CN104007261B - Fowl three kinds of breathing problem three quick detection kit and application - Google Patents
Fowl three kinds of breathing problem three quick detection kit and application Download PDFInfo
- Publication number
- CN104007261B CN104007261B CN201410092170.7A CN201410092170A CN104007261B CN 104007261 B CN104007261 B CN 104007261B CN 201410092170 A CN201410092170 A CN 201410092170A CN 104007261 B CN104007261 B CN 104007261B
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- xycdy
- aiv
- hybridoma cell
- cctccno
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 71
- 208000030303 breathing problems Diseases 0.000 title abstract description 8
- 238000012360 testing method Methods 0.000 claims abstract description 76
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 52
- 239000010931 gold Substances 0.000 claims abstract description 46
- 229910052737 gold Inorganic materials 0.000 claims abstract description 46
- 241000711450 Infectious bronchitis virus Species 0.000 claims abstract description 43
- 206010064097 avian influenza Diseases 0.000 claims abstract description 43
- 241000700605 Viruses Species 0.000 claims abstract description 25
- 241000712461 unidentified influenza virus Species 0.000 claims abstract description 25
- 239000007788 liquid Substances 0.000 claims abstract description 24
- 208000010359 Newcastle Disease Diseases 0.000 claims abstract description 23
- 239000000084 colloidal system Substances 0.000 claims abstract description 22
- 208000002979 Influenza in Birds Diseases 0.000 claims abstract description 20
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 18
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 18
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 11
- 238000010521 absorption reaction Methods 0.000 claims abstract description 11
- 238000007865 diluting Methods 0.000 claims abstract description 9
- 239000000427 antigen Substances 0.000 claims description 46
- 102000036639 antigens Human genes 0.000 claims description 46
- 108091007433 antigens Proteins 0.000 claims description 46
- 210000004408 hybridoma Anatomy 0.000 claims description 41
- 238000002360 preparation method Methods 0.000 claims description 23
- 230000028327 secretion Effects 0.000 claims description 14
- 241000271566 Aves Species 0.000 claims description 6
- 230000003612 virological effect Effects 0.000 claims description 6
- 206010014599 encephalitis Diseases 0.000 claims description 4
- 241000894007 species Species 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 201000010099 disease Diseases 0.000 abstract description 39
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 39
- 241000287828 Gallus gallus Species 0.000 abstract description 27
- 206010006451 bronchitis Diseases 0.000 abstract description 19
- 208000015181 infectious disease Diseases 0.000 abstract description 18
- 230000002458 infectious effect Effects 0.000 abstract description 15
- 239000000203 mixture Substances 0.000 abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 9
- 244000052769 pathogen Species 0.000 abstract description 5
- 230000001717 pathogenic effect Effects 0.000 abstract description 5
- 206010048282 zoonosis Diseases 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 42
- 238000000034 method Methods 0.000 description 38
- 210000004027 cell Anatomy 0.000 description 23
- 241000711404 Avian avulavirus 1 Species 0.000 description 21
- 239000006228 supernatant Substances 0.000 description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 239000013612 plasmid Substances 0.000 description 13
- 108090000623 proteins and genes Proteins 0.000 description 13
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 12
- 239000013604 expression vector Substances 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 238000003745 diagnosis Methods 0.000 description 10
- 238000001556 precipitation Methods 0.000 description 10
- 238000003756 stirring Methods 0.000 description 10
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 9
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 9
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 239000012530 fluid Substances 0.000 description 9
- 230000008569 process Effects 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 8
- 244000061458 Solanum melongena Species 0.000 description 8
- 230000023555 blood coagulation Effects 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- 235000015097 nutrients Nutrition 0.000 description 8
- 238000012797 qualification Methods 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 206010035226 Plasma cell myeloma Diseases 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000011248 coating agent Substances 0.000 description 7
- 238000000576 coating method Methods 0.000 description 7
- 201000000050 myeloid neoplasm Diseases 0.000 description 7
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 7
- 101150008820 HN gene Proteins 0.000 description 6
- 239000006180 TBST buffer Substances 0.000 description 6
- 210000000349 chromosome Anatomy 0.000 description 6
- 230000009089 cytolysis Effects 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 230000004927 fusion Effects 0.000 description 6
- 238000004321 preservation Methods 0.000 description 6
- 238000003757 reverse transcription PCR Methods 0.000 description 6
- 229920000742 Cotton Polymers 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 5
- 229960000723 ampicillin Drugs 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 230000036039 immunity Effects 0.000 description 5
- 230000001524 infective effect Effects 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000003259 recombinant expression Methods 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000013016 damping Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000010369 molecular cloning Methods 0.000 description 4
- 238000012856 packing Methods 0.000 description 4
- 208000023504 respiratory system disease Diseases 0.000 description 4
- 210000004988 splenocyte Anatomy 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 241001260012 Bursa Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000204031 Mycoplasma Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 238000012817 gel-diffusion technique Methods 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 3
- 230000000521 hyperimmunizing effect Effects 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000012460 protein solution Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 101710154606 Hemagglutinin Proteins 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 2
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 101710176177 Protein A56 Proteins 0.000 description 2
- 206010057190 Respiratory tract infections Diseases 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 108020005038 Terminator Codon Proteins 0.000 description 2
- 208000035199 Tetraploidy Diseases 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 210000003837 chick embryo Anatomy 0.000 description 2
- 210000003555 cloaca Anatomy 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000004945 emulsification Methods 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 230000035931 haemagglutination Effects 0.000 description 2
- 238000001631 haemodialysis Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000000185 hemagglutinin Substances 0.000 description 2
- 230000000322 hemodialysis Effects 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 239000003547 immunosorbent Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 238000005096 rolling process Methods 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 238000010023 transfer printing Methods 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 210000001835 viscera Anatomy 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- AXAVXPMQTGXXJZ-UHFFFAOYSA-N 2-aminoacetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound NCC(O)=O.OCC(N)(CO)CO AXAVXPMQTGXXJZ-UHFFFAOYSA-N 0.000 description 1
- 240000001592 Amaranthus caudatus Species 0.000 description 1
- 235000009328 Amaranthus caudatus Nutrition 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- 206010015719 Exsanguination Diseases 0.000 description 1
- 101150034814 F gene Proteins 0.000 description 1
- 241000701063 Gallid alphaherpesvirus 1 Species 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 108010053070 Glutathione Disulfide Proteins 0.000 description 1
- 206010021703 Indifference Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000204022 Mycoplasma gallisepticum Species 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229920002538 Polyethylene Glycol 20000 Polymers 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 101710198474 Spike protein Proteins 0.000 description 1
- 206010044302 Tracheitis Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000012735 amaranth Nutrition 0.000 description 1
- 239000004178 amaranth Substances 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- GEWYFWXMYWWLHW-UHFFFAOYSA-N azanium;octanoate Chemical compound [NH4+].CCCCCCCC([O-])=O GEWYFWXMYWWLHW-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229960002319 barbital Drugs 0.000 description 1
- 229960000796 barbital sodium Drugs 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- -1 each 5mim Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 235000021393 food security Nutrition 0.000 description 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 201000009837 laryngotracheitis Diseases 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- TZBAVQKIEKDGFH-UHFFFAOYSA-N n-[2-(diethylamino)ethyl]-1-benzothiophene-2-carboxamide;hydrochloride Chemical compound [Cl-].C1=CC=C2SC(C(=O)NCC[NH+](CC)CC)=CC2=C1 TZBAVQKIEKDGFH-UHFFFAOYSA-N 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 210000004279 orbit Anatomy 0.000 description 1
- YPZRWBKMTBYPTK-UHFFFAOYSA-N oxidized gamma-L-glutamyl-L-cysteinylglycine Natural products OC(=O)C(N)CCC(=O)NC(C(=O)NCC(O)=O)CSSCC(C(=O)NCC(O)=O)NC(=O)CCC(N)C(O)=O YPZRWBKMTBYPTK-UHFFFAOYSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000007119 pathological manifestation Effects 0.000 description 1
- 210000002976 pectoralis muscle Anatomy 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000565 sealant Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- RGHFKWPGWBFQLN-UHFFFAOYSA-M sodium;5,5-diethylpyrimidin-3-ide-2,4,6-trione Chemical compound [Na+].CCC1(CC)C([O-])=NC(=O)NC1=O RGHFKWPGWBFQLN-UHFFFAOYSA-M 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 230000002277 temperature effect Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 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
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—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
-
- 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
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- 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
-
- 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
- G01N2333/11—Orthomyxoviridae, e.g. influenza virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
Abstract
The invention belongs to zoonosis detection field, be specifically related to fowl three kinds of breathing problem three quick detection kit and application.This kit is by test strips, test is got stuck and sample diluting liquid composition, described test strips is by sample pad, absorption pad, be coated with anti-avian influenza virus monoclonal antibody-colloid gold label thing, the pad of anti-new castle disease virus monoclonal antibody-colloid gold label thing and anti-avian infectious bronchitis virus monoclonal antibody-colloid gold label thing potpourri, be coated with anti-avian influenza monoclonal antibody, anti-ewcastle disease monoclonal antibody, the detection line T1 of infectious bronchitis of chicken monoclonal antibody, T2, T3, be coated with the nitrocellulose filter of the nature controlling line of rabbit anti-mouse igg, press absorption pad successively, nitrocellulose filter, gold mark pad, the order of sample pad is pasted onto on the support slice that do not absorb water and forms test strips.The Pathogen test of bird flu H5 type, H9 hypotype, Newcastle Disease and infectious bronchitis of chicken can be completed, this kit high specificity simultaneously, highly sensitive.
Description
Technical field
The invention belongs to animal immune applied technical field.Be specifically related to three quick detection kit and and the application of fowl three kinds of breathing problems (bird flu H5 hypotype and H9 hypotype, ewcastle disease and infectious bronchitis of chicken) Pathogen test.Kit shell prepared by the present invention once completes fowl three kinds of breathing problems as bird flu H5 hypotype and the former quick detection of H9 hypotype, ewcastle disease and infectious bronchitis simultaneously.
Background technology
The respiratory tract infection of fowl is the very common disease of a class in avian production practice, and especially the generation of such disease is complicated and changeable, and provisions fowl industry brings huge economic loss.The cause of disease causing the respiratory tract infection of fowl mainly comprises virus, bacterium and mycoplasma, wherein by three kinds of disease bird flu (Avianinfluenza that RNA virus causes, AI), ewcastle disease (Newcastledisease, ND) and infectious bronchitis of chicken (Infectiouslaryngotrache, IB) be comparatively common in chicken farm.Three kinds of diseases have similar respiratory symptom clinically, in addition clinical manifestation mostly is atypical, disease infects and mostly is Combination, only be difficult to make a definite diagnosis according to clinical symptoms and pathological manifestations, therefore how making diagnosis is rapidly and accurately effective prophylactic treatment disease, avoids causing serious financial consequences basic.
At present, the method made a definite diagnosis bird flu, ewcastle disease and infectious bronchitis of chicken is mainly diagnosis of molecular biology method and serodiagnosis method.Diagnostic technique in molecular biology mainly contains RT-PCR, Nucleic Acid Probe Technique, Restrictive fragment length polymorphism analysis etc.Serodiagnosis mainly contains agar gel diffusion test (AGID), hemagglutination test and hemagglutination-inhibition test (HA/HI), immunofluorescence technique, euzymelinked immunosorbent assay (ELISA) (ELISA) and immunochromatographic method etc.Diagnosis of molecular biology method has high specificity, sensitivity high, but has higher requirement to experimental facilities, experimenter.Agar gel diffusion test in serodiagnosis method, hemagglutination test and hemagglutination-inhibition test operate comparatively simple, but its specificity and sensitivity are not high, and other are as immunofluorescence, and euzymelinked immunosorbent assay (ELISA) is equally to equipment, and personnel and operation have high request.Said method is mostly consuming time longer (as RT-PCR needs 1.5 ~ 2 hours; Agar gel diffusion test needs 24 ~ 48 hours; Hemagglutination-inhibition test needs 2 ~ 3 hours; ELISA test needs 3 ~ 5 hours), be not easy in good time and quick diagnosis, so all cannot promote the use of on a large scale in basic unit.
Colloidal gold immunochromatographimethod (gold-immunochromatographyassayGICA) is using collaurum as tracer, is applied to a kind of Novel immune labelling technique of antigen-antibody reaction based on colloidal gold-labeled method.Its core technology take nitrocellulose filter as solid phase carrier, impel sample solution swimming on chromatography strip because of capillarity, and make the immune response that the thing to be checked in sample and chromatographic film occur at short notice for the acceptor (as antigen or antibody) of thing to be checked for high specific, high-affinity.It has easy, fast, high specificity, highly sensitive, the advantage that expense is low.
Based on this technology, cure disease surveillance both at home and abroad people, environmental hazard is monitored, and the fields such as food security guarantee and livestock and poultry detection, all have developed multiple colloidal gold immunochromatographimethod and detected test card fast.In chicken disease diagnosis and context of detection, newcastle disease, infectious bronchitis of chicken, infections chicken cloacal bursa and mycoplasma Gallisepticum immune body immune colloidal gold quick detection kit obtain the Patents (patent No.: 200710169103.0; 200710169105.X; 200710169104.5; 200710169106.4).
But mentioned reagent box is the detection to antibody horizontal after relevant disease or vaccine immunity, and can accurately react chicken group and whether infect epidemic disease.Although also have part at present for the immune colloid gold detection method of single cause of disease, as " avian influenza virus antigen detection method and golden label fast diagnosis reagent box and preparation method " thereof that number of patent application is 200510042631.0, number of patent application be 200510057023.7 document " bird flu immune colloid gold diagnosis test paper and test card " disclose the preparation method of several detection avian influenza virus antigen, but not good for the diagnosis effect of the case of mixed infection, use multiple single inspection colloidal gold strip to carry out detecting and causing the waste of time and resource.And multivariate detection can disposablely detect for multiple cause of disease, thus well solve this problem.The combined detection kit (test card) of ternary can make consume and be reduced to 1/3rd, thus save ample resources, for the effective fast treating of disease provides the more sufficient time for society.
The present invention is by preparing each two strains of bird flu, ewcastle disease and infectious bronchitis of chicken monoclonal antibody respectively, whether utilize double antibodies sandwich immunochromatography technique, synchronously can detect in sample with single test card is that bird flu, ewcastle disease and infectious bronchitis are former.
Summary of the invention
The object of the invention is to the defect overcoming existing detection technique, three quick detection kit providing one to be applicable to fowl three kinds of breathing problems (bird flu H5 hypotype and H9 hypotype, ewcastle disease and infectious bronchitis of chicken) Pathogen test and application, kit high specificity of the present invention, highly sensitive, easy and simple to handle, can be used for the detection synchronously completing bird flu H5 and H9 hypotype, ewcastle disease and avian infectious bronchitis virus three kinds of respiratory disease of birds cause of diseases.
The invention still further relates to structure and the exploitation of the hybridoma cell strain of six strain secretion monoclonal antibody specifics.
General technical route map of the present invention as shown in Figure 1.
The present invention is achieved through the following technical solutions:
In order to realize the present invention, applicant has prepared two kinds respectively for different antigen site and can divide anti-avian influenza virus (be called for short AIV) hybridoma cell strain XYCSJL-AIV-2B8 and XYCSJL-AIV-4B7, two kinds of secretion anti-new castle disease virus (being called for short NDV) hybridoma cell strain XYCDY-NDV-3E11 and XYCDY-NDV-4B10 and the two kinds of anti-avian infectious bronchitis viruses of secretion (being called for short IBV) hybridoma cell strain XYCDY-1BV-3G5 and XYCDY-1BV-1E6.Above-mentioned six strain cells deliver China on March 11st, 2014 all. Wuhan. and Wuhan University's China typical culture collection center (CCTCC) preservation, corresponding preservation information is summarized in as table 1.
The preservation information of six strain of hybridoma strains prepared by table 1 the present invention
Applicant provide on this basis three fast fast bird flus (H5 and H9 hypotype) of fowl three kinds of breathing problems,
The kit of ewcastle disease and avian infectious bronchitis virus, this kit comprises test card and sample diluting liquid, wherein test card is by sample pad (1), gold mark pad (2), nitrocellulose filter (3), absorption pad (4), PVC backing (9) and test get stuck (10) composition, its concrete structure is: be stained with successively in order on PVC backing (9) sample pad (1), gold mark pad (2), nitrocellulose filter (3), absorption pad (4); Test strips is loaded test to get stuck in (10) and form test card.Described gold mark pad (2) is coated with three kinds of described gold mark monoclonal antibody mixed solution (this solution contains anti-avian influenza virus monoclonal antibody XYCSJL-AIV-2B8-colloid gold label thing, anti-new castle disease virus monoclonal antibody XYCDY-NDV-3E11-colloid gold label thing and anti-avian infectious bronchitis virus monoclonal antibody XYCDY-1BV-3G5-colloid gold label thing); Described nitrocellulose filter (3) is coated with anti-avian influenza virus monoclonal antibody XYCSJL-AIV-4B7, the detection line T1 (5) that anti-new castle disease virus monoclonal antibody XYCDY-NDV-4B10 and anti-avian infectious bronchitis virus monoclonal antibody XYCDY-1BV-1E6 is formed respectively, T2 (6) and T3 (7); Nature controlling line (8) is containing rabbit anti-mouse igg.
Applicant provides a kind of quick preparation to be applicable to the method for three quick detection kit of bird flu H5 hypotype and H9 hypotype, ewcastle disease and avian infectious bronchitis virus cause of disease, and concrete steps are as follows:
1) avian influenza virus restructuring hemagglutinin HA1 albumen (being called for short AIV-HA1), the expression preparation of newcastle disease virus restructuring hemagglutinin HN albumen (being called for short NDV-HN) and avian infectious bronchitis virus recombinant nuclear capsid N protein (being called for short IBV-NP).By the sequence clone to bird flu HA1 gene, ewcastle disease HN gene and infectious bronchitis of chicken N gene, and connect respective carrier thus build three kinds of expression of recombinant proteins carrier pKG-HA1, pKG-HN and pKG-NP.Wherein the physical build-up collection of illustrative plates of expression vector pKG-HA1 is see document: middle National IP Network Dissertations Database: Wu Renwei, foundation [D] Hua Zhong Agriculture University of the research of avian influenza virus recombinant protein mucosal immunity and N-ELISA antibody detection method, 2006http: //epub3.cnki.net/KCMS/detail/detail.aspx? dbcode=CDFD & QueryID=9 & CurRec=1 & dbname=CDFD9908 & filename=2006190169.nh & urlid=& yx=& uid=WEEvREcwSlJHSldTTGJhYkhsd0Z6ODd6RU81ZVo0ODlGZ2hITVNL ZlMwRHM5RzdZNG1mdWJmU3NZZFBoeTAwbg==& v=MTM0NThSOGVYMUx1eFlTN0RoMVQzcVRyV0.And the physical build-up collection of illustrative plates of expression vector pKG-HN and pKG-NP as shown in Figures 4 and 5.
2) purifying of AIV-HA1, NDV-HN and IBV-NP tri-kinds of albumen.Recombinant protein purification chromatographic column used is GSTrap
tMfFcolumns (buying from GEHealthcare.USA).Purification of recombinant proteins presses GSTrap
tMfFcolumns instructions carries out.
3) with kind of the recombinant protein A IV-HA1 of three after purifying, NDV-HN and IBV-NP be immune Balb/C mouse (purchased from Wuhan Biological Products Inst.'s Experimental Animal Center) respectively, obtain anti-avian influenza virus (AIV) hybridoma cell strain XYCSJL-AIV-2B8 and XYCSJL-AIV-4B7, anti-new castle disease virus (NDV) hybridoma cell strain XYCDY-NDV-3E11 and XYCDY-NDV-4B10, anti-avian infectious bronchitis virus (IBV) hybridoma cell strain XYCDY-1BV-3G5 and XYCDY-1BV-1E6, hybridoma cell strain is injected processed Balb/C mouse, obtain the ascites containing monoclonal antibody, through caprylic acid-ammonium, (Zhu Liping edits " immunology common experimental method ", People's Medical Officer Press, 2000 editions) obtain the monoclonal antibody of purifying.Six strain cells deliver China on March 11st, 2014 all. Wuhan. and in Wuhan University's China typical culture collection (CCTCC), preservation information is respectively: hybridoma cell strain XYCSJL-AIV-2B8, its preserving number: CCTCCNO:C201449; Hybridoma cell strain XYCSJL-AIV-4B7, its preserving number: CCTCCNO:C201453; Hybridoma cell strain XYCDY-NDV-3E11, its preserving number: CCTCCNO:C201451; Hybridoma cell strain XYCDY-NDV-4B10, its preserving number: CCTCCNO:C201455; Hybridoma cell strain XYCDY-IBV-3G5, its preserving number: CCTCCNO:C201450; Hybridoma cell strain XYCDY-IBV-1E6, its preserving number: CCTCCNO:C201454;
4) extract mouse IgG immune health new zealand rabbit (purchased from Wuhan Biological Products Inst.'s Experimental Animal Center), obtain rabbit anti-mouse igg antibody;
5) react with trisodium citrate and gold chloride (purchased from sigma company) and prepare collaurum;
6) by step 3) the anti-AIV antibody (XYCSJL-AIV-2B8) of preparing, anti-NDV antibody (XYCDY-NDV-3E11) and anti-IBV antibody (XYCDY-1BV-3G5) add step 5 respectively) in the collaurum prepared, obtain three kinds of monoclonal antibodies-colloid gold label thing concentrated by certain volume mixing after form gold mark potpourri;
7) three kinds of gold mark potpourris are coated on gold mark pad (2);
8) by step 3) the anti-AIV antibody (XYCSJL-AIV-4B7) of preparing, anti-NDV antibody (XYCDY-NDV-4B10) and anti-IBV antibody (XYCDY-1BV-1E6) are coated on successively on nitrocellulose filter (3) and form detection line T1 (5) respectively, T2 (6) and T3 (7); And rabbit anti-mouse igg is coated on nitrocellulose filter (3) and forms nature controlling line (8);
9) on described PVC backing (9), adhere to described sample pad (1), gold mark pad (2), nitrocellulose filter (3), absorption pad (4) in order successively, obtain described AIV (H5 or H9), the multivariate detection immunity colloidal gold test paper strip of NDV and IBV.
10) by 9) described in test strips load test and get stuck in (10) and form test card.
Obviously, the hybridoma that the present invention prepares six specific monoclonal antibodies of secretion can be divided into three compositions to making by viral species of the same race, uses with namely forming double-antibody sandwich, wherein:
It is paired use that hybridoma XYCSJL-AIV-2B8 and XYCSJL-AIV-4B7 of secretion anti-avian influenza virus monoclonal antibody detects in avian influenza virus H5 and H9 hypotype antigen in preparation.
It is paired use that hybridoma XYCDY-NDV-3E11 and XYCDY-NDV-4B10 of secretion anti-new castle disease virus monoclonal antibody detects in Avian pneumo-encephalitis virus antigen in preparation.
Hybridoma XYCDY-1BV-3G5 and XYCDY-1BV-1E6 secreting anti-avian infectious bronchitis virus monoclonal antibody is paired use in detection Avian pneumo-encephalitis virus antigen.
Because this kit have employed above-mentioned double antibody sandwich method coated antibody, the Detection results of kit of the present invention thus can be substantially increased.
Principle of the present invention adopts double antibody sandwich method (conventional method for report), wrap to be detected in sample by anti-AIV antibody (XYCSJL-AIV-4B7) for detection line T1 and whether be described containing avian influenza virus (AIVH5 or AIVH9), when containing AIV (H5 or H9) in measuring samples, AIV (H5 or H9) in sample can react at gold mark pad position and the anti-AIV antibody (XYCSJL-AIV-2B8) of gold mark, when reaction compound chromatography is to detection line position, be coated on anti-AIV antibody on detection line T1 can and compound in AIV (H5 or H9) react, thus form " golden labeling antibody-AIV-coated antibody " double-antibody sandwich compound in detection line position, colloid gold label owing to there being a kind of antibody in the composite, so just there will be macroscopic red stripes in detection line position, so just obtain positive findings.Have neither part nor lot in the golden labeling antibody chromatography of reaction to nature controlling line position, the lgG of coated rabbit against murine catches and develops the color.On the contrary, if when not containing AIV (H5 or H9) in sample, in detection line position not containing double-antibody sandwich compound, would not red stripes be occurred, so just obtain negative findings.Test strip (structural drawing as shown in Figure 2) in kit of the present invention to be sticked to successively by illustrated order by sample pad (1), gold mark pad (2), nitrocellulose filter (3), absorption pad (4) and PVC backing (9) assembles, and test strips loads test and gets stuck in (10) and form test card.
Compared with prior art, the present invention has following outstanding advantage:
1, polynary quick detection bird flu H5 of the present invention and H9 hypotype, ewcastle disease and avian infectious bronchitis virus colloidal gold kit, have high specificity, highly sensitive, and detection time, short (10-15 minute) judged the advantages such as directly perceived.
2, kit of the present invention is without any need for specific apparatus, equipment, and testing cost is low.Simultaneously because the present invention realizes detecting while three kinds of fowl Major respiratory diseases (bird flu H5 and H9 hypotype, ewcastle disease and avian infectious bronchitis virus) cause of disease, thus detection time and testing cost are more effectively saved.
3, kit of the present invention is easy and simple to handle.
4, kit containment of the present invention is convenient, and less demanding to storage temperature, the shelf-life at 4 DEG C is at least 90 days.
Accompanying drawing explanation
Fig. 1: general technical route map of the present invention.
Fig. 2: the assembling schematic diagram of test strip of the present invention.
1-sample pad in figure, 2-gold mark pad, 3-nitrocellulose filter, 4-absorption pad, 5-detection line T1,6-detection line T2,7-detection line T3,8-nature controlling line, 9-PVC backing, 10-test is got stuck.
Fig. 3: the present invention examines test card result and judges schematic diagram.
In figure: 11: represent that detecting AIV, NDV, IBV is positive findings; 12: represent that it is positive for detecting AIV, NDV and IBV is negative findings; 13: represent that it is positive for detecting NDV, AIV and IBV is negative findings; 14: represent that it is positive for detecting IBV, AIV and NDV is negative findings; 15: represent that detecting AIV, NDV, IBV is weak positive findings; 16,17: represent that test card lost efficacy.
Fig. 4: the physical build-up figure that the present invention relates to the expression of recombinant proteins plasmid of NDV-HN.
Fig. 5: the physical build-up figure that the present invention relates to the expression of recombinant proteins plasmid of IBV-NP.
Embodiment
Embodiment 1
1.AIV-HA1 albumen, the preparation of NDV-HN albumen and IBV-NP albumen
AIV-HA1 albumen, the preparation method of NDV-HN albumen and IBV-NP albumen is mainly see document: Pehanorm Brooker J, not Ritchie EF, Manny A Disi T edits (Jin Dongyan, Li Mengfeng etc. translate). Molecular Cloning: A Laboratory guide. the second edition, Science Press, the method provided in 1992 is carried out.
The choning and sequencing of 1.1 HA 1 Gene of AIVs and the structure of expression vector pKG-NS1
The construction method of the HA 1 Gene of AIV that the present invention relates to and recombinant expression plasmid pKG-HA1 thereof and build collection of illustrative plates and known in China in 2006 and publish on the net, see document: middle National IP Network Dissertations Database: Wu Renwei, foundation [D] Hua Zhong Agriculture University of the research of avian influenza virus recombinant protein mucosal immunity and N-ELISA antibody detection method, 2006; See network address: http://epub3.cnki.net/KCMS/detail/detail.aspx? dbcode=CDFD & QueryID=9 & CurRec=1 & dbname=CDFD9908 & filename=2006190169.nh & urlid=& yx=& uid=WEEvREcwSlJHSldTTGJhYkhsd0Z6ODd6RU81ZVo0ODlGZ2hITVNL ZlMwRHM5RzdZNG1mdWJmU3NZZFBoeTAwbg==& v=MTM0NThSOGVYMUx1eFlTN0RoMVQzcVRyV0;
The choning and sequencing of 1.2 ewcastle disease HN genes and the structure of expression vector pKG-HN
The sequence of ewcastle disease HN gene that the present invention relates to and the construction method list of references of cloning process and expression vector pKG-HN: middle National IP Network Dissertations Database: Sun Shuna, the clonal expression of F gene of NDV strain and HN gene and the preliminary foundation [D] of ELISA antibody detection method thereof, Hua Zhong Agriculture University, 2006; See network address: http://epub3.cnki.net/KCMS/detail/detail.aspx? dbcode=CMFD & QueryID=7 & CurRec=1 & dbname=CMFD9908 & filename=2006190369.nh & urlid=& yx=& uid=WEEvREcwSlJHSldTTGJhYlNcGZ2OHlzRkwxL0k2RVBNdlRoYWMzM llid1d5bUVYOXE3ZU9jcm11MC91WmJUeA==& v=MjI3OTh6TVYxMjdHTEt4SHRMS3BwRWJQSVI.
Specific implementation method is as follows: the strain ewcastle disease NDVLaSota system strain preserved with Hua Zhong Agriculture University microorganism and the freeze-drying of immunization experiment room inoculates NDV in healthy SPF (no-special pathogen) chicken embryo (purchased from Wuhan Biological Products Inst.) of 9-11 age in days in allantoic cavity, abandon dead germ before 24 hours, collect the chick embryo allantoic liquid of 24-96 hour; Under 4 DEG C of conditions, 4,000rpm centrifugal 30min, get supernatant; 30,000rpm centrifugal 60min concentrating virus under 4 DEG C of conditions.With PBS (preparation of the DEPC process water) dissolution precipitation of pH7.2, after packing ,-70 DEG C save backup.Primer is designed and synthesized, amplification HN gene according to the NDVLaSota system separated strain HN gene order (accession number: AJ629060.1) that GenBank includes.Upstream primer P1:ATA
gGATCCgGGGCTAGCACACTTA; Downstream primer P2:AGC
aAGCTTcTAGCCAGACTTGGCT.P1 is positioned at promoter upstream, and be added with BamHI site, P2 includes terminator codon, adds HindIII site, and two restriction enzyme site underscores mark.For lacking the HN gene of cytoplasmic region and transmembrane domain between two primers.Get appropriate viral suspension and extract RNA, by the method provided in TaKaRaRNAPCRKit (AMV) Ver.2.1 instructions, amplify DNA, adopt UNIQ-10 pillar DNA glue to reclaim kit (E.Z.N.AGelExtractionKit) and reclaim purifying RT-PCR amplified production, the RT-PCR product of recovery is directly carried pMD18-T with the clone purchased from TaKaRa company and carries out coupled reaction, then product conversion will be connected in the middle competent cell bacillus coli DH 5 alpha of kit (the commercial reagents box purchased from TaKaRa company), picking positive colony, adopt alkaline lysis (Pehanorm Brooker J, not Ritchie EF, Manny A Disi T edits (Jin Dongyan, Li Mengfeng etc. translate). Molecular Cloning: A Laboratory guide. the second edition, Beijing Science Press, 1992 editions) extract recombinant plasmid dna, and recombinant plasmid dna is identified.Will the correct recombinant plasmid called after pMT-HN of qualification, and serve Hai Shenggong bioengineering company limited and check order, then the relevant data in sequencing results and GeneBank is carried out tetraploid rice and divides rolling over.
PMT-HN BamHI and HindIII enzyme are cut, and reclaim the fragment of about 1590pb, are then connected by the expression vector pGEX-KG of this fragment with the AmershamBiosciences company of cutting with same enzyme enzyme, construction of expression vector.Recombinant expression carrier transforms the DH5 α competent cell of fresh preparation, and by transformed bacteria coating LB agar plate 37 DEG C of overnight incubation containing ampicillin.Prepare plasmid in a small amount with alkaline lysis after choosing several single bacterium colony overnight incubation, carry out restriction analysis and pcr amplification qualification.By the positive colony called after pKG-HN obtained.In a large number prepared by plasmid DNA to positive colony alkaline lysis, and packing is frozen in-20 DEG C.Recombinant expression plasmid pKG-HN builds flow process as shown in Figure 4.
The choning and sequencing of 1.3 infectious bronchitis of chicken N genes and the structure of expression vector pKG-NP
The ewcastle disease that the present invention relates to, the sequence of infectious bronchitis of chicken N gene and the construction method list of references of cloning process and expression vector pKG-NP: middle National IP Network Dissertations Database: Li Zhonghua, the clone of IBV spike protein, nucleoprotein gene, expression and the Preliminary Applications in antibody test [D] thereof, Hua Zhong Agriculture University, 2006; See network address: http://epub3.cnki.net/KCMS/detail/detail.aspx? dbcode=CMFD & QueryID=14 & CurRec=1 & dbname=CMFD9908 & filename=2006190367.nh & urlid=& yx=& uid=WEEvREcwSlJHSldTTGJhYlNPcGZ2OHlzRkwxL0k2RVBNdlRoYWMz Mllid1d5bUVYOXE3ZU9jcm11MC91WmJUeA==& v=MjQ5NDY2ZlpPZHVGeXpnVnJ6SVYxMjdHT.Specific implementation method is as follows: strain infectious bronchitis of chicken IBV (H52strain) strain preserved with Hua Zhong Agriculture University's veterinary microorganism and the freeze-drying of immunization experiment room inoculates NDV in healthy SPF (no-special pathogen) chicken embryo (purchased from Wuhan Biological Products Inst.) of 9-11 age in days in allantoic cavity, abandon dead germ before 24 hours, collect the chick embryo allantoic liquid of 24-96 hour; Under 4 DEG C of conditions, 4,000rpm centrifugal 30min, get supernatant; 30,000rpm centrifugal 60min concentrating virus under 4 DEG C of conditions.With PBS (preparation of the DEPC process water) dissolution precipitation of pH7.2, after packing ,-70 DEG C save backup.The N gene order (number of logining: EF602459.1) of the IBV included according to GenBank to sequences Design comparatively conservative in its N gene and synthetic primer, amplification N gene.Upstream primer P1:
gGATCCaTGGCAAGCGGTAAAGCAG; Downstream primer P2:CC
gAGCTCtCAAAGTTCATTCTCTCC.P1 is positioned at promoter upstream, and be added with BamHI point, P2 includes terminator codon, adds Sca I site, and two restriction enzyme site underscores mark.It is fragment comparatively conservative in N gene between two primers.Get appropriate viral suspension and extract RNA, by the method provided in TaKaRaRNAPCRKit (AMV) Ver.2.1 instructions, amplify DNA, adopt UNIQ-10 pillar DNA glue to reclaim kit (E.Z.N.AGelExtractionKit) and reclaim purifying RT-PCR amplified production, the RT-PCR product of recovery is directly carried pMD18-T with the clone purchased from TaKaRa company and carries out coupled reaction, then product conversion will be connected in the middle competent cell bacillus coli DH 5 alpha of kit (the commercial reagents box purchased from TaKaRa company), picking positive colony, adopt alkaline lysis (Pehanorm Brooker J, not Ritchie EF, Manny A Disi T edits (Jin Dongyan, Li Mengfeng etc. translate). Molecular Cloning: A Laboratory guide. the second edition, Beijing Science Press, 1992 editions) extract recombinant plasmid dna, and recombinant plasmid dna is identified.Will the correct recombinant plasmid called after pMT-N of qualification, and serve Hai Sheng work biotech firm and check order, then the relevant data in sequencing results and GeneBank is carried out tetraploid rice and divides rolling over.
PMT-N BamHI and Sca I enzyme is cut, and reclaims the fragment of about 1.2kb, is then connected by the expression vector pGEX-KG of this fragment with the AmershamBiosciences company of cutting with same enzyme enzyme, construction of expression vector.Recombinant expression carrier transforms the DH5 α competent cell of fresh preparation, and by transformed bacteria coating LB agar plate 37 DEG C of overnight incubation containing ampicillin.Prepare plasmid in a small amount with alkaline lysis after choosing several single bacterium colony overnight incubation, carry out restriction analysis and pcr amplification qualification.By the positive colony called after pKG-NP obtained.In a large number prepared by plasmid DNA to positive colony alkaline lysis, and packing is frozen in-20 DEG C.Recombinant expression plasmid pKG-NP builds flow process as shown in Figure 5.
The expression of 1.4AIV-HA1, NDV-HN and IBV-NP tri-kinds of albumen
With recombinant plasmid pKG-HA1, pKG-HN and pKG-NP and expression vector pGEX-KG transformation of E. coli BL21 (DE3) respectively, be coated with LB flat board (1% (W/V) Tryptone containing ampicillin (Amp), 0.5% (W/V) YeastExtract, 0.5% (W/V) NaCl, 0.1mg/mLAmp and 1.5% (W/V) Agar), put 37 DEG C of constant temperature ovens to cultivate, picking list bacterium colony, is inoculated in 2.0mL LB liquid medium (1% (W/V) Tryptone containing ampicillin (Amp); 0.5% (W/V) YeastExtract; 0.5% (W/V) NaCl and 0.1mg/mLAmp) in, 37 DEG C of 250-300r/min shaken cultivation, when OD600nm reaches 0.6-1.0.Inoculate 1.0mL bacterium liquid respectively to 10.0mL containing in the LB liquid medium of ampicillin (Amp), 37 DEG C of 250-300r/min shaken cultivation 3h, then add the derivant IPTG (isopropyl-beta D-thio galactopyranoside) that final concentration is 0.024mg/mL, induce 4 hours, collected by centrifugation thalline carries out SDS-PAGE detection.
The purifying of 1.5AIV-HA1, NDV-HN and IBV-NP tri-kinds of albumen
(1) purifying of supernatant
According to a large amount of abduction delivering of condition in above-mentioned 1.4, by the bacterial cultures after induction in 8, the centrifugal 10min of 000r/min, precipitation PBS (phosphate) damping fluid (140mMNaCl, 2.4mMKCl, 10mMNa2HPO4.12H2O, 1.8mMKH2PO4pH7.4) (being about the l/10 of original bacteria liquid volume) resuspended, adding DTT (dithiothreitol (DTT)) to final concentration is 10mmol/L, being crushed to liquid with pressure breaking instrument becomes transparent, 4 DEG C, 12, the centrifugal 15min of 000r/min, with the supernatant after the membrane filtration fragmentation of 0.22 μm, with GSTrapTMFFcolumns chromatographic column through AKTA protein purification system AKTAexplorer10 (purchased from GEHealthcare company, USA) purifying is carried out to above-mentioned supernatant.
(2) purifying of inclusion body and renaturation
Precipitation PBS (pH7.4) after centrifugal for fragmentation in above-mentioned (1) is washed 2 times, add 19.7mL buffer A (50mMTris-Cl, 0.5mMEDTA, 50mMNaCl, 5% (W/V) Glycerol) and 0.3mL20% (W/V) SKL (sarcosyl) store liquid, vigorous agitation, make it dissolve, leave standstill 2h, in 4 DEG C of centrifugal 10min of 12000r/min, abandon precipitation, get supernatant, adding 20% (W/V) PEG-4000 (Macrogol 4000) to final concentration is 0.2%, oxidized glutathione to the final concentration adding 50mmol/L is 1mmol/L, reduced glutathione to the final concentration adding 100mmol/L is 2mmol/L, leave standstill 2h, dialyse 2 ~ 3 days with PBS (pH7.4),-70 DEG C save backup.
(3) mensuration of protein concentration and qualification
Measuring the absorbance value of protein solution under 280nm and 260nm wavelength respectively with nucleic acid-protein analyzer is A280, A260.Protein concentration is gone out according to formulae discovery, the protein solution reaching requirement concentration is distributed into 100 μ L/ to manage, get 50 μ L and carry out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) qualification and Western-blot analysis, all the other are put-70 DEG C and save backup.
SDS-PAGE detects: the thalline getting above-mentioned collection, with the abundant resuspended thalline of 40.0 μ LddH2O, add isopyknic sample loading buffer (250mMTris-HCl, 10% (W/V) SDS, 0.5% (W/V) bromophenol blue, 50% (V/V) glycerine, 5% (V/V) beta-mercaptoethanol), 3-5min is boiled in water-bath, and then ice bath waits for application of sample electrophoresis.By Pehanorm Brooker J, not Ritchie EF, Manny A Disi T edits, Jin Dongyan, Li Mengfeng etc. translate, " Molecular Cloning: A Laboratory guide ", the second edition, 1992 editions described method preparative separation glue and spacer gel, add 1 × Tris-glycine running buffer (25mMTris-base, 0.1% (W/V) SDS, 0.25MGlycine), get the sample point sample of process, electrophoresis 2h, take off gel, coomassie brilliant blue R250 dyeing liquor (0.1% (W/V) coomassie brilliant blue R250, 25% (V/V) isopropyl alcohol, 10% (V/V) glacial acetic acid) dye 2h, then SDS-PAGE destainer (5% (V/V) ethanol is used, 10% (V/V) glacial acetic acid) decolouring, observe and take a picture.And by the foreign protein content expressed by gel thin-layer scanning analysis.
Western-blot analyzes: in order to analyze the immunologic competence of fusion AIV-HA1, NDV-HN and IBV-NP of expressed band GST label, method carries out SDS-PAGE electrophoresis as described above, gel after electrophoresis, not dyed, direct transfer device is transferred on NC film by charged for albumen, with 15V electricity transfer printing 1.5h.After transfer printing terminates, by NC film in TBST (150mMNaCl, 20mMTris-HCl, 0.05%Tween20), rinsing once, again NC film is proceeded to and in the polybag of heated sealant, confining liquid (TBST containing 1% (W/V) bovine serum albumin(BSA)) can be added, after draining the bubble in bag as far as possible by 0.1mL-0.15mL/cm2 (NC membrane area), heat airtight sack, room temperature is shaken 1-2h or 4 DEG C gently and is spent the night.Then confining liquid is abandoned, film is taken out, film is proceeded in new polybag, the anti-AIV hyper-immune serum of chicken through TBST dilution (1:150) is added by the amount of 0.1mL-0.15mL/cm2, the same eliminating bubble also seals sack, room temperature effect 1h on shaking table, 6 times are washed with TBST, each 5mim, film is proceeded in new plastic bag, the anti-chicken IgG-HRP of rabbit (purchased from GenScript company) through TBST dilution (1:5000) is added by 0.1mL-0.15mL/cm2, room temperature reaction 1h, TBST washes 6 times, each 5min, finally use TBS rinsing again 2 times, add substrate solution (DAB-H2O2) colour developing of preparing with 0.01mol/LTris-Cl (pH7.6), once there is protein band, use ddH immediately
2o stops, i.e. observable taking a picture.
The target protein concentration obtaining purifying according to formulae discovery (protein concentration (mg/mL)=l.45 × A280-0.74 × A260) is respectively: HA1--0.45mg/mL; HN--0.53mg/mL; N--0.90mg/mL.Western-blot analysis result is utilized to show the AIV-HA1 albumen (62KDa) that the present invention recombinates, NDV-HN albumen (84KDa), IBV-NP albumen (72 and 60KDa) there occurs specific reaction with corresponding positive serum respectively, confirms that this several expression product all has good antigenicity.
2. anti-AIV antibody, the preparation of anti-NDV antibody and anti-IBV antibody:
Utilize three kinds of recombinant protein A IV-HA1 prepared by applicant, NDV-HN and IBV-NP be immune Balb/C mouse (purchased from Wuhan Biological Products Inst.'s Experimental Animal Center) respectively, immune programme for children is solution and isopyknic Freund's complete adjuvant (purchased from the sigma company) emulsification of getting protein content 100 μ g, injection mouse peritoneal, booster immunization after 21 days, use incomplete Freund's adjuvant (purchased from sigma company) emulsification instead, finally in first three sky of fusion, (best and immunity last time is separated by more than 4 weeks), abdominal cavity reinforced immunological, antigen amount doubles (200 μ g), do not add adjuvant.During fusion, the Balb/C mouse of last reinforced immunological of learning from else's experience, eye socket sacrificed by exsanguination (collect serum, be positive serum), soaks 5min sterilization in 75% alcohol.Aseptic taking-up mouse spleen, isolate splenocyte, mix in 50mL centrifuge tube in the ratio of 1 ~ 2 × 107 SP2/0 and 108 immunocytes (1:10 ~ 1:15) with SP2/0 myeloma cell's (SP2/0 myeloma cell is so kind as to give by Wuhan Biological Products Inst., Ministry of Public Health) of recovery, 1500rpm, centrifugal 10min.Evacuation supernatant (filter paper of available sterilizing blots), knocks at the bottom of pipe gently, makes cell precipitation loosening slightly.The centrifuge tube that cell mixture is housed is put in 37 DEG C of water-baths.Then in 1min, slowly instill the 50%PEG0.8mL (purchased from sigma company) of pre-temperature to 37 DEG C, limit edged stirs with pipette tip gently, continues stirring 1min.Then the 10mLRPMI-1640 basic culture solution (purchased from GIBCO company) of 37 DEG C of pre-temperature is slowly added.Concrete grammar is: within first minute, dropwise instill 1mL, within second minute, adds 1ml, within 3rd ~ 4 minutes, adds 3mL, and within the 5th minute, add remaining 5mL, each added-time need slowly add, and constantly stirs lightly.Finally add 30mL1640 liquid, also need slowly to add.The centrifugal 5min of 800rpm, removes supernatant, places 5 ~ 8min in 37 DEG C.Suspend with HAT (purchased from GIBCO company) nutrient culture media, simultaneously also with HAT nutrient culture media suspend the raising splenocyte for preparing and with fusion after mixing with cells, add appropriate HAT nutrient culture media (the RPMI-1640 basic culture solution of the composition of nutrient culture media: 80mL as required, 20mL sterilizing calf serum, the HAT of 1mL100%, 1mL 10,000U/mL mycillin dual anti-), divide and plant in 96 well culture plates, about 250 μ L/ holes.Single cell fusion can inoculate 4 ~ 8 piece of 96 orifice plate.Also can plant less as required, the cell number of generally pressing SP2/0 myeloma cell calculates, and every hole inoculum concentration is about containing about 104 SP2/0 myeloma cells.In 37 DEG C, cultivate in 5%CO2 incubator.Merge and within latter second day, start observation and have pollution-free, added 1 HAT nutrient culture media in the 4th day, within 8th ~ 10 days, suck 100 μ L nutrient culture media and change HT (purchased from GIBCO company) nutrient culture media 100 μ L.Cell colony to be fused grows to culture hole 1/4, when nutrient culture media slightly turns yellow, carries out antibody test.Hole AIV-HA1 fusion and AIV allantoic fluid are merged respectively as coating antigen for AI, it is NDV-HN fusion and NDV allantoic fluid that ND merges hole coating antigen, it is that IBV-NP merges and IBV allantoic fluid that IB merges hole coating antigen, all use two selective mechanisms, utilize conventional ELISA method to filter out the positive hole of secretion corresponding monoclonal antibody.To the positive hole screened use at once limiting dilution assay (with reference to Xue Qingshan, " philosophy and technique of in vitro culture ", Science Press, calendar year 2001 version) carry out cloning, screening.Through 3 ~ 4 time clonings, finishing screen is selected secretion and is obtained anti-avian influenza virus (AIV) antibody hybridoma cell XYCSJL-AIV-2B8 and XYCSJL-AIV-4B7, and anti-new castle disease virus (NDV) antibody hybridoma cell XYCDY-NDV-3E11 and XYCDY-NDV-4B10 and anti-avian infectious bronchitis virus (IBV) antibody hybridoma cell XYCDY-1BV-3G5 and XYCDY-1BV-1E6 be totally 6 strain cells (biological deposits information is see " summary of the invention " part of this instructions).Carry out chromosome counting to above-mentioned 6 strain cells, result shows, and the chromosomal average of SP2/0 myeloma cell is 70, and splenocyte chromosome number is 40, and the chromosome number of hybridoma is between 80 ~ 94.All higher than the chromosome number of two parental cells, the SP2/0 myeloma cell really of fused cell and the hybrid product of splenocyte are described, the chromosome number of hybridoma is obviously more than the chromosome of SP2/0 myeloma cell.Two strain clones of same cause of disease are resisted to be all the antibody produced for different epitope with being added ELISA qualification.6 strain cells (1 × 106) of the present invention are injected Balb/C mouse peritoneal, manufacture order clonal antibody respectively.Adopt mouse source monoclonal antibody hypotype identification kit (MouseMabIsotypingTestKit) of ROCKLAND company to carry out hypotype qualification to the monoclonal antibody that the present invention obtains, result is mouse IgG 2b subclass.
3. the purifying of monoclonal antibody
With reference to Zhu Li equality, " immunology common experimental method ", People's Medical Officer Press, the method of report in 2000 editions: the mouse ascites 5mL getting gained mixes with appropriate silicon dioxide, add isopyknic barbitol buffer solution (formula: sodium chloride 85.00g, barbital 5.75g, barbital sodium 3.75g, Sodium azide 2.00g, is settled to 2000mL with distilled water), after shaken at room temperature 1h, at room temperature leave standstill 30min, get supernatant in clean centrifuge tube, in 4 DEG C, the centrifugal 10min of 3000rpm; Get supernatant 8mL, add 16mL0.06mol sodium-acetate buffer, with HCl adjust pH to 4.5, after slowly adding sad 132 μ L under fully stirring, stirring at room temperature 30min, then proceed to 4 DEG C of refrigerators and fully precipitate 2h, 4 DEG C, the centrifugal 30min of 15000rpm, obtains supernatant 22mL, the phosphate buffer adding 2.2mL0.1M (is called for short PB, formula: 10mMNa
2hPO
4.12H
2o and 1.8mMKH
2pO
4pH7.2), with NaOH adjust pH to 7.6, slowly adding ammonium sulfate to final concentration under stirring is 0.277g/mL, after 4 DEG C of refrigerators fully precipitate 2h, in 4 DEG C, the centrifugal 30min of 12000rpm, abandon supernatant, PBS damping fluid (the formula: 140mMNaCl, 2.4mMKCl, 10mMNa of precipitation 5mL0.01M
2hPO
4.12H
2o, 1.8mMKH
2pO
4pH7.2) resuspended, load bag filter, after 5000mL0.01MpH7.2PBS damping fluid enough hemodialysis, then to 2000mL distill water dialysis, finally ionized water dialysis is boiled off to 3000mL tri-, protein solution PEG-20000 good for enough hemodialysis is concentrated into 3mL, then in 4 DEG C, the centrifugal 30min of 12000rpm, abandons precipitation, collect supernatant, record six strain monoclonal antibody concentration between 1.0-2.2mg/mL.Be accredited as the monoclonal antibody of purifying through SDS-PAGE, its purity is greater than 95%.This monoclonal antibody can be used for preparing immune colloid gold.
4. the preparation of rabbit anti-mouse igg antibody:
Utilize Balb/C mouse IgG immune health new zealand white rabbit, prepare the rabbit anti-mouse igg hyper-immune serum of high specific, high-titer, saturated ammonium sulphate method (list of references: Zhu Li equality is adopted to hyper-immune serum, " immunology common experimental method ", People's Medical Officer Press, 2000 editions) slightly carry, cross after post through G-200 and obtain highly purified rabbit anti-mouse igg antibody.We utilize this antibody to be the core reagent of the nature controlling line as kit.
5. the preparation of monoclonal antibody-colloid gold label thing
(1) preparation of collaurum:
With two ionized water that boils off, 1% gold chloride is diluted to 0.01%, put stirring on magnetic force heating stirrer to boil, add 1.5mL1% trisodium citrate by every 100mL0.01% gold chloride, continue to boil, heat until liquid is orange red namely stopping, after being cooled to room temperature, supply dehydration.The collaurum outward appearance prepared should pure, bright, without precipitation and floating thing, put 4 DEG C of preservations.
(2) preparation of monoclonal antibody-colloid gold label thing:
The anti-AIV antibody XYCSJL-AIV-2B8 of gained, anti-NDV antibody XYCDY-NDV-3E11 and anti-IBV antibody XYCDY-1BV-3G5 are marked colloid gold particle respectively.Concrete steps are as follows: under magnetic agitation, the pH value to 8.0 of collaurum is adjusted with 0.1M solution of potassium carbonate, above-mentioned three kinds of monoclonal antibodies (anti-AIV antibody XYCSJL-AIV-2B8 is added by 5 ~ 7.2 μ g antibody/mL collaurums, anti-NDV antibody XYCDY-NDV-3E11 and anti-IBV antibody XYCDY-1BV-3G5), continue to stir and evenly mix 30min, adding 10%BSA (bovine serum albumin(BSA)) to final concentration is 1%, leaves standstill 30min.12000rpm, 4 DEG C of centrifugal 30min, abandon supernatant, borate buffer solution (the formula: boric acid 0.1237g of precipitation 0.02MpH9.0, PEG-200001g, is settled to 1000mL with tri-distilled water, adjusts pH to 9.0) wash twice, with the borate buffer solution (formula: boric acid 0.1237g of the 0.02MpH9.0 of 1/20th initial colloid gold volume, PEG-200001g, is settled to 1000mL with tri-distilled water, adjust pH to 9.0) will precipitate resuspended, put 4 DEG C for subsequent use, 60 days shelf-lifves.
6. the bag quilt of gold mark pad
Gold is marked pad and be soaked in confining liquid (formula: 2%BSA, 2.5% sucrose, 0.3%PVPK-30,0.02%NaN3,0.5%Teewn-20,0.5%PEG-2000,10mMNa
2hPO
4.12H
2o and 1.8mMKH
2pO
4pH7.2) in after 30min, in 37 DEG C of oven dry.Get three kinds of monoclonal antibodies (anti-AIV antibody XYCSJL-AIV-2B8 of isopyknic above-mentioned preparation, anti-NDV antibody XYCDY-NDV-3E11 and anti-IBV antibody XYCDY-1BV-3G5) antibody gold label thing fully mix rear concentrated, with Biodot point film instrument, the monoclonal antibody prepared-colloid gold label thing is evenly coated on gold mark pad, every centimetre of gold mark pad bag is by 9 μ L antibody-colloidal gold labels, vacuum drying (according to a conventional method), Vacuum Package (according to a conventional method), put 4 DEG C for subsequent use.
7. the process of sample pad
Sample pad is soaked in confining liquid (containing 2%BSA, 2.5% sucrose, 0.3%PVPK-30,0.02%NaN3,0.5%Teewn-20,0.5%PEG-2000,10mMNa
2hPO
4.12H
2o and 1.8mMKH
2pO
4pH6.4) in after 30min, in 37 DEG C of oven dry, Vacuum Package, put 4 DEG C for subsequent use.
8. the bag quilt of nitrocellulose filter
With coating buffer (containing 3% methyl alcohol, the 0.01MpH7.4PBS damping fluid of 1% sucrose) by anti-AIV antibody XYCSJL-AIV-4B7, anti-NDV antibody XYCDY-NDV-4B10 and anti-IBV antibody XYCDY-1BV-1E6 dilutes 10-18 μ g/mL, with Biodot point film instrument it is coated on nitrocellulose filter successively that (detection line is followed successively by T1 as detection line, T2 and T3), package amount is 0.7 μ L/cm, and this detection line, near gold mark pad end, holds about 5mm apart from gold mark pad pad; With coating buffer by rabbit anti-mouse igg antibody dilution to 500 μ g/mL, be coated in nitrocellulose filter as nature controlling line with Biodot point film instrument, package amount is 0.7 μ L/cm, and this nature controlling line is near absorption pad, be about 5mm apart from absorption pad, nature controlling line or detection line between any two distance are 3 ~ 4mm.Dry 30-40min for 37 DEG C, for subsequent use.
9. the assembling of kit
Sample pad (1), gold mark pad (2), cellulose nitrate rope film (3), absorption pad (4) are sticked on PVC backing (7) by the order shown in Fig. 2 successively, be cut into the little bar that 4mm is wide, be packaged in test to get stuck composition test card in (10), by the test card Vacuum Package in this kit after having prepared.In 4 DEG C of preservations, the shelf-life is at least 90 days.
Embodiment 2 (Application Example)
The using method of fowl Major respiratory disease three quick detection kit
1. the composition of kit, comprising:
1. test card 25
2. sample diluting liquid one bottle (10mL/ bottle)
2. the preparation of sample
2.1 sample diluting liquids: sample diluting liquid is 0.85% sodium chloride solution.Compound method: 8.5gNaCl, adding distil water is settled to
1000mL。
2.2 sample preparation
The detailed method of operation of sample collection and process is as follows:
(1) the pharyngeal sample of tracheae: first push aside with the howl of hand by chicken, then with hand or tweezers, the tongue of chicken is pulled out, expose pharyngeal, cotton swab is inserted pharyngeal tracheal strips, stirs several lower taking-up, then put in advance that oneself adds the sample hose of 500 μ L sample diluting liquids, firmly stir, extrude, make the sample elution on cotton swab get off as far as possible, after leaving standstill 15min, liquid in pipe supernatant is measuring samples;
(2) the excrement stain sample on cloaca sample or fortune fowl vehicle: direct cotton swab samples from the cloaca of chicken or dips the excrement stain of transporting fowl vehicle, then cotton swab is put in advance that oneself adds the sample hose of 500 μ L sample diluting liquids, the same elution samples, discard cotton swab, the supernatant after leaving standstill in pipe is measuring samples;
(3) muscle or internal organ sample: get chest muscle or thigh place muscle one fritter or internal organs one fritter (l-2g), add after 1-3mL sample diluting liquid fully grinds in mill, freeze thawing is once, 4,000r/min, centrifugal 5min, supernatant is measuring samples.
3. detect: take out kit, equilibrium at room temperature 20 minutes; Open the package, take out test card, get in the well of the sample instillation test card that 100-120 μ L prepares, result of determination in 10 ~ 15 minutes.
4. result judges: as shown in Figure 4, and the detection line T1 on NC film, T2 and T3 are corresponding respectively detects AIVH5 or AIVH9, NDV and IBV.When macroscopic aubergine appears in the nature controlling line on NC film in test card, and there is not macroscopic aubergine in corresponding detection line such as T1 (or T2 or T3), detect in sample not containing AIVH5 or AIVH9 (or NDV or IBV), result is judged to feminine gender, is designated as "-"; When macroscopic aubergine nature controlling line appears in test card, there is macroscopic aubergine in corresponding detection line such as T1 (or T2 or T3) simultaneously, detect containing AIVH5 or AIVH9 (NDV or IBV) in sample, result is judged to the positive, is designated as "+"; Virus quantity in detection line color depth and tested serum is proportionate, and color illustrates that the viral level of detected sample is higher more deeply, and nature controlling line occurs then being judged to without band and detects test card and lost efficacy.
Embodiment 3
The applicating example of 1 fowl Major respiratory disease three quick detection kit
The detection of 1.1 chicken standard antigens
1. specific test: the bird flu H5 hypotype HI antigen will be purchased respectively, bird flu H9 hypotype HI antigen, newcastle disease HI antigen, infective bronchitis HI antigen (above-mentioned biomaterial is purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture), egg drop syndrome (EDSV-76) HI antigen, infections chicken cloacal bursa (IBD) agp antigen, mycoplasma gallisepticyum antigen, infectious laryngotracheitis of chicken (AILT) viral antigen (above-mentioned biomaterial is purchased from Beijing. China Veterinery Drug Inspection Office), 0.85% sodium chloride solution and negative allantoic fluid are tested by method (GICA) described in the embodiment of the present invention 2.When detection test card in kit of the present invention detects bird flu H5 hypotype HI antigen, bird flu H9 hypotype HI antigen, newcastle disease HI antigen, infective bronchitis HI antigen, all there is obvious aubergine band in the detection line of nature controlling line and correspondence, the detection line of non-corresponding then not displaing amaranth band (during as detected bird flu H5 hypotype HI antigen, there is aubergine band in nature controlling line and detection line T1, and band does not appear in detection line T2 and T3 simultaneously; And detect 3 kinds of above-mentioned antigens simultaneously, be nature controlling line and detection line T1, there is aubergine band in T2 and T3 simultaneously); And detect 0.85% sodium chloride solution (blank), negative allantoic fluid, egg drop syndrome (EDSV-76) HI antigen, infections chicken cloacal bursa (IBD) agp antigen, mycoplasma gallisepticyum antigen and avian infectious laryngotracheitis virus antigen, only there is aubergine band in nature controlling line.This shows that the test card specificity in kit of the present invention is high, shows with other important diseases antigen of fowl without any cross reaction.
Table 2 applies test card of the present invention to fowl standard antigen specific detection result
Note: "+" detection line develops the color, "-" represents that detection line does not develop the color.
2. sensitivity tests: doubling dilution after first 10 times being diluted respectively to bird flu H5 (H9) hypotype HI antigen, newcastle disease HI antigen, infective bronchitis HI antigen with 0.85% sodium chloride solution, final dilutability is 1: 10240, respectively gets the sample that 100-120 μ L diluted and tests by kit test card of the present invention.The results are shown in Table 3.When bird flu H5 (or H9) hypotype HI antigen doubling dilution is to 1:2560, when newcastle disease HI antigen doubling dilution is to 1:1280, infective bronchitis HI antigen doubling dilution is still positive to 1:640 test card, shows that the test card sensitivity in this kit is higher.Prepare AIV, the test card that the individual event of NDV and IBV tri-kinds of cause of diseases detects tests its susceptibility simultaneously, and result shows that the list of test card in kit of the present invention and three kinds of cause of diseases examines test card and indifference.
Table 3 test card of the present invention detects the sensitivity tests result of three kinds of standard antigens
1.2 kits of the present invention to the detection of submitted sample and with the comparing of virus purification detection method
Gather 118 parts of tracheal swab samples with kit of the present invention altogether to periphery chicken farm, Wuhan City, Hubei Province to have carried out detecting (preparation of sample is see embodiment 2), virus isolation and Identification is carried out to sample simultaneously, suppress (HI) to be detected as with blood clotting (HA) with blood clotting to contrast, wherein detect AIV see National Standard of the People's Republic of China GB/T18936-2003, detect NDV see National Standard of the People's Republic of China GB/T16550-2008, detect IBV see National Standard of the People's Republic of China GB/T23197-2008.Therebetween the testing result contrasted is in Table 4-6, and as can be seen from Table 4, when detecting AIVH5 (or H9) hypotype, the positive coincidence rate 70% that kit of the present invention and HA and HI detect and negative match-rate 90.7%, total coincidence rate is 92.3%.As can be seen from Table 5, when detecting NDV, the positive coincidence rate 76.5% that kit of the present invention and HA and HI detect and negative match-rate 91.7%, total coincidence rate is 96.2%.And by seeing in table 6, when detecting IBV, the positive coincidence rate 70.6% that kit of the present invention and HA and HI detect and negative match-rate 95.3%, total coincidence rate is 95.7%.Result shows that the testing result of kit of the present invention and HA and HI detection method is basically identical, and coincidence rate is better.
The clinical detection AIV result that table 4 kit of the present invention and blood clotting (HA) and blood clotting suppress (HI) to detect
The clinical detection NDV result that table 5 kit of the present invention and blood clotting (HA) and blood clotting suppress (HI) to detect
The clinical detection IBV result that table 6 kit of the present invention and blood clotting (HA) and blood clotting suppress (HI) to detect
Embodiment 4
The examination of fowl of the present invention three kinds of breathing problem three quick detection kit stability
By the kit test card of the present invention of Vacuum Package placed 4 DEG C and 37 DEG C respectively, took out at the 7th, 16,35,54,70,90 day, detect with after the dilution of bird flu H5 hypotype HI antigen, bird flu H9 hypotype HI antigen, newcastle disease HI antigen and infective bronchitis HI antigen 0.85% sodium chloride solution 30 times.Result is as table 7, and kit of the present invention is longer the storage life of 4 DEG C, and storage life is at least 90 days.
Storage life test findings under the different storage requirement of table 7 kit
Note: "+" detection line develops the color, "-" represents that detection line does not develop the color.
Although content of the present invention is described in conjunction with the present embodiment, can not think limitation of the scope of the invention, scope of the present invention is defined by the appended claims.In addition, those skilled in the art carries out various change or modification to the present invention in the scope that appended claims limits, and these are changed or modified forms drops in protection scope of the present invention equally
Claims (4)
1. detect three kits of the antigen of H_5 subtype and H9 hypotype, Avian pneumo-encephalitis virus and avian infectious bronchitis virus, it comprises box body, be located at test card in box body and sample diluting liquid; It is characterized in that: described test card is got stuck by test strips and test and forms; Test strips is pasted onto successively on PVC backing by sample pad, gold mark pad, nitrocellulose filter and absorption pad and forms; Described gold mark pad is coated with anti-avian influenza virus monoclonal antibody XYCSJL-AIV-2B8-colloid gold label thing, the potpourri of anti-new castle disease virus monoclonal antibody XYCDY-NDV-3E11-colloid gold label thing and anti-avian infectious bronchitis virus monoclonal antibody XYCDY-1BV-3G5-colloid gold label thing; Described nitrocellulose filter is coated with respectively the detection line T1 of anti-avian influenza virus monoclonal antibody XYCSJL-AIV-4B7, anti-new castle disease virus monoclonal antibody XYCDY-NDV-4B10 and anti-avian infectious bronchitis virus monoclonal antibody XYCDY-1BV-1E6 formation, the nature controlling line that T2, T3 and rabbit anti-mouse igg are formed;
Wherein:
The upper bag of gold mark pad is anti-avian influenza virus monoclonal antibody XYCSJL-AIV-2B8-colloid gold label thing, the potpourri of anti-new castle disease virus monoclonal antibody XYCDY-NDV-3E11-colloid gold label thing and anti-avian infectious bronchitis virus monoclonal antibody XYCDY-1BV-3G5-colloid gold label thing, wherein three kinds of monoclonal antibodies are respectively by anti-avian influenza virus hybridoma cell strain XYCSJL-AIV-2B8, secreted by anti-new castle disease virus hybridoma cell strain XYCDY-NDV-3E11 and anti-avian infectious bronchitis virus hybridoma cell strain XYCDY-1BV-3G5, these hybridoma cell strains are deposited in China typical culture collection center, deposit number is followed successively by CCTCCNO:C201449, CCTCCNO:C201451 and CCTCCNO:C201450,
It is anti-avian influenza virus monoclonal antibody XYCSJL-AIV-4B7 that described nitrocellulose filter wraps, anti-new castle disease virus monoclonal antibody XYCDY-NDV-4B10, anti-avian infectious bronchitis virus monoclonal antibody XYCDY-1BV-1E6 forms detection line T1, T2, three kinds of monoclonal antibodies of T3 are respectively by anti-avian influenza virus hybridoma cell strain XYCSJL-AIV-4B7, secreted by anti-new castle disease virus hybridoma cell strain XYCDY-NDV-4B10 and anti-avian infectious bronchitis virus hybridoma cell strain XYCDY-1BV-1E6, these hybridoma cell strains are deposited in China typical culture collection center, deposit number is followed successively by CCTCCNO:C201453, CCTCCNO:C201455 and CCTCCNO:C201454.
2. secrete a hybridoma cell strain XYCSJL-AIV-2B8 for anti-avian influenza virus monoclonal antibody, be deposited in China typical culture collection center, deposit number is CCTCCNO:C201449.
3. deposit number is the hybridoma cell strain XYCSJL-AIV-2B8 of the secretion anti-avian influenza virus monoclonal antibody of CCTCCNO:C201449, deposit number is the hybridoma cell strain XYCSJL-AIV-4B7 of the secretion anti-avian influenza virus monoclonal antibody of CCTCCNO:C201453, deposit number is the hybridoma cell strain XYCDY-NDV-3E11 of the secretion anti-new castle disease virus monoclonal antibody of CCTCCNO:C201451, deposit number is the hybridoma cell strain XYCDY-NDV-4B10 of the secretion anti-new castle disease virus monoclonal antibody of CCTCCNO:C201455, deposit number is that to secrete the hybridoma cell strain XYCDY-1BV-3G5 of anti-avian infectious bronchitis virus monoclonal antibody and deposit number be that hybridoma cell strain XYCDY-1BV-1E6 that CCTCCNO:C201454 secretes anti-avian infectious bronchitis virus monoclonal antibody is used in combination in preparation by same viral species and detects H_5 subtype and H9 hypotype to CCTCCNO:C201450, application in three kits of the antigen of Avian pneumo-encephalitis virus and avian infectious bronchitis virus.
4. the hybridoma cell strain XYCSJL-AIV-4B7 of deposit number to be the hybridoma cell strain XYCSJL-AIV-2B8 of the secretion anti-avian influenza virus monoclonal antibody of CCTCCNO:C201449 and deposit number the be secretion anti-avian influenza virus monoclonal antibody of CCTCCNO:C201453 detect H_5 subtype and H9 hypotype antigen in preparation monoclonal antibody in application.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410092170.7A CN104007261B (en) | 2014-03-13 | 2014-03-13 | Fowl three kinds of breathing problem three quick detection kit and application |
| CN201610051942.1A CN105567644A (en) | 2014-03-13 | 2014-03-13 | Monoclonal antibodies capable of resisting Newcastle disease virus |
| CN201610051163.1A CN105602907A (en) | 2014-03-13 | 2014-03-13 | Infectious bronchitis virus resistance monoclonal antibodies |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410092170.7A CN104007261B (en) | 2014-03-13 | 2014-03-13 | Fowl three kinds of breathing problem three quick detection kit and application |
Related Child Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610051163.1A Division CN105602907A (en) | 2014-03-13 | 2014-03-13 | Infectious bronchitis virus resistance monoclonal antibodies |
| CN201610051942.1A Division CN105567644A (en) | 2014-03-13 | 2014-03-13 | Monoclonal antibodies capable of resisting Newcastle disease virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104007261A CN104007261A (en) | 2014-08-27 |
| CN104007261B true CN104007261B (en) | 2016-04-06 |
Family
ID=51368015
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610051942.1A Pending CN105567644A (en) | 2014-03-13 | 2014-03-13 | Monoclonal antibodies capable of resisting Newcastle disease virus |
| CN201610051163.1A Pending CN105602907A (en) | 2014-03-13 | 2014-03-13 | Infectious bronchitis virus resistance monoclonal antibodies |
| CN201410092170.7A Active CN104007261B (en) | 2014-03-13 | 2014-03-13 | Fowl three kinds of breathing problem three quick detection kit and application |
Family Applications Before (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610051942.1A Pending CN105567644A (en) | 2014-03-13 | 2014-03-13 | Monoclonal antibodies capable of resisting Newcastle disease virus |
| CN201610051163.1A Pending CN105602907A (en) | 2014-03-13 | 2014-03-13 | Infectious bronchitis virus resistance monoclonal antibodies |
Country Status (1)
| Country | Link |
|---|---|
| CN (3) | CN105567644A (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104865384A (en) * | 2015-04-23 | 2015-08-26 | 江苏金标世纪生物科技有限公司 | Renal failure rapid detection triple kit and preparation method and application thereof |
| CN105675865B (en) * | 2016-03-10 | 2018-04-03 | 陕西瑞凯生物科技有限公司 | Domestic intelligent detection kit |
| CN106771176A (en) * | 2016-11-18 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of avian influenza virus antigen rapid detection card |
| CN108051591A (en) * | 2017-09-10 | 2018-05-18 | 华中农业大学 | A kind of bird flu H9 subtype virus antibody rapid quantitative detection reagent box and its application |
| CN107840884B (en) * | 2017-11-15 | 2020-12-08 | 郑州大学 | Nano antibody for resisting avian infectious bronchitis virus and preparation method thereof |
| CN107942062A (en) * | 2017-11-29 | 2018-04-20 | 洛阳现代生物技术研究院有限公司 | Test card, preparation and the detection method of synchronous detection 2 toxin of ochracin, vomitoxin and T |
| CN108732347A (en) * | 2018-05-23 | 2018-11-02 | 江苏维尔生物科技有限公司 | A kind of detection kit and preparation method thereof detecting HCV antibody in saliva |
| CN108840911B (en) * | 2018-06-09 | 2021-03-30 | 西北农林科技大学 | Epitope, antibody, identification method and application of newcastle disease virus matrix protein |
| CN110452885B (en) * | 2019-09-03 | 2022-11-04 | 江苏省农业科学院 | Hybridoma cell 4F6 strain secreting monoclonal antibody against newcastle disease virus NP protein |
| CN111077318A (en) * | 2019-12-31 | 2020-04-28 | 贵州省烟草科学研究院 | Rapid test card for simultaneously detecting TMV, PVY and TVBMV and preparation and use methods thereof |
| CN111707821A (en) * | 2020-07-28 | 2020-09-25 | 郑州大学 | Colloidal gold test paper for detecting IBV (infectious bronchitis Virus) and preparation method thereof |
| CN111896749A (en) * | 2020-08-07 | 2020-11-06 | 杭州都林生物科技有限公司 | Bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip, preparation method, kit and detection method |
| CN113063939A (en) * | 2021-03-05 | 2021-07-02 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) detection kit and detection method for infectious bronchitis viruses and application |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1300157B1 (en) * | 2001-10-04 | 2005-10-26 | Centrum voor Onderzoek in Diergeneeskunde en Agrochemie | Attenuated mutant newcastle disease virus strains for in ovo vaccination and their use |
| DE602007006471D1 (en) * | 2006-05-18 | 2010-06-24 | Veterinaermedizinische Uni Wie | DETECTION PROCEDURE FOR FLOAT VIRUSES |
| JP5300200B2 (en) * | 2006-08-31 | 2013-09-25 | 大阪府 | Rapid diagnostic method specific to avian influenza virus |
| CN101376677A (en) * | 2008-09-25 | 2009-03-04 | 山东省农业科学院畜牧兽医研究所 | Monoclonal antibody IE5 for detecting new castle disease virus variation strain |
| KR101211593B1 (en) * | 2010-09-06 | 2013-01-07 | 유한회사 바이오노트 | Diagnostic kit for H9 type avian influenza virus using rapid immunochromatography and the method for diagnosing H9 type avian influenza by using the same |
| CN202693594U (en) * | 2012-07-11 | 2013-01-23 | 郑州大学 | Colloidal gold test strip for detecting H5-subtype avian influenza virus antibodies, Newcastle disease virus antibodies and chicken bursal disease virus antibodies |
-
2014
- 2014-03-13 CN CN201610051942.1A patent/CN105567644A/en active Pending
- 2014-03-13 CN CN201610051163.1A patent/CN105602907A/en active Pending
- 2014-03-13 CN CN201410092170.7A patent/CN104007261B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN105602907A (en) | 2016-05-25 |
| CN105567644A (en) | 2016-05-11 |
| CN104007261A (en) | 2014-08-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104007261B (en) | Fowl three kinds of breathing problem three quick detection kit and application | |
| CN105950563B (en) | The monoclonal antibody and application of hybridoma cell strain 7E3 and its resistant to foot and mouth disease A type virus of secretion | |
| CN104593332A (en) | Kit and method for detecting Tilletia controversa Kuhn | |
| CN101130765B (en) | Reagent kit for detecting syncytial virus of respiratory passage | |
| CN109187967B (en) | Duplex rapid detection card for detecting and distinguishing O-type and A-type foot-and-mouth disease viruses and preparation method thereof | |
| CN105542014A (en) | TP recombinant antigen and preparing method and application thereof | |
| CN106226518A (en) | Canine distemper virus colloidal gold immunochromatographydetection detection test paper bar and preparation method thereof | |
| CN107365364A (en) | A kind of quick detection kit of Adenovirus Antigen preparation method and the detection adenovirus antibody prepared using the antigen | |
| CN103134927B (en) | For detecting method and the kit of Shigella | |
| CN100526452C (en) | Colloidal gold immune chromatography fast differential diagnosis kit of chicken avian influenza vaccine immunity and virus strain infection and application | |
| CN106636004A (en) | TMV-CMV-PVY three-virus (tobacco mosaic virus, cucumber mosaic virus and potato virus) colloidal gold rapid test strip | |
| CN106596931B (en) | A kind of Tobacco-growing areas in Guangdong's cucumber mosaic virus colloidal gold fast detecting test paper strip | |
| CN111505289A (en) | Peste des petits ruminants detection kit | |
| CN101833003A (en) | Vesicular stomatitis virus antibody colloidal goldtest paper tape and preparation method thereof | |
| CN102033128B (en) | Edwardsiella tarda rapid detection test paper as well as rapid detection method and application | |
| CN104744590B (en) | Anti-H 1 N 1 swine influenza virus hemagglutinin monoclonal antibody and double crush syndrome kit | |
| CN105585633B (en) | The immune chromatography reagent kit of anti-human haemophilus influenzae P6 protein antibodies and the application antibody | |
| CN105753982B (en) | The immune chromatography reagent kit of anti-human streptococcus pneumonia fam1 family PspA protein antibodies and the application antibody | |
| CN109867722A (en) | AIV H5 hypotype monoclonal antibody, hybridoma and chromatograph test strip | |
| CN104459119B (en) | A kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips and preparation method thereof | |
| CN105277694B (en) | Human group A streptococci quantum dot immunochromatography detection card, preparation method and applications | |
| CN202720233U (en) | Test strip for detecting tylosin and tilmicosin | |
| CN106279408A (en) | The monoclonal antibody of resistant to foot and mouth disease O type virus and Antibody Combination and its application in this virus antigen, antibody test | |
| CN110058016A (en) | Birds H9 subtype avian influenza antibody colloidal gold Test paper and test card | |
| CN105585634B (en) | The immune chromatography reagent kit of anti-human streptococcus pneumonia fam2 family PspA protein antibodies and the application antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |