CN104062430B - A kind of kit for detecting influenza virus in sample and detection method thereof and application - Google Patents
A kind of kit for detecting influenza virus in sample and detection method thereof and application Download PDFInfo
- Publication number
- CN104062430B CN104062430B CN201410308507.3A CN201410308507A CN104062430B CN 104062430 B CN104062430 B CN 104062430B CN 201410308507 A CN201410308507 A CN 201410308507A CN 104062430 B CN104062430 B CN 104062430B
- Authority
- CN
- China
- Prior art keywords
- sample
- monoclonal antibody
- kit
- damping fluid
- influenza
- 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 81
- 241000712461 unidentified influenza virus Species 0.000 title claims abstract description 48
- 102000004190 Enzymes Human genes 0.000 claims abstract description 61
- 108090000790 Enzymes Proteins 0.000 claims abstract description 61
- 239000000758 substrate Substances 0.000 claims abstract description 56
- 239000012530 fluid Substances 0.000 claims abstract description 55
- 238000012360 testing method Methods 0.000 claims abstract description 53
- 238000013016 damping Methods 0.000 claims abstract description 42
- 208000037797 influenza A Diseases 0.000 claims abstract description 40
- 108010061100 Nucleoproteins Proteins 0.000 claims abstract description 37
- 102000011931 Nucleoproteins Human genes 0.000 claims abstract description 37
- 238000000034 method Methods 0.000 claims abstract description 36
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 30
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 30
- 238000002372 labelling Methods 0.000 claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- 241000700605 Viruses Species 0.000 claims description 33
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 32
- 239000007788 liquid Substances 0.000 claims description 26
- 206010022000 influenza Diseases 0.000 claims description 24
- 239000007853 buffer solution Substances 0.000 claims description 19
- 239000008363 phosphate buffer Substances 0.000 claims description 18
- 238000012545 processing Methods 0.000 claims description 13
- 230000008569 process Effects 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 230000000274 adsorptive effect Effects 0.000 claims description 11
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 10
- 241001494479 Pecora Species 0.000 claims description 7
- 239000004094 surface-active agent Substances 0.000 claims description 7
- 238000011282 treatment Methods 0.000 claims description 6
- 238000013508 migration Methods 0.000 claims description 5
- 230000005012 migration Effects 0.000 claims description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 4
- 241000272165 Charadriidae Species 0.000 claims description 4
- -1 neopelex Chemical compound 0.000 claims description 4
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 claims description 3
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 3
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 3
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims description 2
- 235000021355 Stearic acid Nutrition 0.000 claims description 2
- 230000002421 anti-septic effect Effects 0.000 claims description 2
- 150000001720 carbohydrates Chemical class 0.000 claims description 2
- 125000005456 glyceride group Chemical group 0.000 claims description 2
- 239000000787 lecithin Substances 0.000 claims description 2
- 235000010445 lecithin Nutrition 0.000 claims description 2
- 229940067606 lecithin Drugs 0.000 claims description 2
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 claims description 2
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 claims description 2
- 239000008117 stearic acid Substances 0.000 claims description 2
- 230000008901 benefit Effects 0.000 abstract description 5
- 238000004587 chromatography analysis Methods 0.000 description 22
- 241000699666 Mus <mouse, genus> Species 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 15
- 230000035945 sensitivity Effects 0.000 description 13
- 244000005700 microbiome Species 0.000 description 11
- 210000003800 pharynx Anatomy 0.000 description 10
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 241000287828 Gallus gallus Species 0.000 description 8
- 206010064097 avian influenza Diseases 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 7
- 238000011081 inoculation Methods 0.000 description 7
- 230000009182 swimming Effects 0.000 description 7
- 230000003612 virological effect Effects 0.000 description 7
- 241000282465 Canis Species 0.000 description 6
- 241000282898 Sus scrofa Species 0.000 description 6
- 241000725681 Swine influenza virus Species 0.000 description 6
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 6
- 238000012797 qualification Methods 0.000 description 6
- 241000271566 Aves Species 0.000 description 5
- 241000283073 Equus caballus Species 0.000 description 5
- 241001529936 Murinae Species 0.000 description 5
- 230000027455 binding Effects 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 210000003128 head Anatomy 0.000 description 5
- 230000002458 infectious effect Effects 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000003757 reverse transcription PCR Methods 0.000 description 5
- 229960005486 vaccine Drugs 0.000 description 5
- 241000282472 Canis lupus familiaris Species 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 210000004408 hybridoma Anatomy 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 230000009870 specific binding Effects 0.000 description 4
- 238000011895 specific detection Methods 0.000 description 4
- 230000006641 stabilisation Effects 0.000 description 4
- 238000011105 stabilization Methods 0.000 description 4
- 206010003445 Ascites Diseases 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 241000712431 Influenza A virus Species 0.000 description 3
- 208000002979 Influenza in Birds Diseases 0.000 description 3
- 241000158526 Nasalis Species 0.000 description 3
- 206010029719 Nonspecific reaction Diseases 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 3
- 239000005030 aluminium foil Substances 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000011091 antibody purification Methods 0.000 description 3
- 210000001669 bursa of fabricius Anatomy 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 210000003555 cloaca Anatomy 0.000 description 3
- 206010014599 encephalitis Diseases 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000036512 infertility Effects 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000012925 reference material Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 241001473385 H5N1 subtype Species 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 241001597008 Nomeidae Species 0.000 description 2
- 102000007999 Nuclear Proteins Human genes 0.000 description 2
- 108010089610 Nuclear Proteins Proteins 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- 206010044302 Tracheitis Diseases 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 201000009837 laryngotracheitis Diseases 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 239000006916 nutrient agar Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920000573 polyethylene Polymers 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 201000010740 swine influenza Diseases 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 101000621943 Acholeplasma phage L2 Probable integrase/recombinase Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 101000618348 Allochromatium vinosum (strain ATCC 17899 / DSM 180 / NBRC 103801 / NCIMB 10441 / D) Uncharacterized protein Alvin_0065 Proteins 0.000 description 1
- 101000781117 Autographa californica nuclear polyhedrosis virus Uncharacterized 12.4 kDa protein in CTL-LEF2 intergenic region Proteins 0.000 description 1
- 101000708323 Azospirillum brasilense Uncharacterized 28.8 kDa protein in nifR3-like 5'region Proteins 0.000 description 1
- 101000770311 Azotobacter chroococcum mcd 1 Uncharacterized 19.8 kDa protein in nifW 5'region Proteins 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 101000748761 Bacillus subtilis (strain 168) Uncharacterized MFS-type transporter YcxA Proteins 0.000 description 1
- 101000765620 Bacillus subtilis (strain 168) Uncharacterized protein YlxP Proteins 0.000 description 1
- 101000916134 Bacillus subtilis (strain 168) Uncharacterized protein YqxJ Proteins 0.000 description 1
- 101000754349 Bordetella pertussis (strain Tohama I / ATCC BAA-589 / NCTC 13251) UPF0065 protein BP0148 Proteins 0.000 description 1
- 101000827633 Caldicellulosiruptor sp. (strain Rt8B.4) Uncharacterized 23.9 kDa protein in xynA 3'region Proteins 0.000 description 1
- 241000251204 Chimaeridae Species 0.000 description 1
- 101000947628 Claviceps purpurea Uncharacterized 11.8 kDa protein Proteins 0.000 description 1
- 101000686796 Clostridium perfringens Replication protein Proteins 0.000 description 1
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 1
- 206010051625 Conjunctival hyperaemia Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 101000788129 Escherichia coli Uncharacterized protein in sul1 3'region Proteins 0.000 description 1
- 101000788370 Escherichia phage P2 Uncharacterized 12.9 kDa protein in GpA 3'region Proteins 0.000 description 1
- 241000701063 Gallid alphaherpesvirus 1 Species 0.000 description 1
- 101000787096 Geobacillus stearothermophilus Uncharacterized protein in gldA 3'region Proteins 0.000 description 1
- 108010056741 H1N1 virus hemagglutinin Proteins 0.000 description 1
- 101000976889 Haemophilus phage HP1 (strain HP1c1) Uncharacterized 19.2 kDa protein in cox-rep intergenic region Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 101900222562 Influenza A virus Nucleoprotein Proteins 0.000 description 1
- 101000827627 Klebsiella pneumoniae Putative low molecular weight protein-tyrosine-phosphatase Proteins 0.000 description 1
- 101001130841 Middle East respiratory syndrome-related coronavirus (isolate United Kingdom/H123990006/2012) Non-structural protein ORF5 Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 229920002535 Polyethylene Glycol 1500 Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 206010039101 Rhinorrhoea Diseases 0.000 description 1
- 101000974028 Rhizobium leguminosarum bv. viciae (strain 3841) Putative cystathionine beta-lyase Proteins 0.000 description 1
- 101000756519 Rhodobacter capsulatus (strain ATCC BAA-309 / NBRC 16581 / SB1003) Uncharacterized protein RCAP_rcc00048 Proteins 0.000 description 1
- 101000948219 Rhodococcus erythropolis Uncharacterized 11.5 kDa protein in thcD 3'region Proteins 0.000 description 1
- 101000936711 Streptococcus gordonii Accessory secretory protein Asp4 Proteins 0.000 description 1
- 101000929863 Streptomyces cinnamonensis Monensin polyketide synthase putative ketoacyl reductase Proteins 0.000 description 1
- 101000788468 Streptomyces coelicolor Uncharacterized protein in mprR 3'region Proteins 0.000 description 1
- 101000845085 Streptomyces violaceoruber Granaticin polyketide synthase putative ketoacyl reductase 1 Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 101000711771 Thiocystis violacea Uncharacterized 76.5 kDa protein in phbC 3'region Proteins 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 101000711318 Vibrio alginolyticus Uncharacterized 11.6 kDa protein in scrR 3'region Proteins 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 210000001728 clone cell Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 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 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 208000021760 high fever Diseases 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 208000010753 nasal discharge Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000003024 peritoneal macrophage Anatomy 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000036387 respiratory rate Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- VUYXVWGKCKTUMF-UHFFFAOYSA-N tetratriacontaethylene glycol monomethyl ether Chemical compound COCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO VUYXVWGKCKTUMF-UHFFFAOYSA-N 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
Classifications
-
- 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
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- 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
Abstract
The invention provides a kind of kit for detecting influenza virus in sample, wherein, this kit comprises damping fluid feeding unit and test strip; This test strip comprises substrate supply area in the vertical successively, sample supply area, detection zone; This substrate supply area comprises substrate pad (3), and it is adsorbed with dry zymolyte, this substrate pad (3) contacts with nitrocellulose filter (1); This sample supply area comprises enzyme mark pad (2), it is adsorbed with mouse-anti influenza A nucleoprotein antibody labeling monoclonal antibody 1, and this zymolyte can produce chromogenic reaction with the enzyme that marks on this labeled monoclonal antibody 1; And have mouse-anti influenza A nucleoprotein antibody immobilized monoclonal antibody 2 in this detection zone immobilization.Present invention also offers the method using this kit to detect influenza virus in sample.Kit of the present invention and detection method thereof have fast, accurately, the advantage of high specificity, sensitive, good stability.
Description
Technical field
The invention belongs to biological technical field.
Background technology
Influenza A comprises avian influenza virus (AvianInfluenzaVitus, AIV), swine influenza virus (SwineInfluenzaVitus, SIV), equine influenza virus (EquineInfluenzaVitus, and canine influenza virus (CanineInfluenzaVitus EIV), CIV) etc., OIE (OfficeofInternationalEducation, OIE) disease caused by influenza A is classified as category-A animal epidemic, China is classified as a class animal epidemic.
Wherein, avian influenza virus can cause bird flu, and bird flu is a kind of hyperinfection disease, and be pathogenicly divided into highly pathogenic bird flu and low pathogenicity bird flu according to it, highly pathogenic bird flu provisions fowl industry brings crushing blow especially; Swine influenza virus can cause swine flu, and even cause pig dead, it is healthy that swine flu not only endangers pig body, affects the Time To Market of growing and fattening pigs, also add feeding cost, cause the loss that can not be ignored to pig industry; Equine influenza virus can cause equine influenza, and make horse breathing, the increase of pulse frequency, appetite reduces, and spirit is tired, and conjunctival congestion oedema, sheds tears in a large number, often occurs muscular tremor at fb dur, also add and raises and train cost; Canine influenza virus causes dog influenza, shows as persistent cough, with the nasal discharge of yellow, there will be the symptom of the similar pneumonia such as high fever, respiratory rate quickening, also cause tremendous influence to public health time serious.
In the preventing and controlling of influenza A, diagnosis, monitoring are vital links.At present, influenza A detection method mainly comprises nucleic acid detection method and collaurum method for quick.Though nucleic acid detection method specificity is good, highly sensitive, but it detects needs expensive PCR instrument and technical professional, and can only at the labs possessing detection of nucleic acids ability, and most basic unit clinical condition does not possess the ability of detection of nucleic acids, the sample gathered needs to deliver to the laboratory possessing detectability and detects, lack ageing, be unfavorable for the control of epidemic situation, also limit its application in extensive examination.On the quick detection that the advantage such as collaurum quick detection reagent is quick with it, accurate, easy, naked eyes interpretation, experimental result are easily preserved is widely used in influenza A and field diagnostic, but its sensitivity is lower.Therefore, quick, accurate, highly sensitive, easy A type influenza test method is the emphasis of current research.
Summary of the invention
In order to realize influenza A quick, easy, detect and detect the lower problem of medium sensitivity exactly, the invention provides the enzyme chromatography detection kit of two kinds of anti-influenza type A virus nucleoprotein monoclonal antibodies and preparation thereof, this kit not only has higher sensitivity than the colloidal gold fast detecting test paper strip of routine, and detect quick, easy and simple to handle, be applicable to the quick detection of influenza A.
Fundamental purpose of the present invention is to provide a kind of kit for detecting influenza A in sample, and wherein, described kit comprises damping fluid feeding unit and test strip; Described damping fluid feeding unit is used for damping fluid to supply described test strip; Described test strip comprises nitrocellulose filter (1), and described test strip comprises substrate supply area in the vertical successively, sample supply area, detection zone; Described substrate supply area comprises substrate pad (3), it is adsorbed with dry zymolyte, described substrate pad (3) contacts with nitrocellulose filter (1), and described zymolyte to be dissolved in damping fluid and to the distal migration apart from described damping fluid feeding unit on nitrocellulose filter (1); Described sample supply area comprises enzyme mark pad (2), it is adsorbed with mouse-anti influenza A nucleoprotein antibody labeling monoclonal antibody 1, described zymolyte can produce chromogenic reaction with the enzyme that marks on described labeled monoclonal antibody 1, described enzyme mark pad (2) contacts with nitrocellulose filter (1), and described labeled monoclonal antibody 1 to be dissolved in damping fluid and to the distal migration apart from described damping fluid feeding unit on nitrocellulose filter (1); And the immobilization of described detection zone has mouse-anti influenza A nucleoprotein antibody immobilized monoclonal antibody 2.
Preferably, described damping fluid feeding unit comprises expansion fluid cushion (5), substrate buffer liquid bath (8), substrate buffer solution (9) and damping fluid button, described substrate buffer solution (9) is placed in substrate buffer liquid bath (8), described damping fluid button is positioned at the top of substrate buffer solution groove (8), presses and can immerse in damping fluid (9) by described expansion fluid cushion (5); Described detection zone comprises detection line (6), nature controlling line (7), wherein, described nature controlling line (7) comparatively detection line (6) further from described sample supply area, mouse-anti influenza A nucleoprotein antibody immobilized monoclonal antibody 2 is had in described detection line (6) immobilization, have sheep anti mouse to resist in described nature controlling line (7) immobilization, and the labeled monoclonal antibody 1 be adsorbed on enzyme mark pad (2) is excessive for being fixed on the immobilized monoclonal antibody 2 of detection line (6) more; The full section of described nitrocellulose filter (1) sticks on above support (10), stilt (10) connects described damping fluid feeding unit, substrate supply area, sample supply area, detection zone and adsorptive pads (4), described adsorptive pads (4) is in the distalmost end apart from described damping fluid feeding unit; And detect the position that sample (11) position that adds is described enzyme mark pad (2).
As one embodiment of the present invention, described enzyme chromatography test strip comprise solid phase nitrocellulose filter 1, enzyme mark pad 2 containing labelled reagent, substrate pad 3, adsorptive pads 4, launch fluid cushion 5, detection line 6, nature controlling line 7, substrate buffer liquid bath 8, substrate buffer solution 9, stilt 10, wherein 2 belong to sample supply area, 3 belong to substrate supply area, 5,8 belong to damping fluid feeding unit, and 6,7 belong to detection zone.The position that detection sample 11 adds is the position of enzyme mark pad 2.Enzyme chromatography test strip is fixing in a plastic housing, it is from left to right fixed successively and launches fluid cushion 5, substrate pad 3, enzyme mark pad 2, adsorptive pads 4.Nitrocellulose filter 1 sticks to the full section of stilt 10; Adsorptive pads 4 is stuck in the top of nitrocellulose filter 1, and has overlapping with nitrocellulose filter 1; Enzyme mark pad 2 is positioned at the stage casing of nitrocellulose filter 1, above the dry mouse-anti influenza A nucleoprotein antibody 1 having enzyme labeling; Substrate pad 3 is stuck in the bottom of nitrocellulose filter 1, it has dry zymolyte.The upper end of launching fluid cushion 5 covers substrate pad 3, and its lower end is positioned at the bottom of substrate buffer solution groove 8.Cover one deck aluminium-foil paper above substrate buffer liquid bath 8, to prevent substrate buffer solution 9 seepage, it has damping fluid button, press damping fluid button and can puncture aluminium-foil paper, and the lower end of launching fluid cushion 5 is immersed in substrate buffer solution 9.Detection line 6 is arranged in the upper end of nitrocellulose filter 1, and on it, immobilization has mouse-anti influenza A nucleoprotein antibody 2.Nature controlling line 7 is arranged in the upper end of nitrocellulose filter 1, the upstream of detection line 6, and on it, immobilization has sheep anti mouse to resist more.
Preferably, the amino acid sequence of described its variable region of heavy chain of labeled monoclonal antibody 1 is SEQIDNo.2, the amino acid sequence of variable region of light chain is SEQIDNo.3; And the amino acid sequence of described its variable region of heavy chain of immobilized monoclonal antibody 2 be SEQIDNo.4, the amino acid sequence of variable region of light chain is SEQIDNo.5.
Term used herein " monoclonal antibody " refers to the antibody available from the antibody population of homology substantially, and the antibody individuality namely forming this colony is all identical, except there is spontaneous mutation possible on a small quantity.Therefore, modifier " monoclonal " refers to that the character of this antibody is not the potpourri of discrete antibody.Preferably, that described monoclonal antibody comprises unit price or the derivant of single-chain antibody, double-chain antibody, chimeric antibody, humanized antibody and above-mentioned antibody, function equivalent and homologue, also comprise antibody fragment and any polypeptide containing antigen-binding domains.
" antibody " of the present invention should be interpreted as containing any Specific binding members with required specific binding structural domain.Thus, this term covers function equivalent and the homologue of the antibody fragment of homology with it, derivant, humanized antibody and antibody, also comprises any polypeptide containing antigen-binding domains, no matter is natural or synthesis produces.The example of antibody is immunoglobulin (Ig) hypotype (as IgG, IgE, IgM, IgD and IgA) and isotype sub-classes thereof; Also can be that the fragment comprising antigen-binding domains is as Fab, scFv, Fv, dAb, Fd; With double-strand anti-(diabodies).Merge to another polypeptide, the chimer molecules that comprises antigen-binding domains or equivalent be also included within wherein.The cloning and expression of chimeric antibody describes in EP.A.0120694 and EP.A.0125023.
" antibody " of the present invention can be modified by many modes, can produce retain original antibody other antibody specific or chimeric molecule with DNA recombinant technique.This technology can comprise the constant region of the DNA of the immune globulin variable region of encoding antibody or complementarity-determining region (CDRs) being introduced different immunoglobulin (Ig) or constant region adds framework region.See, EP.A.184187, GB2188638A or EP.A.239400.Can also carry out genetic mutation or other change to other cell of hybridoma or generation antibody, this can change or not change the binding specificity of produced antibody.
For " monoclonal antibody " of the present invention, also using hybridoma method is obtained, because the DNA sequence dna of code book invention humanized antibody can use conventional means well known to those skilled in the art, as obtained according to amino acid sequence Prof. Du Yucang disclosed by the invention or with the amplification of PCR method, thus also can use recombinant DNA method, this sequence is connected in suitable expression vector by available various method well known in the art.Finally, under the condition of applicable antibody expression of the present invention, cultivate the host cell transforming gained, then those skilled in the art apply the conventional separation and purification means purifying known and obtain monoclonal antibody of the present invention.
" variable region " of term used herein heavy chain of antibody or light chain is that the N of this chain holds ripe region.All regions, CDRs and residue numbering are all by sequence alignment, define by based on existing structure knowledge.The qualification of framework region and CDRs residue and numbering press Chothia and (Chothia described in other people, Structuraldeterminantsinthesequencesofimmunoglob μ linvariabledomain.J.Mol.Biol., 1998:278,457).
Those of ordinary skill in the art obviously know, in the present invention in the heavy chain of concrete disclosed monoclonal antibody and chain variable region amino acid sequence basis, the modifications such as one or more amino acid whose interpolation, deletion, replacement can be carried out by conventional gene engineering and protein engineering method, obtain conservative variant or its fragment, and still can keep and influenza A specific binding.
Term used herein " nucleoprotein " (NuclearProtein, NP) is a kind of polypeptide of monomer phosphorylation, and molecular weight is about 60kD, is the primary protein component forming virus nucleocapsid; This albumen has specificity, according to its antigenic difference, influenza virus is divided into A, B, C three types (animal medicine is in progress for Liu Hongqi etc., influenza A albumen progress, 2002,23 (2): 6-9).Described nucleoprotein is also its conservative variant or active fragment, compared with influenza A ORF5 encoding nuclear proteins amino acid sequence SEQIDNO.1 of the present invention, one or more conservative amino acid can be there is and replace, add or delete but still can keep and influenza A specific binding in described " its conservative variant or active fragment ".
Preferably, the enzyme of described mark comprises any one of horseradish peroxidase, alkaline phosphatase and beta-D-galactosidase.
Preferably, in described solid phase, anti-influenza type A virus nucleoprotein monoclonal antibody is that the amino acid sequence of variable region of heavy chain is SEQIDNo.4, the amino acid sequence of variable region of light chain is the monoclonal antibody of SEQIDNo.5, and in described labelled reagent, anti-influenza type A virus nucleoprotein monoclonal antibody is that the amino acid sequence of variable region of heavy chain is SEQIDNo.2, the amino acid sequence of variable region of light chain is the monoclonal antibody of SEQIDNo.3.Described labelled reagent obtains by enzyme labeling; Preferably, described enzyme comprises any one in horseradish peroxidase (HRP), alkaline phosphatase (ALP) and beta-D-galactosidase.
Preferably, described substrate buffer solution is aqueous solvent, comprises phosphate buffer, surfactant, antiseptic and carbohydrate one or more.
Preferably, described kit also comprises sample processing tube, described sample processing tube contains sample treatment liquid, and described sample treatment liquid is surfactant solution, and described surfactant comprises any one of stearic acid, neopelex, lecithin, fatty glyceride and CHAPS.
The material that term used herein " surfactant " can make target solution surface tension significantly decline, can reduce the surface tension between two kinds of liquid or liquid-solid.
Preferably, described kit also comprises Sample storage bottle, and described Sample storage bottle contains Sample storage liquid, and described Sample storage liquid is phosphate buffer, is preferably the phosphate buffer of pH7.4.
Term used herein " phosphate buffer " refers to containing phosphoric acid or its salt and is adjusted to the solution of desired pH, is the most widely used a kind of damping fluid in biochemical research.Usually, phosphate buffer is prepared from phosphoric acid or phosphate (including but not limited to sodium and sylvite).Some phosphate have been known in this area, such as sodium dihydrogen phosphate and potassium dihydrogen phosphate, sodium hydrogen phosphate and dipotassium hydrogen phosphate, sodium phosphate and potassium phosphate.Know that phosphate exists with the hydrate forms of salt.Due to the secondary dissociation of damping fluid, the pH value range of buffering is very wide, the scope of such as about pH4 to about pH10, and the preferably scope of about pH5 to pH9 more has the scope selecting about pH6 to about pH8, most preferably from about pH7.4.Further preferably, described phosphate buffer is the phosphate buffer of sodium chloride-containing and potassium chloride.
Another object of the present invention is to provide a kind of method using described kit to detect influenza virus in sample, described method comprises: (1) uses described sample processing tube pretreatment sample; (2) pretreatment sample in described step (1) is added the position of described enzyme mark pad 2; (3) damping fluid button is pressed, observations; And (4) are if nature controlling line place there is no band, no matter whether detection line place has band, and testing process is all invalid, when there is band at nature controlling line place, if there is band at detection line place, is then positive, otherwise is negative.
As one embodiment of the present invention, the detection method of influenza A enzyme chromatography of the present invention, for: the microorganism swab gathered is inserted in sample processing tube by (1), and sample is dissolved in the solution as far as possible, finally makes microorganism swab head fracture and stays with bottle; (2) the sample processing tube lid head containing sample is fractureed, extrude a sample and add well, press rapidly damping fluid button simultaneously; In the detection zone observed and recorded result of described test strip after (3) 30 minutes.
Another object of the present invention is to provide described kit detecting the application in sample in influenza virus.
Present invention also offers a kind of anti-influenza type A virus nucleoprotein monoclonal antibody 1, the amino acid sequence of described its variable region of heavy chain of monoclonal antibody 1 is SEQIDNo.2, the amino acid sequence of variable region of light chain is SEQIDNo.3.
Present invention also offers a kind of anti-influenza type A virus nucleoprotein monoclonal antibody 2, the amino acid sequence of described its variable region of heavy chain of monoclonal antibody 2 is SEQIDNo.4, the amino acid sequence of variable region of light chain is SEQIDNo.5.
Based on this, outstanding advantages of the present invention is:
(1) enzyme chromatography test strip completes ELISA testing process on nitrocellulose filter, achieve enzyme linked immunological Cascaded amplification effect, therefore its sensitivity can reach ELISA level, higher than colloidal gold strip more than 10 times, suitable with PCR detection sensitivity in the detection of some cause of disease;
(2) there is the feature that result is simple, easy to use, detection efficiency is high.
Accompanying drawing explanation
Fig. 1 is the side schematic view of enzyme chromatography test strip, in figure: 1-nitrocellulose filter, 2-enzyme mark pad, 3-substrate pad, 4-adsorptive pads, 5-launches fluid cushion, 6-detection line, 7-nature controlling line, 8-substrate buffer liquid bath, 9-substrate buffer solution, 10-stilt, 11-detects sample.
Fig. 2 is the purity that 2 strain anti-influenza type A virus nucleoprotein monoclonal antibodies are identified in SDS-PAGE gel electrophoresis, wherein swimming lane 1 is monoclonal antibody 2, comprises the heavy chain of upper end and the light chain of lower end, and swimming lane 2 is monoclonal antibody 1, comprise the heavy chain of upper end and the light chain of lower end, swimming lane 3 is albumen Maker.
in sequence table:
Sequence 1 is the amino acid sequence of influenza A nucleoprotein;
Sequence 2 is the heavy chain variable amino acid sequence of the anti-influenza type A virus nucleoprotein monoclonal antibody 1 of mark;
Sequence 3 is the chain variable region amino acid sequence of the anti-influenza type A virus nucleoprotein monoclonal antibody 1 of mark;
Sequence 4 is the heavy chain variable amino acid sequence of fixing anti-influenza type A virus nucleoprotein monoclonal antibody 2;
Sequence 5 is the chain variable region amino acid sequence of fixing anti-influenza type A virus nucleoprotein monoclonal antibody 2.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Carbonate buffer solution used in the embodiment of the present invention, pH value is 9.6, and its 1L volume formula is: Na
2cO
31.59g, NaHCO
32.93g, but no matter this embodiment does not under any circumstance all form limitation of the invention.
Sample storage liquid used in example of the present invention is phosphate buffer, and pH value is 7.4, and its 1L volume formula is: NaC18.0g, KCl0.2g, Na
2hPO
412H
2o2.9g, KH
2pO
40.24g, final concentration is the kanamycins of the penicillin of 10000IU/mL, the streptomysin of 8000IU/mL and 5000IU/mL, but no matter this embodiment does not under any circumstance all form limitation of the invention.
Enzyme labelled antibody in the embodiment of the present invention marks for horseradish peroxidase (HRP), but no matter this embodiment does not under any circumstance all form limitation of the invention.
In the embodiment of the present invention, A type avian flu strain A/Ck/HK/Yu22/02 strain is open in patented claim CN1814623A.
In the embodiment of the present invention, A type avian flu strain A/California/04/2009 strain is open in patented claim CN101974489A.
In the embodiment of the present invention, Type B strains of influenza viruses VR788 strain is open in patented claim CN102337351A.
It is pure that chemical reagent used in the present invention is analysis, purchased from traditional Chinese medicines group.
The criterion of influenza A enzyme chromatography used in this example is: whether effectively the presence or absence of nature controlling line determines testing process, if nature controlling line place there is no band, no matter whether detection line place has band, and testing process is all invalid; When there is band at nature controlling line place, if there is band at detection line place, be then positive, otherwise be negative.
For making the present invention easier to understand, below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for the present invention to limit the scope of the invention.Experimental technique of the present invention, if without specified otherwise, is conventional method; Described biomaterial, if without specified otherwise, all can obtain from commercial channels.
The preparation of embodiment 1 anti-influenza type A virus nucleoprotein monoclonal antibody, purifying, qualification and inspection
The preparation of 1.1 anti-influenza type A virus nucleoprotein monoclonal antibodies and purifying
The preparation of anti-influenza type A virus nucleoprotein monoclonal antibody and purifying, comprise the steps:
1.1.1 animal immune: the inactivation of viruses 1mL of influenza A A/California/04/2009 (H1N1) strain is added equivalent complete Freund's adjuvant and through fully emulsified, the 6-8 BALB/c healthy mice in age in week of myeloma cell's homology immune and used, influenza A A/California/04/2009 (H1N1) strain 500 μ L after every multi-point injection emulsification, interval is strengthened once for 2 weeks; Its antiserum is measured with indirect ELISA:
Adopt indirect ELISA to measure antiserum after carrying out, the method is made up of following operation steps:
A, bag quilt:
Diluted by influenza A nucleoprotein 1:2000 with the carbonate buffer solution of 20mmol/L, pH9.6, wrap by 96 hole polyethylene boards, vacuum is drained, and seals 4 DEG C and saves backup.
B, close:
The phosphate buffer 200 μ L that every hole adds pH7.4 washs, includes 10% (V/V) calf serum;
C, application of sample:
Every hole adds the clear 50 μ L of mouse peripheral blood that the third time immunity 1:5000 (V/V) of latter 3 days dilutes, and every plate establishes a normal control, positive control and phosphate buffer blank, washing;
D, add enzyme mark sheep anti mouse two and resist every hole 100 μ L, washing;
E, colour developing:
Every hole adds substrate 100 μ L;
F, colorimetric:
With blank zeroing, 450nm wavelength measures optical density (OD);
G, result judge: P/N=measures sample OD average/negative serum OD average, and P/N >=2.1 are positive.
Polyethylene board specification in step a is 200 μ L/ holes, to drain temperature be 4 DEG C to vacuum, and the Tween-20 phosphate buffer of washing employing 0.05% washs 3 times.
Technological parameter in step b is that every hole adds pH7.4, containing 10% calf serum phosphate buffer 200 μ L, temperature is 37 DEG C, time 2 h, washs 1 time.
Process conditions in step c are the third time immunity clear 50 μ L of mouse peripheral blood of latter 3 days after every hole adds 1:5000 (V/V) dilution, every plate establishes a normal control, positive control and phosphate buffer blank, temperature is 37 DEG C, the time is 1 hour, washs 3 times.
Process conditions in steps d add enzyme mark sheep anti mouse two and resist every hole 100 μ L, and temperature is 37 DEG C, the time is 1 hour, washs 3 times.
Process conditions in step e are room temperature, the time is 15 minutes, then use stop buffer cessation reaction, through microplate reader in wavelength 450nm place reading optical density value.
1.1.2 separating Morr. cell: get non-immune BALB/c healthy mice peritoneal macrophage, spread 96 well culture plates, is adjusted to 9 × 10 by the Sp2/0 cell of 15 hours after changing liquid
5/ mL cell suspension, the mouse boosting cell of separating immune also makes cell suspension;
1.1.3 Fusion of Cells: the Sp2/0 myeloma cell being in logarithmic phase is mixed in 1: 10 (V/V) ratio with splenocyte suspension, adding PEG-1500 makes cell fusion together, in the mixed cell suspension of two kinds of cells, within the 1st minute, drip 4.5mL nutrient solution; Interval 2 minutes drips 5mL nutrient solution, then adds nutrient solution 50mL, with HAT Selective agar medium by 36% hole be that 1 cells/well carries out cell chulture;
1.1.4 hybridoma is screened: when cell chulture to be fused was to the 7th day, namely cell chulture is to when covering at the bottom of 10% hole, draw in the hole of 96 well culture plates and occur that the culture supernatant indirect ELISA of clone cell bunch detects antibody content, three subclone screenings are carried out through limiting dilution, secretion situation according to antibody filters out the cell line of high-titer, high specific, and expansion cultivation is also frozen.
1.1.5 monoclonal antibody-purified preservation: select BALB/c mouse or its parent mouse, first with the capable mouse peritoneal injection of whiteruss, by 5 × 10 after one week
5hybridoma is inoculated in mouse peritoneal, and collect the ascites of mouse after one week in inoculation, every mouse can collect the ascites of 10mL, uses AKTA protein purification instrument and DE-52 ion exchange column by mouse IgG monoclonal antibody ascites solution at A
280collect during nm.
Finally obtain two strain of hybridoma systems, respectively secrete monoclonal antibody 1 and monoclonal antibody 2.
According to (Li Huijin etc. such as Li Huijin, the nested PCR amplification of H1N1virus hemagglutinin monoclonal antibody light chain and heavy chain variable region gene and sequential analysis, biotechnology communication, 2013,24 (1): 61-64) the method for operating determination monoclonal antibody 1 of document and the variable region of heavy chain of monoclonal antibody 2 and variable region of light chain.
Result: the amino acid sequence of the variable region of heavy chain of monoclonal antibody 1 is SEQIDNo.2, the amino acid sequence of variable region of light chain is SEQIDNo.3; The amino acid sequence of the variable region of heavy chain of monoclonal antibody 2 is SEQIDNo.4, the amino acid sequence of variable region of light chain is SEQIDNo.5.
The qualification of 1.2 anti-influenza type A virus nucleoprotein monoclonal antibodies
1.2.1 the qualification of monoclonal antibody type and subclass
1.2.1.1 according to (Fang Shisong etc. such as Fang Shisong, the clonal expression of influenza A virus nucleoprotein gene and purifying, China's experiment and clinical virology magazine, 2005,19 (2): 165-168) method of operating of document prepares influenza A nucleoprotein.
1.2.1.2 with the influenza A nucleoprotein after the restructuring of carbonic acid buffer dilution gene engineering, by 100 μ g/ hole bag quilts, the cells and supernatant of monoclonal antibody is detected, according to KoenigR. (KoenigR.IndirectELISAmethodsforthebroadspecificitydetect ionofplantviruses.JournalofGeneralVirology, 1981, 55 (1): 53-62) indirect elisa method in document carries out, the enzyme added is respectively the HRP-goat against murine IgM (HRP-IgM) of dilution, HRP-goat against murine IgG1 (HRP-IgG1), HRP-goat against murine IgG2a (HRP-IgG2a), HRP-goat against murine IgG2b (HRP-IgG2b) and HRP-goat against murine IgG3 (HRP-IgG3) enzyme-labeled antibody two resist, the results are shown in Table 1.
The qualification of each monoclonal antibody type of table 1
Note :+represent positive ,-represent negative.
As shown in Table 1: the subclass of monoclonal antibody 1 is IgG1, the subclass of monoclonal antibody 2 is IgG2b.
1.2.2 the qualification of monoclonal antibody specificity
1.2.2.1 the reactivity of monoclonal antibody 1, monoclonal antibody 2 and influenza A nucleoprotein is measured respectively with above-mentioned indirect ELISA method.
1.2.2.2 measure monoclonal antibody 1, monoclonal antibody 2 and Type B influenza nucleoprotein respectively with above-mentioned indirect ELISA method and whether there is cross reactivity.
Measurement result shows: 2 strain monoclonal antibodies and influenza A nucleoprotein have good reactivity, and no cross reaction equal to Type B influenza nucleoprotein, show that this 2 strain monoclonal antibody is all monoclonal antibody specifics of anti-influenza type A virus nucleoprotein.
The inspection of 1.3 monoclonal antibody purifications
1.3.1 the outward appearance of monoclonal antibody purification
Under light-illuminating, the visible antibody purification of visual inspection is achromaticity and clarification state, has no floccus precipitation.
1.3.2 sterility test
Adopt Sterility Test, after getting purifying, monoclonal antibody raw material is seeded to the nutrient agar slant medium of 10mL/ pipe, improvement Martin's nutrient culture media and each 2 pipes (inoculum concentration be 0.5mL/ pipe) of sulphur glycollate culture medium.Postvaccinal sulphur glycollate culture medium and each pipe of nutrient agar slant medium are placed in 30-35 DEG C of cultivation, and another pipe is in 20-25 DEG C of cultivation.Improvement Martin nutrient culture media puts 20-25 DEG C of cultivation.Do negative control with 0.9% aseptic NaCl solution with method operation simultaneously.Cultivate all not observe in nutrient culture media after 14 days and have growth of microorganism, show that monoclonal antibody raw material meets sterility requirements.
1.3.3 monoclonal antibody purity
Use 12%SDS-PAGE protein electrophoresis, the applied sample amount of every swimming lane is 10 μ g, with coomassie brilliant blue staining after electrophoresis, through the scanning of gel imaging instrument, and records concentration by software.As shown in Figure 2, wherein swimming lane 1 is monoclonal antibody 2 to testing result, comprises the heavy chain being greater than 44.3kD and the light chain being greater than 20.1kD; Swimming lane 2 is monoclonal antibody 1, comprises the heavy chain being greater than 44.3kD and the light chain being greater than 20.1kD; Swimming lane 3 is albumen Maker.
Result shows: the purity of 2 strain monoclonal antibodies is all not less than 90%.
The pairing of embodiment 2 monoclonal antibody
The selection of 2.1 monoclonal antibody pairing modes
During screening monoclonal antibody combinations of pairs, main consider following factor: one is the activity of monoclonal antibody; Two is whether monoclonal antibody and non-A type influenza virus exist nonspecific reaction; Three is colour developing backgrounds.
2.2 monoclonal antibody activity detect
Select A type avian flu strain A/Ck/HK/Yu22/02, be diluted to 0.01HA, different collocation mode detected, the results are shown in Table 2:
The collocation of table 2 monoclonal antibody and Activity determination
Note :+represent positive ,-represent negative.
Result shows: fixing except monoclonal antibody 1 the Yu22 viral dilution liquid detecting 0.01HA with monoclonal antibody 2 enzyme mark is except feminine gender, other collocation is positive findings, but monoclonal antibody 1, monoclonal antibody 2 itself is fixing and be marked in application certainly infeasible.
The non-specific detection of 2.3 monoclonal antibody
Select Type B strains of influenza viruses VR788 to cultivate, virus titer is 256HA, detects, the results are shown in Table 3 for different collocation mode:
The collocation of table 3 monoclonal antibody and non-specific detection
Note :+represent positive ,-represent negative.
Result shows: monoclonal antibody 1 is fixing and monoclonal antibody 1 marks, monoclonal antibody 2 is fixing marks with monoclonal antibody 2, and these two kinds collocation all occur nonspecific reaction.
According to above experimental result: monoclonal antibody 1, as labelled antibody, selects monoclonal antibody 2 as sessile antibody.
The structure of embodiment 3A type influenza virus enzyme chromatography detection kit and use
Fixing of 3.1 monoclonal antibodies
Be fixed on nitrocellulose filter by the monoclonal antibody 2 that embodiment 1 is prepared by the three-dimensional specking platform of BioDotXYZ3050.
Horseradish peroxidase (HRP) mark of 3.2 monoclonal antibodies
According to monoclonal antibody 1 prepared by embodiment 1, according to (TijssenP such as TijssenP, KurstakE.Highlyefficientandsimplemethodsforthepreparatio nofperoxidaseandactiveperoxidaseantibodyconjugatesforenz ymeimmunoassays (J) .AnalBiochem, 1984,136:451-457) document to anti-influenza type A virus monoclonal antibody carry out horseradish peroxidase (HRP) mark.
The composition of 3.3 detection kit
Detection kit includes enzyme chromatography test strip, sample treatment liquid, sample processing tube, Sample storage liquid.Wherein,
(1) enzyme chromatography test strip, as shown in Figure 1.
(2) sample treatment liquid is in sample preparation bottle, is the phosphate buffer of the pH7.4 containing 0.5%-3.5%CHAPS.
(3) sample processing tube.
(4) Sample storage liquid is in Sample storage bottle, is the phosphate buffer of pH7.4.
Described enzyme chromatography test strip comprises sample supply area, and sample supply area comprises enzyme mark pad 2, it is adsorbed with labeled monoclonal antibody 1 prepared by embodiment 3.2; Substrate supply area, substrate supply area comprises substrate pad 3; Damping fluid feeding unit, damping fluid feeding unit comprises expansion fluid cushion 5, substrate buffer liquid bath 8, substrate buffer solution 9; Detection zone, detection zone comprises detection line 6, nature controlling line 7, at the immobilized monoclonal antibody 2 that detection line 6 immobilization has embodiment 1 to prepare, has sheep anti mouse to resist in nature controlling line 7 immobilization more.Nitrocellulose filter 1 sticks to the full section of above support 10; And detect the position that the position that adds of sample 11 is described enzyme mark pad 2.
Enzyme chromatography test strip is fixing in a plastic housing, it is from left to right fixed successively and launches fluid cushion 5, substrate pad 3, enzyme mark pad 2, adsorptive pads 4.Adsorptive pads 4 is stuck in the top of nitrocellulose filter 1; Enzyme mark pad 2 is positioned at the stage casing of nitrocellulose filter 1; Substrate pad 3 is stuck in the bottom of nitrocellulose filter 1, it has dry zymolyte.The upper end of launching fluid cushion 5 covers substrate pad 3, and its lower end is positioned at the bottom of substrate buffer solution groove 8.Cover one deck aluminium-foil paper above substrate buffer liquid bath 8, to prevent substrate buffer solution 9 seepage, top is damping fluid button (not marking in Fig. 1).Detection line 6 is arranged in the upper end of nitrocellulose filter 1, and nature controlling line 7 is arranged in the upper end of nitrocellulose filter 1, and is positioned at the upstream of detection line 6.
When using detection kit, first use sample processing tube pretreatment sample; Then pretreatment sample is added the position of enzyme mark pad 2, press damping fluid button simultaneously, damping fluid button control damping fluid 9 is by launching the chromatography process of fluid cushion 5 to adsorptive pads 4.If containing influenza A, it will be combined with enzyme labeling monoclonal antibody 1 in pretreatment sample, influenza A and enzyme labeling monoclonal antibody 1 combination and excessive enzyme labeling monoclonal antibody 1 will be moved to adsorptive pads 4 direction under capillarity.When the influenza A of moving and enzyme labeling monoclonal antibody 1 combination arrive detection line 6 position, the immobilized monoclonal antibody 2 being fixed in detection line 6 position is caught, when arriving detection line 6 from the substrate that the migration of substrate supply area is next, produce chromogenic reaction with enzyme labeling monoclonal antibody 1.Meanwhile, excessive enzyme labeling monoclonal antibody 1 continues to move to nature controlling line 7, and resist of the sheep anti mouse being fixed on nature controlling line 7 position catches more, and produces chromogenic reaction with the substrate moved from substrate supply area.
Examination criteria: whole reaction carries out about 30 minutes, after reaction terminates, if nature controlling line place there is no band, no matter whether detection line place has band, and testing process is all invalid, when there is band at nature controlling line place, if there is band at detection line place, is then positive, in interpret sample, there is influenza A, otherwise be negative, in interpret sample, there is not influenza A.
Embodiment 4 sample pre-treatments
The collection of 4.1 samples
The sample of main collection comes from pharynx nasalis, pars oralis pharyngis, cloaca portion, and Details as Follows:
When using microorganism swab to gather pharynx nasalis sample, rotate gently and promote microorganism swab, making microorganism swab head deeply be positioned at the pharynx nasalis of nasal cavity root, light turn a few circle is to obtain the higher Nasopharyngeal swabs sample of viral abundance;
When using the pharyngeal sample of microorganism swab acquisition port, appropriateness firmly wipes pharynx rear wall and both sides, should avoid touching tongue;
When using microorganism swab to gather cloaca portion sample, microorganism swab portion is stretched into cloaca 1.5-2cm place, adherent rotation was taken out after 3 weeks.
The process of 4.2 samples
The microorganism swab gathered is inserted in sample processing tube, rotates near tube wall and repeatedly make sample dissolve as far as possible in the solution, finally make microorganism swab head fracture and stay with bottle.
The application of embodiment 5A type influenza virus enzyme chromatography detection kit
The detection method of A type influenza endonuclease chromatography is:
(1) the sample processing tube lid head containing sample is fractureed, extrude a sample and add well, press rapidly damping fluid button simultaneously;
In view window observed and recorded result after (2) 30 minutes.
5.1 sensitivity and susceptibility
Detect the different dilutability of 70 parts of test samples with the kit of three continuous lot numbers, statistic mixed-state lower limit, in order to evaluate sensitivity and the susceptibility of the method.Simultaneously to A type avian influenza virus (the H5 hypotype: 10 of known viruse content
8.5eID
50/ 100 μ L, H9 hypotypes: 10
9.5eID
50/ 100 μ L) carry out a series of dilution, dilutability is followed successively by 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, then detect, determine its lowest detection lower limit.What the kit of three continuous lot numbers detected 70 parts of different dilutability samples the results are shown in Table 4.
The testing result of the influenza A of table 4 pair different subtype
Note :-represent negative, ND represents and does not detect.
Result shows: it is 0.01-0.1HA that kit detects lower limit, confirms that this kit has higher sensitivity; All can detect 70 increment product, confirm that this kit susceptibility is good.
To A type avian influenza virus (the H5 hypotype: 10 of known viruse content
8.5eID
50/ 100 μ L, H9 hypotypes: 10
9.5eID
50/ 100 μ L) carry out a series of dilution, dilutability is followed successively by 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, then detect, assay is in table 5.
The Monitoring lower-cut of table 5A type influenza virus enzyme chromatography detection kit
Note :+represent positive ,-represent negative ,-/+represent the weak positive.
As shown in Table 5: the lowest detection lower limit of kit to H5, H9 hypotype A type avian influenza virus of three continuous lot numbers is virus stock solution used dilution 10
4doubly, 10 are respectively through the lowest detection lower limit obtaining this kit that converts
4.5eID
50/ 100 μ L (H5 hypotype) and 10
5.5eID
50/ 100 μ L (H9 hypotype).
The SPF chicken of the H5 subtype influenza virus positive is swallowed, cloacal swab sample; The pharynx of the SPF chicken pharynx of inoculation H9 subtype influenza virus, cloacal swab sample and clinical onset chicken, cloacal swab sample; The Nasal swabs sample of the pig of clinical infection H1, H3 hypotype swine influenza virus; The Nasopharyngeal swabs sample of the dog of clinical infection H3 hypotype canine influenza virus detects, the susceptibility of kits for evaluation.Testing result is in table 6.
Table 6A type influenza virus enzyme chromatography detection kit swab samples sensitivity Detection result
Note :+represent positive ,-represent negative ,-/+representing the weak positive, ND represents and does not detect.
As shown in Table 6: the kit of three continuous lot numbers can both effectively detect above-mentioned swab samples, illustrate that kit has good susceptibility.
5.2 specificity
With three continuous lot number kits to the pharynx of negative SPF chicken, cloacal swab sample, the pharynx of healthy chicken, cloacal swab sample, the Nasopharyngeal swabs sample of health pig, the Nasopharyngeal swabs sample of Healthy Dogs detects, and evaluates its specificity.Three continuous lot number kits are to the pharynx of negative SPF chicken, cloacal swab sample, and the pharynx of healthy chicken, cloacal swab sample, the Nasopharyngeal swabs sample of health pig, the assay of the Nasopharyngeal swabs sample of Healthy Dogs is in table 7.
Table 7A type influenza virus enzyme chromatography detection kit negative swab specific detection result
Note :+represent positive ,-represent negative.
As shown in Table 7: specificity can reach 100%, proved kit has higher specificity.
The avian influenza virus (the H5 subtype avian influenza virus comprising various years and geographical separation represents strain) of the kit of three continuous lot numbers or the Main Subtype that be separated popular to China, H1 and H3 hypotype swine influenza virus represents strain, H3 hypotype canine influenza virus represents strain, H6, H7 and H9 subtype avian influenza virus represents 10 of other avian viral such as strain and Avian pneumo-encephalitis virus, IBV, infectious bursa of Fabricius virus, infectious laryngotracheitis
-1, 10
-2, 10
-3detect Deng dilution series, evaluate its avian influenza virus for Main Subtype and the specificity of other avian viral.Assay is in table 8.
Table 8A type influenza virus enzyme chromatography detection kit specific detection result
Note :+represent positive ,-represent negative ,-/+representing the weak positive, ND represents and does not detect.
As shown in Table 8: three continuous lot number kits all can with China's Major Epidemic or the antigen be separated group (clade0, clade1, clade2.2, clade2.3.2, clade2.3.4 class RE-5 vaccine strain, clade2.5, clade3, clade4, clade5, clade7 class RE-4 vaccine strain, clade9) H5N1 subtype avian influenza virus generation specific reaction; Strain can be represented with H1 and H3 hypotype swine influenza virus, H3 hypotype canine influenza virus represents strain, H6, H7 and H9 subtype avian influenza virus represents strain generation specific reaction; Not with Avian pneumo-encephalitis virus, IBV, infectious bursa of Fabricius virus, infectious laryngotracheitis generation nonspecific reaction, specificity is good.
5.3 repeatable
With three continuous lot number kits by 3 people to the 30 parts of test sample duplicate detection three times comprising strong positive reference material 10 parts (P1-P10), weak positive reference material 10 parts (P11-P20) and specificity reference material 10 parts (N1-N10), coincidence rate between statistic mixed-state result, 30 parts of test samples are detected by 1 people with 5 single lot number kits, in order to evaluate three batches of kits batch between, batch in repeatable, assay is in Table 9-11.
Table 930 part test sample batch between testing result
Note :+represent positive ,-represent negative.
Batch interior testing result of table 1030 part test sample
Note :+represent positive ,-represent negative.
Table 11 three batches of kits criticize interior, batch between coincidence rate
From table 9-11: three continuous lot number kits to 30 parts of test sample duplicate detection three times, through add up three lot number kits criticize between, batch in positive coincidence rate between each testing result be respectively 100%, 100%, negative match-rate is respectively 96.8%, 96.2%, illustrate three batches of goods criticize between, batch in all there is good repeatability.
5.4 with the comparing of other diagnostic method
Select avian influenza virus (H5, H9 hypotype) strong positive, the weak positive, each 3 parts of negative sample, avian influenza virus (H5, H9 hypotype) serial dilution of known viruse content is 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7wait dilutability sample, detect by inoculation SPF chicken embryo virus isolation procedure and RT-PCR method and influenza A virus enzyme chromatography test strip, that compares three kinds of detection methods meets situation.
This kit the results are shown in Table 12-13 with egg inoculation isolated viral method with the parallel comparison test of RT-PCR.
The comparison test 1 of table 12 influenza A virus enzyme chromatography test strip and egg inoculation isolated viral method and RT-PCR
Note :+represent positive ,-represent negative ,-/+represent the weak positive.
The comparison test 2 of table 13 influenza A virus enzyme chromatography test strip and egg inoculation isolated viral method and RT-PCR
Note :+represent positive ,-represent negative ,-/+represent the weak positive.
From table 12-13: detect corresponding strong positive, the weak positive and ' negative ' specimens and avian influenza virus, although egg inoculation virus isolation procedure is more responsive than this kit, but the method takes time and effort, can not realize easy to influenza A, detect fast; Although the sensitivity of RT-PCR is slightly higher than this kit, the method needs expensive instrument and the technical professionals such as PCR instrument, can not meet easy, diagnostic requirements fast.
This kit is compared with the colloidal gold strip of the detection avian influenza virus of certain company domestic, and specificity is consistent, but remolding sensitivity it is high 10 times.
5.5 storage life
Three continuous lot number kits are placed 6 days and laggard line stabilization test in 7 days respectively at 37+1 DEG C, i.e. kit heat stabilization test; In order to prevent there are differences between 37 ± 1 DEG C of heat stabilization tests and 2-8 DEG C of storage condition, kit is deposited 6 months, 9 months, 12 months and 15 months at 2-8 DEG C again by respectively, i.e. the real-time stability test of kit.The kit of different preservation condition is tested with 35 parts of test samples, in order to evaluate the storage life of these goods.
Table 14 kit heat stabilization test result
Table 15 kit real-time stability test findings
This kit is preserved under 37 ± 1 DEG C of conditions, stores 7 days; Preserve under 2-8 DEG C of rated condition, store 15 months, still by finished product kit quality control standard, show kit term of validity duration of test steady quality, storage life at least can reach 12 months.
From above result, kit of the present invention and detection method tool thereof have the following advantages:
1, quick, testing process only needs 30 minutes;
2, accurate, high specificity, not with Type B influenza virus generation non-specific responding, not with Avian pneumo-encephalitis virus, IBV, infectious bursa of Fabricius virus, ILTV generation non-specific responding;
3, susceptibility is high, can detect influenza A and represent strain, comprises the H5N1 hypotype antigen group (clade0 of China's Major Epidemic or separation, clade1, clade2.2, clade2.3.2 class RE-6 vaccine strain, clade2.3.4 class RE-5 vaccine strain, clade2.5, clade3, clade4, clade5, clade7.2 class RE-4 vaccine strain, clade8, clade9) avian influenza virus; Other subtype avian influenza virus such as H6, H7 and H9 hypotype represent strain; H1 and H3 swine influenza virus and equine influenza virus represent strain; H3 hypotype canine influenza virus represents strain;
4, sensitive, its sensitivity can reach ELISA level, higher than colloidal gold strip more than 10 times, and suitable with PCR detection sensitivity in the detection of some cause of disease, Monitoring lower-cut is 0.01HA-0.1HA;
5, easy, do not need to be equipped with expensive device and the technical professionals such as PCR instrument, can detect in the clinical setting, simple to operate, easy to carry;
6, good stability, can preserve for a long time at 2-8 DEG C, storage life is 12 months.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (11)
1. for detecting a kit for influenza virus in sample, wherein, described kit comprises damping fluid feeding unit and test strip;
Described damping fluid feeding unit is used for damping fluid to supply described test strip;
Described test strip comprises nitrocellulose filter (1), and described test strip comprises substrate supply area in the vertical successively, sample supply area, detection zone;
Described substrate supply area comprises substrate pad (3), it is adsorbed with dry zymolyte, described substrate pad (3) contacts with nitrocellulose filter (1), and described zymolyte to be dissolved in damping fluid and to the distal migration apart from described damping fluid feeding unit on nitrocellulose filter (1);
Described sample supply area comprises enzyme mark pad (2), it is adsorbed with mouse-anti influenza A nucleoprotein antibody labeling monoclonal antibody 1, described zymolyte can produce chromogenic reaction with the enzyme that marks on described labeled monoclonal antibody 1, described enzyme mark pad (2) contacts with nitrocellulose filter (1), described labeled monoclonal antibody 1 is dissolved in damping fluid, and to the distal migration apart from described damping fluid feeding unit on nitrocellulose filter (1); And
The immobilization of described detection zone has mouse-anti influenza A nucleoprotein antibody immobilized monoclonal antibody 2;
Wherein, the amino acid sequence of described its variable region of heavy chain of labeled monoclonal antibody 1 is SEQIDNo.2, the amino acid sequence of variable region of light chain is SEQIDNo.3; And
The amino acid sequence of described its variable region of heavy chain of immobilized monoclonal antibody 2 is SEQIDNo.4, the amino acid sequence of variable region of light chain is SEQIDNo.5.
2. kit according to claim 1, wherein, described damping fluid feeding unit comprises expansion fluid cushion (5), substrate buffer liquid bath (8), substrate buffer solution (9) and damping fluid button, described substrate buffer solution (9) is placed in substrate buffer liquid bath (8), described damping fluid button is positioned at the top of substrate buffer solution groove (8), presses and can immerse in damping fluid (9) by described expansion fluid cushion (5);
Described detection zone comprises detection line (6), nature controlling line (7), wherein, described nature controlling line (7) comparatively detection line (6) further from described sample supply area, mouse-anti influenza A nucleoprotein antibody immobilized monoclonal antibody 2 is had in described detection line (6) immobilization, have sheep anti mouse to resist in described nature controlling line (7) immobilization, and the labeled monoclonal antibody 1 be adsorbed on enzyme mark pad (2) is excessive for being fixed on the immobilized monoclonal antibody 2 of detection line (6) more;
Stilt (10) connects described damping fluid feeding unit, substrate supply area, sample supply area, detection zone and adsorptive pads (4), the full section of described nitrocellulose filter (1) sticks on above support (10), and described adsorptive pads (4) is in the distalmost end apart from described damping fluid feeding unit; And
The position that detection sample (11) adds is the position of described enzyme mark pad (2).
3. kit according to claim 1, wherein, the enzyme of described mark comprises any one in horseradish peroxidase, alkaline phosphatase and beta-D-galactosidase.
4. kit according to claim 2, wherein, described substrate buffer solution comprises one or more in phosphate buffer, surfactant, antiseptic and carbohydrate.
5. kit according to claim 1, wherein, described kit also comprises sample processing tube, described sample processing tube contains sample treatment liquid, described sample treatment liquid is surfactant solution, and described surfactant comprises any one in stearic acid, neopelex, lecithin, fatty glyceride and CHAPS.
6. kit according to claim 1, wherein, described kit also comprises Sample storage bottle, and described Sample storage bottle contains Sample storage liquid, and Sample storage liquid is phosphate buffer.
7. kit according to claim 1, wherein, described Sample storage liquid is the phosphate buffer of pH7.4.
8. for non-diagnostic object any one of use claim 1 ~ 7 described in kit detect the method for influenza virus in sample, described method comprises:
Step (1) uses described sample processing tube pretreatment sample;
Pretreatment sample in described step (1) is added the position of described enzyme mark pad (2) by step (2);
Step (3) presses damping fluid button; And
Step (4) is if nature controlling line place there is no band, and no matter whether detection line place has band, and testing process is all invalid, when there is band at nature controlling line place, if there is band at detection line place, is then positive, otherwise is negative.
9. the application of kit in for the detection sample of non-diagnostic object in influenza virus according to any one of claim 1 ~ 7.
10. an anti-influenza type A virus nucleoprotein monoclonal antibody 1, the amino acid sequence of described its variable region of heavy chain of monoclonal antibody 1 is SEQIDNo.2, the amino acid sequence of variable region of light chain is SEQIDNo.3.
11. 1 kinds of anti-influenza type A virus nucleoprotein monoclonal antibodies 2, the amino acid sequence of described its variable region of heavy chain of monoclonal antibody 2 is SEQIDNo.4, the amino acid sequence of variable region of light chain is SEQIDNo.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410308507.3A CN104062430B (en) | 2014-06-30 | 2014-06-30 | A kind of kit for detecting influenza virus in sample and detection method thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410308507.3A CN104062430B (en) | 2014-06-30 | 2014-06-30 | A kind of kit for detecting influenza virus in sample and detection method thereof and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104062430A CN104062430A (en) | 2014-09-24 |
| CN104062430B true CN104062430B (en) | 2016-03-30 |
Family
ID=51550237
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410308507.3A Active CN104062430B (en) | 2014-06-30 | 2014-06-30 | A kind of kit for detecting influenza virus in sample and detection method thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104062430B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105461805B (en) * | 2015-12-17 | 2019-02-15 | 洛阳普莱柯万泰生物技术有限公司 | Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus and its application |
| CN105891491A (en) * | 2016-04-11 | 2016-08-24 | 洛阳普莱柯万泰生物技术有限公司 | Kit and application thereof |
| JP6652262B2 (en) * | 2017-12-28 | 2020-02-19 | 株式会社Icst | Test equipment and test method |
| CN108089013A (en) * | 2018-01-05 | 2018-05-29 | 潍坊科瑞斯生物科技有限公司 | The preparation method of fluorometric reagent in a kind of dog Procalcitonin detection kit and the detection kit |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1081765A (en) * | 1992-03-05 | 1994-02-09 | 曼海姆泊灵格股份公司 | Be used to measure the immunization method of haemoglobin dervative |
| WO1999027364A1 (en) * | 1997-11-24 | 1999-06-03 | Quidel Corporation | Enzyme substrate delivery and product registration in one-step enzyme immunoassays |
| CN1756834A (en) * | 2003-02-24 | 2006-04-05 | 比南股份有限公司 | Dry chemistry, lateral flow-reconstituted chromatographic enzyme-driven assays |
| CN102066932A (en) * | 2008-07-14 | 2011-05-18 | 田中贵金属工业株式会社 | Developing solution for immunochromatography, and measurement method using same |
| CN102388312A (en) * | 2009-04-09 | 2012-03-21 | 爱科来株式会社 | Analysis method, sample analyzing tool, method of preventing backflow of sample solution and method of preventing background rise |
-
2014
- 2014-06-30 CN CN201410308507.3A patent/CN104062430B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1081765A (en) * | 1992-03-05 | 1994-02-09 | 曼海姆泊灵格股份公司 | Be used to measure the immunization method of haemoglobin dervative |
| WO1999027364A1 (en) * | 1997-11-24 | 1999-06-03 | Quidel Corporation | Enzyme substrate delivery and product registration in one-step enzyme immunoassays |
| CN1756834A (en) * | 2003-02-24 | 2006-04-05 | 比南股份有限公司 | Dry chemistry, lateral flow-reconstituted chromatographic enzyme-driven assays |
| CN102066932A (en) * | 2008-07-14 | 2011-05-18 | 田中贵金属工业株式会社 | Developing solution for immunochromatography, and measurement method using same |
| CN102388312A (en) * | 2009-04-09 | 2012-03-21 | 爱科来株式会社 | Analysis method, sample analyzing tool, method of preventing backflow of sample solution and method of preventing background rise |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104062430A (en) | 2014-09-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105695420B (en) | Mouse bone marrow cells hybridoma cell strain and its monoclonal antibody and application generated | |
| CN105461805A (en) | Monoclonal antibody for resisting porcine epidemic diarrhea viruses and application thereof | |
| CN101253198A (en) | Antibody produced using ostrich and method for production thereof | |
| CN105675873B (en) | A kind of detection kit and its application | |
| CN104928258B (en) | Canine parvovirus hybridoma and monoclonal antibody and application | |
| CN105527437A (en) | Detection kit and application thereof | |
| CN104062430B (en) | A kind of kit for detecting influenza virus in sample and detection method thereof and application | |
| Moreno et al. | MAb‐based competitive ELISA for the detection of antibodies against influenza D virus | |
| CN105891491A (en) | Kit and application thereof | |
| CN109232734B (en) | Monoclonal antibody specifically binding canine adenovirus, pharmaceutical composition, kit and application thereof | |
| CN109517802B (en) | Hybridoma cell strain secreting salmonella pullorum IpaJ monoclonal antibody, monoclonal antibody thereof and application of monoclonal antibody | |
| CN108084255A (en) | A kind of preparation of canine recombinant c reactive protein and its monoclonal antibody | |
| CN110272488B (en) | Cat calicivirus monoclonal antibody and application thereof | |
| CN105837686A (en) | Monoclone antibody and application thereof | |
| CN106226518A (en) | Canine distemper virus colloidal gold immunochromatographydetection detection test paper bar and preparation method thereof | |
| CN109705216B (en) | Monoclonal antibody for resisting bovine skeletal muscle troponin I and application thereof | |
| CN101887061A (en) | Colloidal gold immuno-chromatography test paper strip used for detecting escherichia coil F5 pilus and preparation method thereof | |
| CN109810191B (en) | Monoclonal antibody for resisting sheep skeletal muscle troponin I and application thereof | |
| CN109852588B (en) | Monoclonal antibody of anti-tilapia immune globulin IgM, cell strain and application thereof | |
| CN102838676A (en) | Carcino-embryonic antigen monoclonal antibody, chip containing same and application | |
| CN105717293B (en) | A kind of kit for being used to detect porcine circovirus 2 type | |
| Zhou et al. | Development of a novel antibody probe useful for domoic acid detection | |
| CN104744590B (en) | Anti-H 1 N 1 swine influenza virus hemagglutinin monoclonal antibody and double crush syndrome kit | |
| CN109521199A (en) | A kind of gold-immunochromatographyreagent reagent for assay box and its application for detecting avian leukosis virus | |
| CN111220809B (en) | Colloidal gold immunochromatography test strip for detecting chicken or duck skeletal muscle troponin I and preparation method and application thereof |
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 | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: Room 1010, Commune No. 6, Huaxia Road, No. 6, High-tech Zone, Luoyang China (Henan) Free Trade Experimental Zone, Henan Province Patentee after: Luoyang Pu Tai Biotechnology Co., Ltd. Address before: 471000 Cui Wei Road, hi tech Zone, Luoyang, Henan Patentee before: Luoyang Pu Laikewantai Bioisystech Co., Ltd |