double indexes | |
| KR20210116115A (en) | Antibody binding to Porcine Epidemic Diarrhea virus and use thereof | |
| KR20100031369A (en) | Rapid diagnostic kit of hemorrhagic fever with renal syndrome detecting specific igm and igg using nucleocapsid protein derived from soochong virus | |
| WO2023167317A1 (en) | ANTI-SARS-CoV-2 ANTIBODY AGAINST SARS-CoV-2 ANTIGEN IN BODY FLUID, INCLUDING MUTANT; METHOD FOR DETECTING SARS-CoV-2 USING ANTIBODY; AND KIT CONTAINING ANTIBODY | |
| CN115850459B (en) | Antibody targeting novel coronavirus N protein and application thereof | |
| WO2024103504A1 (en) | Marker and method for detecting inflammation-related diseases | |
| KR102008608B1 (en) | Hybridomas that produce specific antibodies to non-structural protein 1 of dengue virus and antibodies produced therefrom, and uses thereof | |
| EP4342909A1 (en) | Anti-norovirus antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 20948520 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 20948520 Country of ref document: EP Kind code of ref document: A1 |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 20948520 Country of ref document: EP Kind code of ref document: A1 |