WO2009148150A1 - インフルエンザウィルス検出用デバイス - Google Patents
インフルエンザウィルス検出用デバイス Download PDFInfo
- Publication number
- WO2009148150A1 WO2009148150A1 PCT/JP2009/060328 JP2009060328W WO2009148150A1 WO 2009148150 A1 WO2009148150 A1 WO 2009148150A1 JP 2009060328 W JP2009060328 W JP 2009060328W WO 2009148150 A1 WO2009148150 A1 WO 2009148150A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antibody
- influenza
- virus nucleoprotein
- influenza virus
- human anti
- Prior art date
Links
- 241000712461 unidentified influenza virus Species 0.000 title claims abstract description 224
- 238000001514 detection method Methods 0.000 title claims abstract description 171
- 206010022000 influenza Diseases 0.000 claims abstract description 252
- 239000007790 solid phase Substances 0.000 claims abstract description 41
- 108010061100 Nucleoproteins Proteins 0.000 claims description 474
- 102000011931 Nucleoproteins Human genes 0.000 claims description 473
- 241000700605 Viruses Species 0.000 claims description 358
- 238000000034 method Methods 0.000 claims description 158
- 208000037797 influenza A Diseases 0.000 claims description 110
- 239000000427 antigen Substances 0.000 claims description 105
- 108091007433 antigens Proteins 0.000 claims description 105
- 102000036639 antigens Human genes 0.000 claims description 105
- 239000003153 chemical reaction reagent Substances 0.000 claims description 55
- 238000011002 quantification Methods 0.000 claims description 54
- 208000037798 influenza B Diseases 0.000 claims description 46
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 claims description 44
- 238000002372 labelling Methods 0.000 claims description 34
- 108010046023 influenza B virus nucleoprotein Proteins 0.000 claims description 32
- 101900222562 Influenza A virus Nucleoprotein Proteins 0.000 claims description 31
- 230000008859 change Effects 0.000 claims description 31
- 241000712431 Influenza A virus Species 0.000 claims description 28
- 239000000126 substance Substances 0.000 claims description 25
- 238000005406 washing Methods 0.000 claims description 21
- 238000010521 absorption reaction Methods 0.000 claims description 12
- 238000012790 confirmation Methods 0.000 claims description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims 31
- 108010015780 Viral Core Proteins Proteins 0.000 abstract 2
- 239000000523 sample Substances 0.000 description 87
- 239000000243 solution Substances 0.000 description 82
- 108090000623 proteins and genes Proteins 0.000 description 79
- 150000001413 amino acids Chemical group 0.000 description 57
- 210000004027 cell Anatomy 0.000 description 53
- 238000006243 chemical reaction Methods 0.000 description 46
- 102000004169 proteins and genes Human genes 0.000 description 44
- 235000018102 proteins Nutrition 0.000 description 43
- 210000003719 b-lymphocyte Anatomy 0.000 description 42
- 239000000872 buffer Substances 0.000 description 41
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 32
- 239000002953 phosphate buffered saline Substances 0.000 description 32
- 108091028043 Nucleic acid sequence Proteins 0.000 description 29
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 28
- 241000699666 Mus <mouse, genus> Species 0.000 description 28
- 238000009739 binding Methods 0.000 description 28
- 230000027455 binding Effects 0.000 description 27
- 239000007788 liquid Substances 0.000 description 24
- 229940098773 bovine serum albumin Drugs 0.000 description 22
- 239000013604 expression vector Substances 0.000 description 22
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 21
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 21
- 239000000758 substrate Substances 0.000 description 21
- 241000713196 Influenza B virus Species 0.000 description 20
- 102000004190 Enzymes Human genes 0.000 description 19
- 108090000790 Enzymes Proteins 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 15
- 230000035945 sensitivity Effects 0.000 description 15
- 239000002299 complementary DNA Substances 0.000 description 14
- 238000003018 immunoassay Methods 0.000 description 14
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 14
- 238000004519 manufacturing process Methods 0.000 description 13
- 239000007983 Tris buffer Substances 0.000 description 12
- 239000012634 fragment Substances 0.000 description 11
- 125000000524 functional group Chemical group 0.000 description 11
- 238000005259 measurement Methods 0.000 description 11
- 108060003951 Immunoglobulin Proteins 0.000 description 10
- 229920001213 Polysorbate 20 Polymers 0.000 description 10
- 239000006172 buffering agent Substances 0.000 description 10
- 239000012228 culture supernatant Substances 0.000 description 10
- 238000011161 development Methods 0.000 description 10
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 10
- 238000003317 immunochromatography Methods 0.000 description 10
- 102000018358 immunoglobulin Human genes 0.000 description 10
- 210000004698 lymphocyte Anatomy 0.000 description 10
- 239000002245 particle Substances 0.000 description 10
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 10
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 10
- 238000002965 ELISA Methods 0.000 description 9
- 241000845082 Panama Species 0.000 description 9
- 239000013598 vector Substances 0.000 description 9
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 230000003100 immobilizing effect Effects 0.000 description 8
- 238000001179 sorption measurement Methods 0.000 description 8
- 239000004094 surface-active agent Substances 0.000 description 8
- 241000486679 Antitype Species 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 7
- DZBUGLKDJFMEHC-UHFFFAOYSA-O acridine;hydron Chemical compound C1=CC=CC2=CC3=CC=CC=C3[NH+]=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-O 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 230000003993 interaction Effects 0.000 description 7
- 238000004020 luminiscence type Methods 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 239000012981 Hank's balanced salt solution Substances 0.000 description 6
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 6
- 239000012980 RPMI-1640 medium Substances 0.000 description 6
- 230000003321 amplification Effects 0.000 description 6
- 239000011575 calcium Substances 0.000 description 6
- 229910052791 calcium Inorganic materials 0.000 description 6
- 239000003593 chromogenic compound Substances 0.000 description 6
- 239000006059 cover glass Substances 0.000 description 6
- 239000012894 fetal calf serum Substances 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 238000000691 measurement method Methods 0.000 description 6
- FSVCQIDHPKZJSO-UHFFFAOYSA-L nitro blue tetrazolium dichloride Chemical compound [Cl-].[Cl-].COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 FSVCQIDHPKZJSO-UHFFFAOYSA-L 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- -1 polyoxyethylene Polymers 0.000 description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 6
- 241000856131 recombinant Influenza A viruses Species 0.000 description 6
- 238000012546 transfer Methods 0.000 description 6
- QRXMUCSWCMTJGU-UHFFFAOYSA-L (5-bromo-4-chloro-1h-indol-3-yl) phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP([O-])(=O)[O-])=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-L 0.000 description 5
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 5
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 5
- QOMNQGZXFYNBNG-UHFFFAOYSA-N acetyloxymethyl 2-[2-[2-[5-[3-(acetyloxymethoxy)-2,7-difluoro-6-oxoxanthen-9-yl]-2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]-n-[2-(acetyloxymethoxy)-2-oxoethyl]-4-methylanilino]acetate Chemical compound CC(=O)OCOC(=O)CN(CC(=O)OCOC(C)=O)C1=CC=C(C)C=C1OCCOC1=CC(C2=C3C=C(F)C(=O)C=C3OC3=CC(OCOC(C)=O)=C(F)C=C32)=CC=C1N(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O QOMNQGZXFYNBNG-UHFFFAOYSA-N 0.000 description 5
- 239000012736 aqueous medium Substances 0.000 description 5
- 230000000903 blocking effect Effects 0.000 description 5
- 239000007850 fluorescent dye Substances 0.000 description 5
- 230000016784 immunoglobulin production Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 4
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 4
- 108010083359 Antigen Receptors Proteins 0.000 description 4
- 241001481710 Cerambycidae Species 0.000 description 4
- OUVXYXNWSVIOSJ-UHFFFAOYSA-N Fluo-4 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=C(C=2)C2=C3C=C(F)C(=O)C=C3OC3=CC(O)=C(F)C=C32)N(CC(O)=O)CC(O)=O)=C1 OUVXYXNWSVIOSJ-UHFFFAOYSA-N 0.000 description 4
- 239000007995 HEPES buffer Substances 0.000 description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 239000002585 base Substances 0.000 description 4
- 150000001720 carbohydrates Chemical class 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 239000005018 casein Substances 0.000 description 4
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 4
- 235000021240 caseins Nutrition 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000003623 enhancer Substances 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 108700010900 influenza virus proteins Proteins 0.000 description 4
- 238000010030 laminating Methods 0.000 description 4
- 239000004816 latex Substances 0.000 description 4
- 229920000126 latex Polymers 0.000 description 4
- 229910021645 metal ion Inorganic materials 0.000 description 4
- 238000002493 microarray Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 210000005259 peripheral blood Anatomy 0.000 description 4
- 239000011886 peripheral blood Substances 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- 230000002335 preservative effect Effects 0.000 description 4
- 238000003127 radioimmunoassay Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 3
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 3
- 125000000954 2-hydroxyethyl group Chemical group [H]C([*])([H])C([H])([H])O[H] 0.000 description 3
- 102000006306 Antigen Receptors Human genes 0.000 description 3
- 102100034349 Integrase Human genes 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 210000004102 animal cell Anatomy 0.000 description 3
- 108010005774 beta-Galactosidase Proteins 0.000 description 3
- 102000005936 beta-Galactosidase Human genes 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 229910001424 calcium ion Inorganic materials 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000003365 glass fiber Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000031146 intracellular signal transduction Effects 0.000 description 3
- 230000002934 lysing effect Effects 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 239000002736 nonionic surfactant Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- 230000009385 viral infection Effects 0.000 description 3
- QAPSNMNOIOSXSQ-YNEHKIRRSA-N 1-[(2r,4s,5r)-4-[tert-butyl(dimethyl)silyl]oxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O[Si](C)(C)C(C)(C)C)C1 QAPSNMNOIOSXSQ-YNEHKIRRSA-N 0.000 description 2
- AJTVSSFTXWNIRG-UHFFFAOYSA-N 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid Chemical compound OCC[NH+](CCO)CCS([O-])(=O)=O AJTVSSFTXWNIRG-UHFFFAOYSA-N 0.000 description 2
- BGFTWECWAICPDG-UHFFFAOYSA-N 2-[bis(4-chlorophenyl)methyl]-4-n-[3-[bis(4-chlorophenyl)methyl]-4-(dimethylamino)phenyl]-1-n,1-n-dimethylbenzene-1,4-diamine Chemical compound C1=C(C(C=2C=CC(Cl)=CC=2)C=2C=CC(Cl)=CC=2)C(N(C)C)=CC=C1NC(C=1)=CC=C(N(C)C)C=1C(C=1C=CC(Cl)=CC=1)C1=CC=C(Cl)C=C1 BGFTWECWAICPDG-UHFFFAOYSA-N 0.000 description 2
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 2
- NUFBIAUZAMHTSP-UHFFFAOYSA-N 3-(n-morpholino)-2-hydroxypropanesulfonic acid Chemical compound OS(=O)(=O)CC(O)CN1CCOCC1 NUFBIAUZAMHTSP-UHFFFAOYSA-N 0.000 description 2
- XCBLFURAFHFFJF-UHFFFAOYSA-N 3-[bis(2-hydroxyethyl)azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound OCCN(CCO)CC(O)CS(O)(=O)=O XCBLFURAFHFFJF-UHFFFAOYSA-N 0.000 description 2
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 2
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- FSVCELGFZIQNCK-UHFFFAOYSA-N N,N-bis(2-hydroxyethyl)glycine Chemical compound OCCN(CCO)CC(O)=O FSVCELGFZIQNCK-UHFFFAOYSA-N 0.000 description 2
- MKWKNSIESPFAQN-UHFFFAOYSA-N N-cyclohexyl-2-aminoethanesulfonic acid Chemical compound OS(=O)(=O)CCNC1CCCCC1 MKWKNSIESPFAQN-UHFFFAOYSA-N 0.000 description 2
- SEQKRHFRPICQDD-UHFFFAOYSA-N N-tris(hydroxymethyl)methylglycine Chemical compound OCC(CO)(CO)[NH2+]CC([O-])=O SEQKRHFRPICQDD-UHFFFAOYSA-N 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 150000001448 anilines Chemical class 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 239000002041 carbon nanotube Substances 0.000 description 2
- 229910021393 carbon nanotube Inorganic materials 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 238000004040 coloring Methods 0.000 description 2
- 230000006957 competitive inhibition Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000004163 cytometry Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- DIOQZVSQGTUSAI-UHFFFAOYSA-N decane Chemical compound CCCCCCCCCC DIOQZVSQGTUSAI-UHFFFAOYSA-N 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 208000018459 dissociative disease Diseases 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 238000012215 gene cloning Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 2
- 238000010324 immunological assay Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 150000002989 phenols Chemical class 0.000 description 2
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 230000008707 rearrangement Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- CWLYDTVACYGEPD-UHFFFAOYSA-M sodium;2-[[3,7-bis(dimethylamino)phenothiazine-10-carbonyl]amino]acetate Chemical compound [Na+].C1=C(N(C)C)C=C2SC3=CC(N(C)C)=CC=C3N(C(=O)NCC([O-])=O)C2=C1 CWLYDTVACYGEPD-UHFFFAOYSA-M 0.000 description 2
- ZPCAZHPYLUKSMY-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate;dihydrate Chemical compound O.O.[Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 ZPCAZHPYLUKSMY-UHFFFAOYSA-M 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000000057 synthetic resin Substances 0.000 description 2
- 229920003002 synthetic resin Polymers 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- PVGATNRYUYNBHO-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(2,5-dioxopyrrol-1-yl)butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O PVGATNRYUYNBHO-UHFFFAOYSA-N 0.000 description 1
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- PHOLIFLKGONSGY-CSKARUKUSA-N (e)-(3-methyl-1,3-benzothiazol-2-ylidene)hydrazine Chemical compound C1=CC=C2S\C(=N\N)N(C)C2=C1 PHOLIFLKGONSGY-CSKARUKUSA-N 0.000 description 1
- CCVYCJAZARDILT-UHFFFAOYSA-N 1,1-dimethyl-2,2-diphenylhydrazine Chemical compound C=1C=CC=CC=1N(N(C)C)C1=CC=CC=C1 CCVYCJAZARDILT-UHFFFAOYSA-N 0.000 description 1
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- ZIRURAJAJIQZFG-UHFFFAOYSA-N 1-aminopropane-1-sulfonic acid Chemical compound CCC(N)S(O)(=O)=O ZIRURAJAJIQZFG-UHFFFAOYSA-N 0.000 description 1
- QZTKDVCDBIDYMD-UHFFFAOYSA-N 2,2'-[(2-amino-2-oxoethyl)imino]diacetic acid Chemical compound NC(=O)CN(CC(O)=O)CC(O)=O QZTKDVCDBIDYMD-UHFFFAOYSA-N 0.000 description 1
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- XUJZNKFIBZHDKL-UHFFFAOYSA-N 2-[[3,7-bis(dimethylamino)phenothiazine-10-carbonyl]amino]acetic acid Chemical compound C1=C(N(C)C)C=C2SC3=CC(N(C)C)=CC=C3N(C(=O)NCC(O)=O)C2=C1 XUJZNKFIBZHDKL-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- LVQFQZZGTZFUNF-UHFFFAOYSA-N 2-hydroxy-3-[4-(2-hydroxy-3-sulfonatopropyl)piperazine-1,4-diium-1-yl]propane-1-sulfonate Chemical compound OS(=O)(=O)CC(O)CN1CCN(CC(O)CS(O)(=O)=O)CC1 LVQFQZZGTZFUNF-UHFFFAOYSA-N 0.000 description 1
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 1
- HSXUNHYXJWDLDK-UHFFFAOYSA-N 2-hydroxypropane-1-sulfonic acid Chemical compound CC(O)CS(O)(=O)=O HSXUNHYXJWDLDK-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- XEZFKMUDMLXWAW-UHFFFAOYSA-N 3,7-bis(dimethylamino)-n-methylphenothiazine-10-carboxamide Chemical compound CN(C)C1=CC=C2N(C(=O)NC)C3=CC=C(N(C)C)C=C3SC2=C1 XEZFKMUDMLXWAW-UHFFFAOYSA-N 0.000 description 1
- MENXRDLDYNLDHE-UHFFFAOYSA-N 3-(3,5-dimethoxyanilino)propane-1-sulfonic acid Chemical compound COC1=CC(NCCCS(O)(=O)=O)=CC(OC)=C1 MENXRDLDYNLDHE-UHFFFAOYSA-N 0.000 description 1
- INEWUCPYEUEQTN-UHFFFAOYSA-N 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid Chemical compound OS(=O)(=O)CC(O)CNC1CCCCC1 INEWUCPYEUEQTN-UHFFFAOYSA-N 0.000 description 1
- BTIDJAQNJLWPTI-UHFFFAOYSA-N 3-(n-ethyl-3,5-dimethoxyanilino)-2-hydroxypropane-1-sulfonic acid Chemical compound OS(=O)(=O)CC(O)CN(CC)C1=CC(OC)=CC(OC)=C1 BTIDJAQNJLWPTI-UHFFFAOYSA-N 0.000 description 1
- NZAVBNVWEPQSBL-UHFFFAOYSA-N 3-(n-ethyl-3,5-dimethoxyanilino)propane-1-sulfonic acid Chemical compound OS(=O)(=O)CCCN(CC)C1=CC(OC)=CC(OC)=C1 NZAVBNVWEPQSBL-UHFFFAOYSA-N 0.000 description 1
- CDGBQMHYFARRCC-UHFFFAOYSA-N 3-(n-ethyl-3,5-dimethylanilino)-2-hydroxypropane-1-sulfonic acid Chemical compound OS(=O)(=O)CC(O)CN(CC)C1=CC(C)=CC(C)=C1 CDGBQMHYFARRCC-UHFFFAOYSA-N 0.000 description 1
- NPROGRQJOGOVDS-UHFFFAOYSA-N 3-(n-ethyl-3,5-dimethylanilino)propane-1-sulfonic acid Chemical compound OS(=O)(=O)CCCN(CC)C1=CC(C)=CC(C)=C1 NPROGRQJOGOVDS-UHFFFAOYSA-N 0.000 description 1
- ZLQNJZKBJSMXCJ-UHFFFAOYSA-N 3-(n-ethyl-3-methoxyanilino)-2-hydroxypropane-1-sulfonic acid Chemical compound OS(=O)(=O)CC(O)CN(CC)C1=CC=CC(OC)=C1 ZLQNJZKBJSMXCJ-UHFFFAOYSA-N 0.000 description 1
- STBWJPWQQLXSCK-UHFFFAOYSA-N 3-(n-ethyl-3-methoxyanilino)propane-1-sulfonic acid Chemical compound OS(=O)(=O)CCCN(CC)C1=CC=CC(OC)=C1 STBWJPWQQLXSCK-UHFFFAOYSA-N 0.000 description 1
- IBSUMVZKDLDAEK-UHFFFAOYSA-N 3-(n-ethyl-3-methylanilino)propane-1-sulfonic acid Chemical compound OS(=O)(=O)CCCN(CC)C1=CC=CC(C)=C1 IBSUMVZKDLDAEK-UHFFFAOYSA-N 0.000 description 1
- HXITYOAFXWBMLL-UHFFFAOYSA-N 3-(n-ethylanilino)propane-1-sulfonic acid Chemical compound OS(=O)(=O)CCCN(CC)C1=CC=CC=C1 HXITYOAFXWBMLL-UHFFFAOYSA-N 0.000 description 1
- NYLUYMWPXIIXDX-UHFFFAOYSA-N 3-(phenylazaniumyl)propane-1-sulfonate Chemical compound OS(=O)(=O)CCCNC1=CC=CC=C1 NYLUYMWPXIIXDX-UHFFFAOYSA-N 0.000 description 1
- RZQXOGQSPBYUKH-UHFFFAOYSA-N 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound OCC(CO)(CO)NCC(O)CS(O)(=O)=O RZQXOGQSPBYUKH-UHFFFAOYSA-N 0.000 description 1
- GIAVHGFPMPSIFI-UHFFFAOYSA-N 3-hydroxy-2,4,6-triiodobenzoic acid Chemical compound OC(=O)C1=C(I)C=C(I)C(O)=C1I GIAVHGFPMPSIFI-UHFFFAOYSA-N 0.000 description 1
- BNKQFTFTSQDHMT-UHFFFAOYSA-N 3-phenyldioxetane;sodium Chemical compound [Na].[Na].C1OOC1C1=CC=CC=C1 BNKQFTFTSQDHMT-UHFFFAOYSA-N 0.000 description 1
- HUDPLKWXRLNSPC-UHFFFAOYSA-N 4-aminophthalhydrazide Chemical compound O=C1NNC(=O)C=2C1=CC(N)=CC=2 HUDPLKWXRLNSPC-UHFFFAOYSA-N 0.000 description 1
- 239000007991 ACES buffer Substances 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 108700016232 Arg(2)-Sar(4)- dermorphin (1-4) Proteins 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 102000006942 B-Cell Maturation Antigen Human genes 0.000 description 1
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 description 1
- 239000008000 CHES buffer Substances 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 101710184216 Cardioactive peptide Proteins 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- SDKQRNRRDYRQKY-UHFFFAOYSA-N Dioxacarb Chemical compound CNC(=O)OC1=CC=CC=C1C1OCCO1 SDKQRNRRDYRQKY-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101710121417 Envelope glycoprotein Proteins 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- OZLGRUXZXMRXGP-UHFFFAOYSA-N Fluo-3 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=C(C=2)C2=C3C=C(Cl)C(=O)C=C3OC3=CC(O)=C(Cl)C=C32)N(CC(O)=O)CC(O)=O)=C1 OZLGRUXZXMRXGP-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- OWXMKDGYPWMGEB-UHFFFAOYSA-N HEPPS Chemical compound OCCN1CCN(CCCS(O)(=O)=O)CC1 OWXMKDGYPWMGEB-UHFFFAOYSA-N 0.000 description 1
- GIZQLVPDAOBAFN-UHFFFAOYSA-N HEPPSO Chemical compound OCCN1CCN(CC(O)CS(O)(=O)=O)CC1 GIZQLVPDAOBAFN-UHFFFAOYSA-N 0.000 description 1
- 101710154606 Hemagglutinin Proteins 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 208000002979 Influenza in Birds Diseases 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000005348 Neuraminidase Human genes 0.000 description 1
- 108010006232 Neuraminidase Proteins 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 239000007990 PIPES buffer Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- 101100431670 Rattus norvegicus Ybx3 gene Proteins 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- UZMAPBJVXOGOFT-UHFFFAOYSA-N Syringetin Natural products COC1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UZMAPBJVXOGOFT-UHFFFAOYSA-N 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 239000007997 Tricine buffer Substances 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 208000034953 Twin anemia-polycythemia sequence Diseases 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- QWXOJIDBSHLIFI-UHFFFAOYSA-N [3-(1-chloro-3'-methoxyspiro[adamantane-4,4'-dioxetane]-3'-yl)phenyl] dihydrogen phosphate Chemical compound O1OC2(C3CC4CC2CC(Cl)(C4)C3)C1(OC)C1=CC=CC(OP(O)(O)=O)=C1 QWXOJIDBSHLIFI-UHFFFAOYSA-N 0.000 description 1
- XYIPYISRNJUPBA-UHFFFAOYSA-N [3-(3'-methoxyspiro[adamantane-2,4'-dioxetane]-3'-yl)phenyl] dihydrogen phosphate Chemical compound O1OC2(C3CC4CC(C3)CC2C4)C1(OC)C1=CC=CC(OP(O)(O)=O)=C1 XYIPYISRNJUPBA-UHFFFAOYSA-N 0.000 description 1
- ZTOJFFHGPLIVKC-CLFAGFIQSA-N abts Chemical compound S/1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C\1=N\N=C1/SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-CLFAGFIQSA-N 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- HAXFWIACAGNFHA-UHFFFAOYSA-N aldrithiol Chemical compound C=1C=CC=NC=1SSC1=CC=CC=N1 HAXFWIACAGNFHA-UHFFFAOYSA-N 0.000 description 1
- 229910001508 alkali metal halide Inorganic materials 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- WOLHOYHSEKDWQH-UHFFFAOYSA-N amantadine hydrochloride Chemical compound [Cl-].C1C(C2)CC3CC2CC1([NH3+])C3 WOLHOYHSEKDWQH-UHFFFAOYSA-N 0.000 description 1
- 229960001280 amantadine hydrochloride Drugs 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000002280 amphoteric surfactant Substances 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000003452 antibody preparation method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 206010064097 avian influenza Diseases 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 239000007998 bicine buffer Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000012767 chemiluminescent enzyme immunoassay Methods 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000009918 complex formation Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- KCFYHBSOLOXZIF-UHFFFAOYSA-N dihydrochrysin Natural products COC1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 KCFYHBSOLOXZIF-UHFFFAOYSA-N 0.000 description 1
- WCIOMKKBXBFVLC-UHFFFAOYSA-L disodium phenoxymethyl phosphate Chemical compound [Na+].[Na+].O(C1=CC=CC=C1)COP([O-])([O-])=O WCIOMKKBXBFVLC-UHFFFAOYSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000002875 fluorescence polarization Methods 0.000 description 1
- YFHXZQPUBCBNIP-UHFFFAOYSA-N fura-2 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=3OC(=CC=3C=2)C=2OC(=CN=2)C(O)=O)N(CC(O)=O)CC(O)=O)=C1 YFHXZQPUBCBNIP-UHFFFAOYSA-N 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000010185 immunofluorescence analysis Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 229960003971 influenza vaccine Drugs 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000002796 luminescence method Methods 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 125000000040 m-tolyl group Chemical group [H]C1=C([H])C(*)=C([H])C(=C1[H])C([H])([H])[H] 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 229910001437 manganese ion Inorganic materials 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 108010077372 mast cell degranulating peptide Proteins 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000012514 monoclonal antibody product Substances 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- JSABTPDNEXHNOQ-UHFFFAOYSA-N n-phenylaniline;sodium Chemical compound [Na].C=1C=CC=CC=1NC1=CC=CC=C1 JSABTPDNEXHNOQ-UHFFFAOYSA-N 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 239000004745 nonwoven fabric Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- VSZGPKBBMSAYNT-RRFJBIMHSA-N oseltamivir Chemical compound CCOC(=O)C1=C[C@@H](OC(CC)CC)[C@H](NC(C)=O)[C@@H](N)C1 VSZGPKBBMSAYNT-RRFJBIMHSA-N 0.000 description 1
- 229960002194 oseltamivir phosphate Drugs 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 229920003217 poly(methylsilsesquioxane) Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- SVLRFMQGKVFRTB-UHFFFAOYSA-M sodium;3-(3,5-dimethoxyanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].COC1=CC(NCC(O)CS([O-])(=O)=O)=CC(OC)=C1 SVLRFMQGKVFRTB-UHFFFAOYSA-M 0.000 description 1
- IRQRBVOQGUPTLG-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 IRQRBVOQGUPTLG-UHFFFAOYSA-M 0.000 description 1
- VRJUJRXWRNPXLV-UHFFFAOYSA-M sodium;3-(n-ethyl-4-fluoro-3,5-dimethoxyanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC(OC)=C(F)C(OC)=C1 VRJUJRXWRNPXLV-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000008362 succinate buffer Substances 0.000 description 1
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
-
- 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
Definitions
- the present invention relates to an influenza virus detection or quantification device, an influenza virus detection or quantification kit, an influenza virus detection or quantification method, a human anti-type A influenza virus nucleoprotein antibody, and a human anti-type B influenza virus nucleoprotein antibody. About.
- Immunoassays using antigen-antibody reactions are widely used to measure analytes in specimens, such as enzyme immunoassay (ELISA), radioimmunoassay (RIA), chemiluminescent enzyme immunoassay Many measurement methods have been developed, such as a turbidimetric immunoassay method and a measurement method using surface plasmon resonance (SPR).
- ELISA enzyme immunoassay
- RIA radioimmunoassay
- SPR surface plasmon resonance
- Immunochromatography has attracted attention from the viewpoint of simplicity and speed, and many products based on this method have already been distributed.
- Immunochromatography is classified into a flow-through type and a lateral flow type based on its measurement principle, but in recent years, lateral flow type immunochromatography has become the mainstream.
- lateral flow type immunochromatography the region where the labeled antibody, which is supported on the support so as to be movable and the label is bound to the antibody that binds to the analyte, is supported on the support so that it can move. And a region in which an antibody that binds to an object to be measured is immobilized and has been reported.
- Influenza pathogen viruses are classified into three types, A type, B type and C type, depending on the antigenicity of soluble nucleoprotein (hereinafter abbreviated as NP) in the virus. Furthermore, it is classified into subtypes depending on the antigenicity of two envelope glycoproteins, hemagglutinin (abbreviated as HA) and neuraminidase (abbreviated as NA) present on the surface of the virus.
- NP soluble nucleoprotein
- HA hemagglutinin
- NA neuraminidase
- anti-influenza drugs have been found and widely used as therapeutic agents in addition to vaccines used for prevention.
- These therapeutic agents include amantadine hydrochloride adapted to influenza B virus, zanavir and oseltamivir phosphate adapted to influenza A virus and A type.
- influenza virus In selecting these therapeutic agents, it is important to detect the influenza virus in the specimen and identify whether the infection is caused by the influenza virus and the virus type is type A or type B.
- influenza A virus is more infectious than influenza B virus and causes serious symptoms. Therefore, it is important to identify the type of infecting virus for early treatment.
- influenza viruses have been detected by anti-influenza virus antibodies.
- an antibody against influenza virus an antibody that recognizes the HA region, an antibody that recognizes the NA region, an antibody that recognizes a matrix protein (such as M1), an antibody that recognizes a non-structural protein (such as NS1), and specifically recognizes NP
- M1 matrix protein
- NS1 non-structural protein
- NP non-structural protein
- the present inventors have found that human anti-influenza virus nucleoprotein antibodies can be used in influenza virus detection or quantification devices and influenza virus detection or quantification kits. Further, the inventors have found that antibodies having high affinity can be obtained from lymphocytes extracted from humans vaccinated with influenza vaccine, and have completed the present invention. That is, the present invention relates to the following [1] to [37].
- a device for detecting or quantifying influenza virus in a sample comprising a detection region, a sample supply region and a sample movement region in which a human anti-influenza virus nucleoprotein antibody is immobilized on a support.
- Anti-influenza virus nucleoprotein antibody (antibody 1) has a detection region immobilized on a support, a sample supply region and a sample transfer region, and a label is bound to the anti-influenza virus nucleoprotein antibody (antibody 2).
- a device for detecting or quantifying influenza virus in a sample wherein the labeled antibody is supplied from a sample supply region, and at least one of antibody 1 and antibody 2 is a human anti-influenza virus nucleoprotein antibody.
- the human anti-influenza virus nucleoprotein antibody is a human anti-influenza A virus nucleoprotein antibody.
- a human anti-influenza A virus nucleoprotein monoclonal antibody that specifically reacts with influenza A virus nucleoprotein but does not react with influenza B virus nucleoprotein [5] A device.
- the amino acid sequence of the heavy chain variable region of the human anti-influenza A virus nucleoprotein monoclonal antibody includes the amino acid sequence represented by any of SEQ ID NOs: 8 to 13, and the amino acid sequence of the light chain variable region is [6]
- a human anti-influenza B virus nucleoprotein monoclonal antibody that specifically reacts with influenza B virus nucleoprotein but does not react with influenza A virus nucleoprotein [8] The device according to item. [10] The amino acid sequence of the heavy chain variable region of the human anti-influenza B virus nucleoprotein monoclonal antibody comprises the amino acid sequence represented by SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is SEQ ID NO: 28 [9] The device according to [9], comprising the amino acid sequence represented. [11] A kit for detecting or quantifying influenza virus, comprising a solid phase in which a human anti-influenza virus nucleoprotein antibody is immobilized on a carrier.
- a kit for detecting or quantifying influenza virus wherein at least one is a human anti-influenza virus nucleoprotein antibody.
- antibody 1 and antibody 2 is a human anti-influenza virus nucleoprotein antibody, an influenza virus detection or quantification kit.
- An influenza virus detection or quantification kit An influenza virus detection or quantification kit.
- Influenza virus detection or quantification kit [16] The kit according to any one of [11] to [15], wherein the human anti-influenza virus nucleoprotein antibody is a human anti-influenza A virus nucleoprotein antibody.
- the heavy chain variable region amino acid sequence of the human anti-influenza A virus nucleoprotein monoclonal antibody comprises the amino acid sequence represented by any of SEQ ID NOs: 8 to 13, and the light chain variable region amino acid sequence comprises:
- a human anti-influenza B virus nucleoprotein monoclonal antibody that specifically reacts with influenza B virus nucleoprotein but does not react with influenza A virus nucleoprotein [19] The kit according to [19]. [21] The amino acid sequence of the heavy chain variable region of the human anti-influenza B virus nucleoprotein monoclonal antibody includes the amino acid sequence represented by SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is SEQ ID NO: 28 [20] The kit according to [20], comprising the amino acid sequence represented.
- [22] (1) reacting a specimen with a human anti-influenza virus nucleoprotein antibody immobilized on a carrier; and (2) A method for detecting or quantifying influenza virus, comprising a step of measuring a physical change generated in step (1).
- an antibody 1 -influenza virus nucleoprotein-labeled antibody 2 complex thereon (3) A step of washing the carrier after step (2) to remove substances not bound on the carrier; (4) a step of measuring the label in the antibody 1-influenza virus nucleoprotein-labeled antibody 2 complex on the carrier after step (3), wherein at least one of antibody 1 and antibody 2 comprises: A method for detecting or quantifying influenza virus, which is a human anti-influenza virus nucleoprotein antibody.
- a sample is reacted with a labeled antibody (labeled antibody 2) in which a label is bound to an anti-influenza virus nucleoprotein antibody (antibody 2), to thereby give a complex of labeled antibody 2-influenza virus nucleoprotein Forming a body;
- the complex formed in the step (1) is reacted with an anti-influenza virus nucleoprotein antibody (antibody 1) immobilized on a carrier, and antibody 1 -influenza virus nucleoprotein-label on the carrier Forming a complex of conjugated antibody 2; (3) A step of washing the carrier after step (2) to remove substances not bound on the carrier; (4) a step of measuring the label in the antibody 1-influenza virus nucleoprotein-labeled antibody 2 complex on the carrier after step (3), wherein at least one of antibody 1 and antibody 2 comprises: A method for detecting or quantifying influenza virus, which is a human anti-influenza virus nucleoprotein antibody.
- a specimen and a human anti-influenza virus nucleoprotein antibody immobilized on a carrier are reacted in the presence of a labeled antigen analog in which the label is bound to the influenza virus nucleoprotein antigen analog.
- Forming a human anti-influenza virus nucleoprotein antibody-influenza virus nucleoprotein complex and a human anti-influenza virus nucleoprotein antibody-labeled antigen analog complex on a carrier (2) A step of washing the carrier after step (1) to remove substances not bound on the carrier; (3) A method for detecting or quantifying influenza virus, comprising a step of measuring a label in the complex on the carrier after step (2).
- [28] (1) reacting a specimen with an influenza virus nucleoprotein antigen analog immobilized on a carrier in the presence of a labeled antibody in which a label is bound to a human anti-influenza virus nucleoprotein antibody; Forming an influenza virus nucleoprotein antigen analog-labeled antibody complex on a carrier; (2) A step of washing the carrier after step (1) to remove substances not bound on the carrier; (3) A method for detecting or quantifying influenza virus, comprising a step of measuring a label in the complex on the carrier after step (2).
- a human anti-influenza A virus nucleoprotein monoclonal antibody that specifically reacts with influenza A virus nucleoprotein but does not react with influenza B virus nucleoprotein [29] The method described. [31]
- the amino acid sequence of the heavy chain variable region of the human anti-influenza A virus nucleoprotein monoclonal antibody comprises the amino acid sequence represented by any of SEQ ID NOs: 8 to 13, and the amino acid sequence of the light chain variable region is [30] The method according to [30], comprising the amino acid sequence represented by any of SEQ ID NOs: 22 to 27.
- the human anti-influenza virus nucleoprotein antibody is a human anti-influenza B virus nucleoprotein antibody.
- a human anti-influenza B virus nucleoprotein monoclonal antibody that specifically reacts with influenza B virus nucleoprotein but does not react with influenza A virus nucleoprotein [32] The method described.
- the amino acid sequence of the heavy chain variable region of the human anti-influenza B virus nucleoprotein monoclonal antibody comprises the amino acid sequence represented by SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is SEQ ID NO: 28 The method according to [33], comprising the amino acid sequence represented.
- the amino acid sequence of the heavy chain variable region includes the amino acid sequence represented by any of SEQ ID NOs: 8 to 13, and the amino acid sequence of the light chain variable region is represented by any of SEQ ID NOs: 22 to 27.
- a human anti-influenza A virus nucleoprotein monoclonal antibody that specifically reacts with influenza A virus nucleoprotein and does not react with influenza B virus nucleoprotein comprising the amino acid sequence of [36]
- a human anti-influenza B virus nucleoprotein monoclonal antibody that reacts specifically with influenza B virus nucleoprotein but does not react with influenza A virus nucleoprotein.
- Influenza B wherein the amino acid sequence of the heavy chain variable region comprises the amino acid sequence represented by SEQ ID NO: 14, and the amino acid sequence of the light chain variable region comprises the amino acid sequence represented by SEQ ID NO: 28 A human anti-influenza B virus nucleoprotein monoclonal antibody that reacts specifically with the virus nucleoprotein and does not react with the influenza A virus nucleoprotein.
- a highly sensitive and widely specific influenza virus detection or quantification device an influenza virus detection or quantification kit, an influenza virus detection or quantification method, and an antibody suitable for influenza virus detection or quantification are provided. Is done.
- FIG. 2-a shows devices using various combinations of an antibody used in the detection region (denoted as a solid phase in the figure) and an antibody used for labeling (denoted as a label in the figure). It is a photograph which shows the immunochromatogram. It is a photograph which shows the immunochromatogram of the device (device of Example 1) of this invention using a human anti-influenza A virus nucleoprotein antibody.
- FIG. 2-a shows devices using various combinations of an antibody used in the detection region (denoted as a solid phase in the figure) and an antibody used for labeling (denoted as a label in the figure). It is a photograph which shows the immunochromatogram. It is a photograph which shows the immunochromatogram of the device (device of Example 1) of this invention using a human anti-influenza A virus nucleoprotein antibody.
- FIG. 2-a shows devices using various combinations of an antibody used in the detection region (denoted as a solid phase in the figure) and an antibody used for labeling (denoted as a label in
- FIG. 2 is a view showing an antibody ⁇ chain expression vector and an antibody ⁇ chain expression vector used in the production of the human anti-influenza virus nucleoprotein antibody of the present invention.
- FIG. 4-a is a diagram showing an antibody heavy chain expression vector.
- FIG. 2 is a view showing an antibody ⁇ chain expression vector and an antibody ⁇ chain expression vector used in the production of the human anti-influenza virus nucleoprotein antibody of the present invention.
- FIG. 4-b shows the antibody light chain expression vector 1.
- FIG. FIG. 2 is a view showing an antibody ⁇ chain expression vector and an antibody ⁇ chain expression vector used in the production of the human anti-influenza virus nucleoprotein antibody of the present invention.
- FIG. 4-c shows the antibody light chain expression vector 2. It is a figure which shows the result of the competitive inhibition experiment (specific binding test) to the influenza virus nucleoprotein antigen of the human anti-influenza A virus nucleoprotein antibody of this invention.
- Underline, double underline, and wavy line represent CDR1, 2, and 3, respectively. It is the figure which showed DNA which codes the human anti-influenza A virus protein antibody 23G272 * H chain (sequence number: 2). Underlined portions indicate DNA sequences corresponding to frameworks 1, 2, and 3, and boxes indicate DNA sequences corresponding to CDRs 1, 2, and 3. It is the figure which showed DNA which codes the human anti-influenza A virus protein antibody 23G272 * L chain (sequence number: 16). Underlined portions indicate DNA sequences corresponding to frameworks 1, 2, and 3, and boxes indicate DNA sequences corresponding to CDRs 1, 2, and 3.
- Underlined portions indicate DNA sequences corresponding to frameworks 1, 2, and 3, and boxes indicate DNA sequences corresponding to CDRs 1, 2, and 3. It is the figure which showed DNA which codes the human anti-influenza A virus protein antibody 23G285 light chain (sequence number: 17). Underlined portions indicate DNA sequences corresponding to frameworks 1, 2, and 3, and boxes indicate DNA sequences corresponding to CDRs 1, 2, and 3. It is the figure which showed the amino acid sequence (sequence number: 10) corresponding to the human anti-influenza A virus protein antibody 23G285 H chain. Underline, double underline, and wavy line represent CDR1, 2, and 3, respectively.
- Underlined portions indicate DNA sequences corresponding to frameworks 1, 2, and 3, and boxes indicate DNA sequences corresponding to CDRs 1, 2, and 3. It is the figure which showed the amino acid sequence (sequence number: 11) corresponding to the human anti-influenza A virus protein antibody 23G312 H chain. Underline, double underline, and wavy line represent CDR1, 2, and 3, respectively. It is the figure which showed the amino acid sequence (sequence number: 25) corresponding to the human anti-influenza A virus protein antibody 23G312 L chain. Underline, double underline, and wavy line represent CDR1, 2, and 3, respectively.
- Underline, double underline, and wavy line represent CDR1, 2, and 3, respectively. It is the figure which showed the amino acid sequence (sequence number: 26) corresponding to the human anti-influenza A virus protein antibody 23G447 * L chain. Underline, double underline, and wavy line represent CDR1, 2, and 3, respectively. It is the figure which showed DNA which codes the human anti-influenza A virus protein antibody 23G494 * H chain (sequence number: 6). Underlined portions indicate DNA sequences corresponding to frameworks 1, 2, and 3, and boxes indicate DNA sequences corresponding to CDRs 1, 2, and 3. It is the figure which showed DNA which codes the human anti-influenza A virus protein antibody 23G494 * L chain (sequence number: 20).
- Underlined portions indicate DNA sequences corresponding to frameworks 1, 2, and 3, and boxes indicate DNA sequences corresponding to CDRs 1, 2, and 3. It is the figure which showed the amino acid sequence (sequence number: 13) corresponding to the human anti-influenza A virus protein antibody 23G494 H chain. Underline, double underline, and wavy line represent CDR1, 2, and 3, respectively. It is the figure which showed the amino acid sequence (sequence number: 27) corresponding to the human anti-influenza A virus protein antibody 23G494 L chain. Underline, double underline, and wavy line represent CDR1, 2, and 3, respectively. It is the figure which showed DNA which codes the human anti-type B influenza virus protein antibody 23G327 * H chain (sequence number: 7).
- Underlined portions indicate DNA sequences corresponding to frameworks 1, 2, and 3, and boxes indicate DNA sequences corresponding to CDRs 1, 2, and 3. It is the figure which showed DNA which codes the human anti-B influenza virus protein antibody 23G327 * L chain (sequence number: 21). Underlined portions indicate DNA sequences corresponding to frameworks 1, 2, and 3, and boxes indicate DNA sequences corresponding to CDRs 1, 2, and 3. It is the figure which showed the amino acid sequence (SEQ ID NO: 14) corresponding to the human anti-B influenza virus protein antibody 23G327 H chain. Underline, double underline, and wavy line represent CDR1, 2, and 3, respectively. It is the figure which showed the amino acid sequence (SEQ ID NO: 28) corresponding to the human anti-B influenza virus protein antibody 23G327 L chain. Underline, double underline, and wavy line represent CDR1, 2, and 3, respectively.
- influenza virus detection or quantification device of the present invention is a device that can be used for detection or quantification of influenza virus, and is specifically a device suitable for immunochromatography and the like.
- the device of the present invention may be either a flow-through type or a lateral flow type.
- influenza virus detection or quantification device of the present invention comprises a detection region, a sample supply region, and a sample movement region, and a human anti-influenza virus nucleoprotein antibody is immobilized on a support in the detection region. It is a device characterized by this.
- the device is suitable for a crystal oscillator microbalance (QCM) sensor or a carbon nanotube (CNT) sensor.
- QCM crystal oscillator microbalance
- CNT carbon nanotube
- influenza virus detection or quantification device of the present invention comprises a detection region, a sample supply region, and a sample movement region, and in the detection region, an anti-influenza virus nucleoprotein antibody (
- the antibody 1) is immobilized, and a labeled antibody in which a label is bound to the anti-influenza virus nucleoprotein antibody (antibody 2) is supplied from the specimen supply region, and at least one of antibody 1 and antibody 2 is a human anti-influenza
- the device does not include a labeling reagent region, but the labeled antibody is supplied from the sample supply region together with the sample.
- influenza virus nucleoprotein-labeled antibody complex produced by the reaction between the influenza virus nucleoprotein and the labeled antibody in the sample reaches the detection region through the sample movement region.
- the complex reacts with the immobilized antibody 1 in the detection region to form an antibody 1 -influenza virus nucleoprotein-labeled antibody complex in the detection region.
- influenza virus By detecting the label in the produced antibody 1-influenza virus nucleoprotein-labeled antibody complex, influenza virus can be detected or quantified.
- Another aspect of the influenza virus detection or quantification device of the present invention comprises a detection region, a sample supply region, a sample movement region, and a labeling reagent region, and in the detection region, an anti-influenza virus is formed on a support constituting the region.
- a nucleoprotein antibody (antibody 1) is immobilized, and a labeled antibody having a label bound to the anti-influenza virus nucleoprotein antibody (antibody 2) is held so as to be movable to the labeling reagent region.
- At least one of the antibodies 2 is a device characterized in that it is a human anti-influenza virus nucleoprotein antibody.
- the specimen supplied to the specimen supply area expands in the specimen movement area and reaches the labeling reagent area.
- the influenza virus nucleoprotein in the sample reacts with the labeled antibody to form an influenza virus nucleoprotein-labeled antibody complex, and the generated complex further expands the sample movement region and detects it. Reach the area.
- the complex reacts with immobilized antibody 1 to form an antibody 1 -influenza virus nucleoprotein-labeled antibody complex in the detection region.
- the sample supply region may also serve as the labeling reagent region [see FIGS. 1 (1) and (2)].
- the influenza virus detection or quantification device of the present invention comprises a detection region in which a human anti-influenza virus nucleoprotein antibody is immobilized on a support, a sample supply region, and a sample transfer region, and an influenza virus nucleoprotein antigen Labeled to a device in which a labeled antigen analog to which a label is bound is supplied from a specimen supply region, a detection region in which a human anti-influenza virus nucleoprotein antibody is immobilized on a support, or an influenza virus nucleoprotein antigen Also included is a device comprising a labeled reagent region, a sample supply region, and a sample moving region that are held on a support so that a labeled antigen analog bound to can be moved.
- influenza virus detection or quantification device of the present invention comprises a detection region in which an influenza virus nucleoprotein antigen analog is immobilized on a support, a sample supply region, and a sample transfer region, and a human anti-influenza virus nucleus.
- the specimen used for detection or quantification of influenza virus using the device of the present invention is not particularly limited as long as it can contain influenza virus collected from humans.
- nasal swab nasal swab
- nasal cavity examples include biological samples such as suction liquid, pharyngeal swab liquid (pharyngeal swab), sputum liquid, and the like.
- these biological samples can be used as they are, but pretreated biological samples can also be used.
- the pretreatment agent used in the pretreatment is not particularly limited as long as it is a pretreatment agent that destroys influenza virus nuclei and enables reaction with anti-influenza virus nucleoprotein antibodies. Etc.
- the surfactant examples include a nonionic surfactant, a cationic surfactant, an anionic surfactant, and an amphoteric surfactant, and a nonionic surfactant is preferable.
- nonionic surfactants include polyoxyethylene surfactants (eg, Nonidet P-40).
- the pretreatment agent may contain an aqueous medium, a buffering agent, a preservative, salts, saccharides, metal ions, proteins, and the like, as necessary.
- the aqueous medium include deionized water, distilled water, and membrane filtered water.
- Buffers include, for example, lactate buffer, citrate buffer, acetate buffer, succinate buffer, phthalate buffer, phosphate buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur tool Buffering agents, imidazole buffering agents, malic acid buffering agents, oxalic acid buffering agents, glycine buffering agents, boric acid buffering agents, carbonate buffering agents, Good buffering agents and the like can be mentioned.
- Good buffering agents include, for example, Tris [tris (hydroxymethyl) aminomethane] buffer, MES (2-morpholinoethanesulfonic acid) buffer, bis-tris [bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane] Buffer, ADA [N- (2-acetamido) iminodiacetic acid] buffer, PIPES [piperazine-N, N′-bis (2-ethanesulfonic acid)] buffer, ACES ⁇ 2- [N- (2- Acetamido) amino] ethanesulfonic acid ⁇ buffer, MOPSO (3-morpholino-2-hydroxypropanesulfonic acid) buffer, BES ⁇ 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid ⁇ buffer Agent, MOPS (3-morpholinopropanesulfonic acid) buffer, TES ⁇ 2- ⁇ N- [tris (hydroxy Methyl) methyl] amino ⁇ ethanes
- Examples of the preservative include sodium azide and antibiotics.
- Examples of the salts include alkali metal halide salts such as sodium chloride and potassium chloride.
- Examples of the saccharide include mannitol, sorbitol, sucrose and the like.
- Examples of the metal ion include magnesium ion, manganese ion, zinc ion and the like.
- Examples of the protein include bovine serum albumin (BSA), casein, Block Ace TM (Dainippon Pharmaceutical Co., Ltd.), animal serum and the like.
- the sample When the sample is added to the sample supply region, the sample develops on the support by capillary action and reaches the labeling reagent region.
- the influenza virus nucleoprotein in the sample reacts with the labeled antibody 2 in which the label is bound to the anti-influenza virus nucleoprotein antibody (antibody 2) in the labeling reagent region, and the influenza virus nucleoprotein-labeled antibody A complex (complex 1) is generated.
- the produced complex 1 further develops on the support by capillary action and reaches the detection region.
- the complex 1 that has reached the detection region reacts with the immobilized anti-influenza virus nucleoprotein antibody (antibody 1) in the region, and the antibody 1-influenza virus nucleoprotein-labeled antibody 2 complex (complex 2) is generated immobilized on the support.
- the influenza virus in the sample can be detected or quantified.
- at least one of antibody 1 and antibody 2 is a human anti-influenza virus nucleoprotein antibody.
- the combination of antibody 1 and antibody 2 includes human anti-influenza virus nucleoprotein antibody-non-human anti-influenza virus nucleoprotein antibody, non-human anti-influenza virus nucleoprotein antibody-human anti-influenza virus nucleoprotein antibody, human anti-influenza virus nucleoprotein An antibody-human anti-influenza virus nucleoprotein antibody combination may be mentioned.
- influenza viruses to be detected or quantified examples include influenza A virus and influenza B virus.
- Influenza virus nucleoprotein is a protein present in the nucleus of the influenza virus, that is, one of the components of the influenza virus. Therefore, detecting or quantifying the influenza virus in a sample by measuring the influenza virus nucleoprotein Can do.
- anti-influenza A virus nucleoprotein antibody (antibody A1) as antibody 1 and anti-influenza A virus nucleoprotein antibody as antibody 2 (Antibody A2) is used.
- antibody A1 and antibody A2 is a human antibody, ie a human anti-influenza A virus nucleoprotein antibody.
- the combination of antibody A1 and antibody A2 includes a combination of human anti-influenza A virus nucleoprotein antibody and human anti-influenza A virus nucleoprotein antibody, human anti-influenza A virus nucleoprotein antibody and non-human anti-type A influenza Examples include a combination with a virus nucleoprotein antibody and a combination of a non-human anti-influenza A virus nucleoprotein antibody and a human anti-influenza A virus nucleoprotein antibody.
- the human anti-influenza A virus nucleoprotein antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
- the site in influenza A virus nucleoprotein recognized by antibody A1 and the site of influenza A virus nucleoprotein recognized by antibody A2 may be the same or different, but different. It is preferable.
- a movable influenza A virus nucleoprotein-labeled antibody A2 complex (complex A1) is generated in the labeled reagent region.
- an immobilized antibody A1-A influenza virus nucleoprotein-labeled antibody A2 complex (complex A2) is produced.
- anti-influenza B virus nucleoprotein antibody as antibody 1
- Antibody B2 anti-influenza B virus nucleoprotein antibody as antibody 2
- antibody B1 and antibody B2 are human antibodies, ie, a human anti-influenza B virus nucleoprotein antibody.
- the combination of antibody B1 and antibody B2 includes a combination of human anti-B influenza virus nucleoprotein antibody and human anti-B influenza virus nucleoprotein antibody, human anti-B influenza virus nucleoprotein antibody and non-human anti-B influenza A combination with a virus nucleoprotein antibody and a combination of a non-human anti-influenza B virus nucleoprotein antibody and a human anti-B influenza virus nucleoprotein antibody can be mentioned.
- the human anti-B influenza virus nucleoprotein antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
- influenza B virus In the case of a monoclonal antibody, the site in influenza B virus nucleoprotein recognized by antibody B1 and the site of influenza B virus nucleoprotein recognized by antibody B2 may be the same or different, but different. It is preferable.
- a complex of influenza B virus nucleoprotein-labeled antibody B2 (complex B1) is generated that is movable in the labeling reagent region. In the detection region, an immobilized antibody B1-B influenza virus nucleoprotein-labeled antibody B2 complex (complex B2) is produced.
- human anti-influenza A virus nucleoprotein antibodies include human anti-influenza A virus nucleoprotein antibodies described below.
- human anti-B influenza virus nucleoprotein antibodies include human anti-B influenza virus nucleoprotein antibodies described below.
- the influenza virus detection or quantification device of the present invention may further include one or more regions selected from the group consisting of a developing solution supply region, a surplus solution absorption region, and a sample supply confirmation region.
- the developing solution added to the developing solution supply region expands the support by capillary action, moves the specimen to the labeling reagent region, and detects the complex 1 (complex A1; complex B1) generated in the labeling reagent region. Move to area.
- the developing solution supply region can also serve as the sample supply region. Excess liquid other than the complex 2 (complex A2; complex B2) generated in the detection area is absorbed in the excess liquid absorption area.
- the addition of the sample can be confirmed by providing a sample supply confirmation region containing the immobilized anti-human IgG antibody on the support.
- the support in the device of the present invention is not particularly limited as long as it is a material in which the liquid can be developed in the specimen movement region.
- a material in which the liquid can be developed in the specimen movement region For example, glass fiber, cellulose, nylon, crosslinked dextran, various chromatographic papers, nitrocellulose And metals such as gold.
- the size of the support is not limited, but is preferably a strip having a width of about 3 mm to 10 mm and a length of about 30 mm to 100 mm. A support having a thickness of 100 ⁇ m to 1 mm can be used.
- the support is partially or wholly blocked with, for example, animal serum such as bovine serum albumin (BSA) or casein, casein, sucrose, or the like in order to prevent non-specific adsorption of the sample-derived protein to the support during measurement. Can be used.
- BSA bovine serum albumin
- an anti-influenza virus nucleoprotein antibody is immobilized.
- the detection region may be formed separately from the support, but is preferably formed on the support.
- the anti-influenza virus nucleoprotein antibody immobilized on the detection region may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
- At least one of an anti-influenza virus nucleoprotein antibody immobilized in the detection region and an anti-influenza virus nucleoprotein antibody that forms a labeled anti-influenza virus nucleoprotein antibody held movably in the labeling reagent region Are human antibodies, preferably both antibodies are monoclonal antibodies.
- anti-influenza virus nucleoprotein antibodies immobilized in the detection region include human anti-influenza A virus nucleoprotein antibodies, human anti-influenza B virus nucleoprotein antibodies described below, and fragments of these human antibodies [Fab, F ( ab ′) 2 , F (ab ′)] and the like.
- Examples of the method for immobilizing the anti-influenza virus nucleoprotein antibody in the detection region include a method in which the anti-influenza virus nucleoprotein antibody is directly physically adsorbed on the support, a method in which the antibody is immobilized on the support by a chemical bond such as a covalent bond, etc. Can be mentioned.
- an anti-influenza virus nucleoprotein antibody may be bound to insoluble carrier particles and contained in the support.
- examples of insoluble carrier particles include polystyrene latex particles, magnetic particles, and glass fibers.
- Examples of the method for binding the anti-influenza virus nucleoprotein antibody to the insoluble carrier particles include the aforementioned physical adsorption and chemical binding.
- the detection area is present downstream of the labeling reagent area, the specimen supply area, the specimen movement area, and the developing liquid supply area, and is present upstream of the surplus liquid absorption area.
- Labeling reagent region In the labeling reagent region of the device of the present invention, a labeled antibody in which a label is bound to an anti-influenza virus nucleoprotein antibody is held so as to be movable.
- the labeling reagent region may be formed separately from the support, but is preferably formed on the support.
- the anti-influenza virus nucleoprotein antibody that forms a labeled anti-influenza virus nucleoprotein antibody that is movably retained in the labeling reagent region may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
- Examples of the anti-influenza virus nucleoprotein antibody retained so as to be movable to the labeling reagent region include human anti-influenza A virus nucleoprotein antibodies, human anti-influenza B virus nucleoprotein antibodies described below, and fragments of these human antibodies. [Fab, F (ab ′) 2 , F (ab ′)] and the like.
- Examples of the method for constructing the labeled reagent region include a method of spotting a labeled antibody-containing reagent on a support, a method of laminating a water-absorbing pad containing a labeled antibody-containing reagent on a support, and the like. It is done.
- Examples of the water-absorbing pad include a water-absorbing pad used in a specimen supply region described later.
- Examples of the label that forms the labeled antibody include enzymes such as peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, metal colloid particles such as gold colloid particles and selenium colloid particles, colored latex particles, luminescent substances, and fluorescent substances. It is done.
- Examples of the preparation method of the labeled antibody include a method of binding the label and the antibody by covalent bond, a method of binding the label and the antibody by non-covalent bond, and the like. Specific examples include known methods such as the glutaraldehyde method, periodate method, maleimide method, pyridyl disulfide method, methods using various cross-linking agents, etc. ["Protein Nucleic Acid Enzyme", Vol. 31, 37-45 (1985)].
- the crosslinking agent for example, N-succinimidyl-4-maleimidobutyric acid (GMBS), N-succinimidyl-6-maleimidohexanoic acid, N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid, etc. may be used.
- GMBS N-succinimidyl-4-maleimidobutyric acid
- N-succinimidyl-6-maleimidohexanoic acid N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid, etc.
- a functional group present in the antibody can be used.
- a functional group such as an amino group, a carboxyl group, a hydroxyl group, or a sulfhydryl group is introduced by a conventional method, and then a labeled antibody is prepared by the above
- the sample supply region in the device of the present invention is located upstream of the detection region, and the sample is supplied through this region.
- the specimen supply region is formed on the support, but may be formed by laminating a water absorbing pad on the support.
- the support and the water-absorbing pad that form the specimen supply region may appropriately contain salts, surfactants, and the like. Examples of the salts and surfactants include the aforementioned salts and surfactants.
- a water absorbing pad glass fiber, a cellulose, a nonwoven fabric, polyvinyl alcohol etc. are mentioned, for example.
- the sample supply region may also serve as the labeling reagent region described above.
- the sample can be efficiently collected by laminating a water-absorbing pad containing a labeled antibody-containing reagent on the support. Can be absorbed into.
- the water-absorbing pad is not particularly limited as long as it is a material that absorbs labeled antibody and influenza virus nucleoprotein and has little adsorption, and examples thereof include those described above.
- sample moving area in the device of the present invention is an area where the sample, the labeled antibody, and a developing solution described later move, which exist throughout the support.
- the specimen supplied to the specimen supply area is carried to the detection area together with the labeled antibody through the specimen movement area.
- the specimen can be transported to the detection area through the specimen moving area by the developing liquid supplied from the developing liquid supply area described later.
- region in the device of this invention is an area
- an anti-influenza virus nucleoprotein antibody-influenza virus nucleoprotein-labeled antibody complex is produced.
- the developing solution is an aqueous medium for developing the specimen on the support. If necessary, the aqueous medium includes the above-mentioned buffer, surfactant, preservative, salt, saccharide, metal ion, protein, and the like. An additive may be included.
- sample supply confirmation region in the device of the present invention is a region provided upstream of the surplus liquid absorption region and for confirming that the sample has been reliably added to the sample supply region of the device.
- the influenza virus nucleoprotein in the specimen is labeled antibody (labeled anti-influenza virus nucleus).
- the unreacted labeled antibody develops the specimen movement region together with the influenza virus nucleoprotein-labeled antibody complex formed by reaction with the protein antibody. That is, when the sample is reliably supplied, the unreacted labeled antibody develops the detection movement region.
- the specimen supply confirmation area is an area for capturing the unreacted labeled antibody, and can be constructed by immobilizing and immobilizing the antibody against the labeled antibody.
- Examples of the antibody that reacts with the labeled antibody include an antibody against an anti-influenza virus nucleoprotein antibody and an antibody against a label.
- Examples of the method for immobilizing an antibody that reacts with a labeled antibody include the method for immobilizing an antibody in the detection region described above.
- an antibody against a substance (for example, an enzyme) commonly contained in a specimen can be immobilized and immobilized to construct a specimen supply confirmation region (see (3) in FIG. 1).
- an antibody against a substance for example, an enzyme
- a labeled antigen analog labeled influenza virus nucleoprotein antigen analog
- the antibody against the antibody and the antibody against the labeled antibody can be immobilized and immobilized to construct a specimen supply confirmation region.
- the surplus liquid absorption region in the device of the present invention is provided on the most downstream side of the support, and if necessary on the support, the sample and the labeled antibody developed together with the developing solution are captured in the detection region.
- This is a region for producing a complex of anti-influenza nucleoprotein antibody-influenza nucleoprotein-labeled antibody and absorbing excess liquid after the complex is produced.
- the excess liquid absorption region can be formed from a support, but can also be formed by attaching a water-absorbing substance to the support, and can also be formed by laminating a water-absorbing substance on the support. . Examples of the water-absorbing substance include filter paper and sponge having high water absorption.
- Detection and quantification of influenza virus using the device of the present invention can be performed, for example, as follows.
- influenza virus nucleoprotein in the sample reacts with the human anti-influenza virus nucleoprotein antibody to produce an influenza virus nucleoprotein-human anti-influenza virus nucleoprotein antibody complex.
- Influenza virus can be detected or quantified by measuring physical changes associated with this complex formation. As a physical change here, a mass change etc. are mentioned, for example.
- the specimen and labeled antibody (labeled anti-influenza virus nucleoprotein antibody) supplied to the specimen supply area develops the detection movement area and reaches the detection area.
- the influenza virus nucleoprotein-labeled antibody complex produced at the time of supply or development reacts with the anti-influenza virus nucleoprotein antibody in the detection region, and the anti-influenza virus nucleoprotein antibody-influenza virus nucleoprotein-labeled antibody Create a complex.
- the influenza virus can be detected or quantified.
- the sample supplied to the sample supply region reacts with the labeled anti-influenza virus nucleoprotein antibody (labeled antibody 2) in the labeling reagent region, and the complex of influenza virus nucleoprotein-labeled antibody 2 (complex 1) is produced.
- the complex 1 produced and then developed through the specimen movement region to the detection region reacts with the anti-influenza virus nucleoprotein antibody (antibody 1) in the detection region, and antibody 1-anti-influenza virus nucleoprotein-labeled antibody 2
- This complex (complex 2) is immobilized and produced in the detection region, and the influenza virus can be detected or quantified by measuring the label of the complex 2 produced in the detection region.
- At least one of the anti-influenza virus nucleoprotein antibody (antibody 2) constituting the labeled anti-influenza virus nucleoprotein antibody (labeled antibody 2) and the antibody 1 is a human antibody.
- a developing solution can also be used for the development of the specimen supplied to the detection supply area and the development of the complex 1 generated in the labeling reagent area to the detection area.
- the specimen and labeled antigen analog (labeled influenza virus nucleoprotein antigen analog) supplied to the specimen supply area develops the detection movement area and reaches the detection area.
- the influenza virus nucleoprotein antigen and the labeled antigen analog in the sample react competitively with the human anti-influenza virus nucleoprotein antibody, and human anti-influenza virus nucleoprotein antibody-influenza virus nucleoprotein antigen And the human anti-influenza virus nucleoprotein antibody-labeled antigen analog complex.
- Influenza virus can be detected or quantified by measuring the label in the complex produced in this detection region.
- the specimen supplied to the specimen supply area expands the specimen movement area and reaches the labeling reagent area.
- the sample that has reached the labeling reagent region further develops the sample movement region together with the labeled antigen analog (labeled influenza virus nucleoprotein antigen analog) and reaches the detection region.
- the influenza virus nucleoprotein antigen and the labeled antigen analog in the sample react competitively with the human anti-influenza virus nucleoprotein antibody, and human anti-influenza virus nucleoprotein antibody-influenza virus nucleoprotein antigen And the human anti-influenza virus nucleoprotein antibody-labeled antigen analog complex.
- Influenza virus can be detected or quantified by measuring the label in the complex produced in this detection region.
- the specimen and labeled antibody (labeled human anti-influenza virus nucleoprotein antibody) supplied to the specimen supply area develops the detection movement area.
- the influenza virus nucleoprotein-labeled antibody complex produced at the time of supply or development reaches the detection region together with the unreacted labeled antibody.
- the unreacted labeled antibody reacts with the influenza virus nucleoprotein antigen analog to produce an influenza virus nucleoprotein antigen analog-labeled antibody complex.
- influenza virus can be detected or quantified.
- influenza virus nucleoprotein reacts with a labeled antibody (labeled human anti-influenza virus nucleoprotein antibody) to form an influenza virus nucleoprotein-labeled antibody complex.
- a labeled antibody labeled human anti-influenza virus nucleoprotein antibody
- the generated complex further develops the specimen movement region together with the unreacted labeled antibody, and reaches the detection region.
- the unreacted labeled antibody reacts with the influenza virus nucleoprotein antigen analog to produce an influenza virus nucleoprotein antigen analog-labeled antibody complex.
- the label in the complex generated in the detection region can be measured using a known method.
- the label is gold colloid particles or colored latex particles, it can be carried out by measuring the absorbance of the band formed on the support due to the formation of the complex.
- the label is an enzyme, the label can be measured by allowing the enzyme substrate to act on the enzyme.
- the detection method can be selected depending on the enzyme used and the substrate of the enzyme.
- the enzyme substrate can be contained in the developing solution, but can also be supported so as to be movable in the substrate reagent region provided in the device.
- the substrate include a chromogenic substrate and a luminescent substrate.
- the chromogenic substrate include a combination of a leuco chromogen and hydrogen peroxide, a combination of a coupling chromogen comprising two compounds and hydrogen peroxide, and the like.
- leuco chromogens include 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3 ′, 5,5′-tetramethylbenzidine (TMB).
- Diaminobenzidine (DAB), 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7-bis (dimethylamino)- 10H-phenothiazine (MCDP), N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine sodium salt (DA-64), 10-N- (carboxymethylaminocarbonyl) -3,7- Bis (dimethylamino) -10H-phenothiazine sodium salt (DA-67), 4,4'-bis And (dimethylamino) diphenylamine, bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine (BCMA), and the like.
- DAB 10-N-carboxymethylcarbamoyl-3,7-bis (
- Examples of the coupling type chromogen comprising a combination of two compounds include a combination of a coupler and anilines or phenols.
- Examples of the coupler include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazone.
- Examples of anilines include N-ethyl-N- (3-methylphenyl) -N′-succinylethylenediamine (EMSE) and N- (3,5-dimethoxyphenyl) -N′-succinylethylenediamine sodium salt (DOSE).
- phenols include phenol and 3-hydroxy-2,4,6-triiodobenzoic acid.
- chromogenic substrate examples include a combination of luminol and hydrogen peroxide, a combination of isoluminol and hydrogen peroxide, and the like.
- examples of the substrate include a chromogenic substrate, a fluorescent substrate, and a luminescent substrate. Detection or quantification is possible by measuring absorbance when the substrate is a chromogenic substrate, measuring fluorescence intensity when the substrate is a fluorescent substrate, and measuring luminescence intensity when the substrate is a luminescent substrate.
- examples of the chromogenic substrate include 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 4-nitrophenyl phosphate and the like.
- Examples of the fluorescent substrate include 4-methylum berylphenyl-phosphate (4MUP) ⁇ -D-galactosidase: 4-methylum berylphenyl- ⁇ -D-galactoside (4MUG).
- Examples of the luminescent substrate include 3- (2′-spiroadamantane) -4-methoxy-4- (3′-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD), 2-chloro-5- ⁇ 4-Methoxyspiro [1,2-dioxetane-3,2 ′-(5′-chloro) tricyclo [3.3.1.13,7] decan] -4-yl ⁇ phenyl phosphate disodium salt (CDP -Star TM ), 3- ⁇ 4-methoxyspiro [1,2-dioxetane-3,2 '-(5'-chloro) tricyclo [3.3.1.13,7] decan] -4-yl ⁇ phenyl
- the substrate of the enzyme is, for example, 3- (2′-spiroadamantane) -4-methoxy-4- (3 ′′ - ⁇ -D-galactopyranosyl) phenyl
- luminescent substrates such as -1,2-dioxetane (AMGPD).
- the device of the present invention only needs to contain a human anti-influenza virus nucleoprotein antibody as a constituent element, and the detection method in the detection region is not particularly limited.
- the detection method the color development method (absorbance method) described above is used.
- a method for detecting mass change can also be used. Examples of the method for detecting the mass change include a surface plasmon resonance (SPR) method, a method using a piezoelectric vibrator (for example, refer to WO2005 / 015217 pamphlet), and the like.
- Quantification of influenza virus in a sample using the device of the present invention can be performed as follows. First, using an influenza virus nucleoprotein prepared from a known concentration of influenza virus as a sample, the sample is supplied to the sample supply region of the device of the present invention, and information (for example, absorbance) in the detection region is measured. Create a calibration curve that represents the relationship between influenza virus concentration and information content. Thereafter, the same measurement is performed using the actual target specimen, and the concentration is determined by comparing the obtained information amount with the calibration curve created earlier.
- influenza virus detection or quantification method of the present invention is a method characterized by using at least one human anti-influenza virus nucleoprotein antibody. Influenza virus can be detected or quantified using the influenza virus detection or quantification kit of the present invention described later.
- the influenza virus detection or quantification method of the present invention is not particularly limited as long as it is a method capable of detecting or quantifying influenza virus.
- radioimmunoassay RIA
- immunoradiometric assay IRMA
- enzyme immunoassay ELISA
- Homogeneous enzyme immunoassay fluorescence immunoassay
- FIA fluorescence immunoassay
- IFMA immunofluorescence analysis
- fluorescence polarization fluorescence polarization
- CLIA chemiluminescence immunoassay
- CLIA chemiluminescence enzyme immunoassay
- turbidimetric immunoassay turbidimetric immunoassay
- SPR surface plasmon resonance Law
- influenza virus detection or quantification method of the present invention include the following methods.
- Method 1 (1) reacting a specimen with a human anti-influenza virus nucleoprotein antibody immobilized on a carrier; and (2) A method for detecting or quantifying influenza virus, comprising a step of measuring a physical change generated in step (1).
- Examples of the physical change of the physical change amount in the method include turbidity change and mass change.
- This method can be applied to a turbidimetric immunoassay method for measuring a turbidity change of a reaction solution accompanying an antigen-antibody reaction, a surface plasmon resonance method (SPR) for measuring a mass change accompanying an antigen-antibody reaction, or the like.
- SPR surface plasmon resonance method
- Method 2 (1) reacting a specimen with an anti-influenza virus nucleoprotein antibody (antibody 1) immobilized on a carrier to form an antibody 1-influenza virus complex on the carrier; (2) reacting the complex formed on the carrier in step (1) with an anti-influenza virus nucleoprotein antibody (antibody 2); and (3) An influenza virus comprising a step of measuring a physical change amount generated in step (2), wherein at least one of antibody 1 and antibody 2 is a human anti-influenza virus nucleoprotein antibody Detection or quantification method.
- antibody 1 and antibody 2 is a human anti-influenza virus nucleoprotein antibody Detection or quantification method.
- Antibody 1 and antibody 2 may be monoclonal antibodies or polyclonal antibodies, but both are preferably monoclonal antibodies.
- Examples of the physical change of the physical change amount in the method include turbidity change and mass change.
- This method can be applied to a turbidimetric immunoassay method for measuring a turbidity change of a reaction solution accompanying an antigen-antibody reaction, a surface plasmon resonance method (SPR) for measuring a mass change accompanying an antigen-antibody reaction, or the like.
- SPR surface plasmon resonance method
- Method 3 (1) reacting a specimen with an anti-influenza virus nucleoprotein antibody (antibody 1) immobilized on a carrier to form an antibody 1-influenza virus nucleoprotein complex on the carrier; (2) In step (1), the complex formed on the carrier is reacted with a labeled antibody (labeled antibody 2) in which a label is bound to the anti-influenza virus nucleoprotein antibody (antibody 2).
- an antibody 1 -influenza virus nucleoprotein-labeled antibody 2 complex thereon (3) A step of washing the carrier after step (2) to remove substances not bound on the carrier; (4) a step of measuring the label in the antibody 1-influenza virus nucleoprotein-labeled antibody 2 complex on the carrier after step (3), wherein at least one of antibody 1 and antibody 2 comprises: A method for detecting or quantifying influenza virus, which is a human anti-influenza virus nucleoprotein antibody.
- each of antibody 1 and antibody 2 in the above method may be a monoclonal antibody or a polyclonal antibody, but both are preferably monoclonal antibodies.
- the cleaning step (3) can be omitted (homogeneous method). Further, the step (1) and the step (2) may be performed simultaneously, and a cleaning step may be inserted between the step (1) and the step (2).
- Examples of the method for measuring the label in step (4) of the above method include the above-described methods.
- Method 4 (1) A sample is reacted with a labeled antibody (labeled antibody 2) having a label bound to an anti-influenza virus nucleoprotein antibody (antibody 2) to form a complex of labeled antibody 2-influenza virus nucleoprotein The step of causing; (2) The complex formed in the step (1) is reacted with an anti-influenza virus nucleoprotein antibody (antibody 1) immobilized on a carrier, and antibody 1 -influenza virus nucleoprotein-label on the carrier Forming a complex of conjugated antibody 2; (3) A step of washing the carrier after step (2) to remove substances not bound on the carrier; (4) a step of measuring the label in the antibody 1-influenza virus nucleoprotein-labeled antibody 2 complex on the carrier after step (3), wherein at least one of antibody 1 and antibody 2 comprises: A method for detecting or quantifying influenza virus, which is a human anti-influenza virus nucleoprotein antibody.
- each of antibody 1 and antibody 2 in the above method may be a monoclonal antibody or a polyclonal antibody, but both are preferably monoclonal antibodies.
- the cleaning step (3) can be omitted (homogeneous method). Further, the step (1) and the step (2) may be performed simultaneously, and a cleaning step may be inserted between the step (1) and the step (2).
- Examples of the method for measuring the label in step (4) of the above method include the above-described methods.
- Method 5 A sample and a human anti-influenza virus nucleoprotein antibody immobilized on a carrier are reacted in the presence of a labeled antigen analog in which the label is bound to the influenza virus nucleoprotein antigen analog, Forming a human anti-influenza virus nucleoprotein antibody-influenza virus nucleoprotein complex and a human anti-influenza virus nucleoprotein antibody-labeled antigen analog complex above; (2) A step of washing the carrier after step (1) to remove substances not bound on the carrier; (3) A method for detecting or quantifying influenza virus, comprising a step of measuring a label in the complex on the carrier after step (2).
- the above method is one aspect of the competitive method.
- the human anti-influenza virus nucleoprotein antibody in the above method may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
- Examples of the method for measuring the label in step (3) of the above method include the above-described methods.
- Method 6 (1) A sample and an influenza virus nucleoprotein antigen analog immobilized on a carrier are reacted in the presence of a labeled antibody in which a label is bound to a human anti-influenza virus nucleoprotein antibody, Forming an influenza virus nucleoprotein antigen analog-labeled antibody complex; (2) A step of washing the carrier after step (1) to remove substances not bound on the carrier; (3) A method for detecting or quantifying influenza virus, comprising a step of measuring a label in the complex on the carrier after step (2).
- the above method is another aspect of the competition method.
- the human anti-influenza virus nucleoprotein antibody in the above method may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
- Examples of the method for measuring the label in step (3) of the above method include the above-described methods.
- Reaction conditions in the method of the present invention can be appropriately set by those skilled in the art.
- the reaction temperature is 0 ° C. to 50 ° C., preferably 4 ° C. to 40 ° C.
- the reaction time is 1 minute to 24 hours, preferably 3 minutes to 12 hours.
- the carrier can be washed using, for example, phosphate buffered saline (PBS) when it includes a washing step.
- PBS phosphate buffered saline
- the solid phase used in the method of the present invention is one in which an anti-influenza virus nucleoprotein antibody or labeled antigen analog is immobilized and immobilized on a carrier.
- a carrier an anti-influenza virus nucleoprotein antibody or a labeled antigen analog can be immobilized and immobilized, and is not particularly limited as long as it is insoluble in water.
- a polystyrene plate such as a microtiter plate, glass or synthetic Examples include resin granules (beads), glass or synthetic resin spheres (balls), latex, magnetic particles, nitrocellulose membranes, nylon membranes, synthetic resin tubes, and the like.
- Examples of the method for immobilizing and immobilizing an anti-influenza virus nucleoprotein antibody or labeled antigen analog on a carrier include a direct method and an indirect method.
- Examples of the direct method include a method in which a functional group on a carrier is covalently bound to a functional group in an anti-influenza virus nucleoprotein antibody or labeled antigen analog.
- examples of the combination of the functional group on the carrier and the functional group in the anti-influenza virus nucleoprotein antibody or the labeled antigen analog include amino group-carboxyl group, carboxyl group-amino group and the like.
- a method in which a functional group on a carrier and a functional group in an anti-influenza virus nucleoprotein antibody or labeled antigen analog are indirectly bonded via a linker, or an interaction between substances is used. And the like.
- the functional group on the carrier and the functional group in the anti-influenza virus nucleoprotein antibody or the labeled antigen analog for example, amino group-amino group, amino group-carboxyl group, amino group-sulfhydryl group Carboxyl group-amino group, carboxyl group-carboxyl group, carboxyl group-sulfhydryl group and the like.
- the functional group may be introduced by chemical modification.
- Examples of the method utilizing the interaction between substances include a method utilizing the avidin-biotin interaction, a method utilizing the sugar-lectin interaction, and the like.
- the avidin-biotin interaction for example, by reacting a carrier on which avidin has been adsorbed with a biotinylated anti-influenza virus nucleoprotein antibody or a biotinylated labeled antigen analog, the anti-influenza on the carrier Viral nucleoprotein antibodies or labeled antigen analogs can be immobilized and immobilized.
- a solid carrier prepared by immobilizing an immobilized anti-influenza virus nucleoprotein antibody or labeled antigen analog with bovine serum albumin, casein or the like is used.
- a phase can also be used.
- human anti-influenza virus nucleoprotein antibodies used in the method of the present invention include human anti-influenza A virus nucleoprotein antibodies and human anti-B influenza virus nucleoprotein antibodies described below.
- Examples of the label used in the method of the present invention include the above-mentioned label.
- Examples of the label measurement method in the method of the present invention include the label measurement method described above.
- the labeled anti-influenza virus nucleoprotein antibody used in the method of the present invention can be prepared in accordance with the above-described method.
- the influenza virus nucleoprotein antigen analog used in the method of the present invention is one that reacts competitively with the influenza virus nucleoprotein in a sample against a human anti-influenza virus nucleoprotein antibody immobilized on a carrier.
- a carrier for example, not only the influenza virus nucleoprotein itself, which is an antigen, but also a substance containing an epitope in the influenza virus nucleoprotein recognized by the anti-influenza virus nucleoprotein antibody, and the like.
- the labeled antigen analog (labeled influenza virus nucleoprotein antigen analog) used in the method of the present invention can be prepared by the same method as the labeled antibody preparation method described above.
- influenza virus detection or quantification kit of the present invention is a kit used in the influenza virus detection or quantification method of the present invention.
- influenza virus detection or quantification kit of the present invention include the following kits.
- Kit 1 A kit for detecting or quantifying influenza virus, comprising a solid phase in which a human anti-influenza virus nucleoprotein antibody is immobilized on a carrier.
- the human anti-influenza virus nucleoprotein antibody may be a monoclonal antibody or a polyclonal antibody.
- This kit is suitable for methods such as turbidimetric immunoassay and surface plasmon resonance (SPR).
- Kit 2 A solid phase in which an anti-influenza virus nucleoprotein antibody (antibody 1) is immobilized on a carrier; A reagent containing an anti-influenza virus nucleoprotein antibody (antibody 2), wherein at least one of antibody 1 and antibody 2 is a human anti-influenza virus nucleoprotein antibody, or detecting or quantifying an influenza virus For kit.
- Antibody 1 and antibody 2 may be monoclonal antibodies or polyclonal antibodies, but both are preferably monoclonal antibodies.
- This kit is suitable for methods such as turbidimetric immunoassay and surface plasmon resonance (SPR).
- Kit 3 A solid phase in which an anti-influenza virus nucleoprotein antibody (antibody 1) is immobilized on a carrier; A reagent containing a labeled antibody in which a label is bound to an anti-influenza virus nucleoprotein antibody (antibody 2), wherein at least one of antibody 1 and antibody 2 is a human anti-influenza virus nucleoprotein antibody An influenza virus detection or quantification kit.
- Antibody 1 and antibody 2 may be monoclonal antibodies or polyclonal antibodies, but both are preferably monoclonal antibodies. This kit is suitable for immunoassay based on the sandwich method.
- Kit 4 A solid phase in which a human anti-influenza virus nucleoprotein antibody is immobilized on a carrier;
- This kit is suitable for immunological assay based on competitive method.
- Kit 5 A solid phase in which an influenza virus nucleoprotein antigen analog is immobilized on a carrier; A kit for detecting or quantifying influenza virus, comprising a reagent containing a labeled antibody in which a label is bound to a human anti-influenza virus nucleoprotein antibody.
- This kit is suitable for immunological assay based on competitive method.
- Examples of the solid phase used in the influenza virus detection or quantification kit of the present invention include the aforementioned solid phase.
- human anti-influenza virus nucleoprotein antibodies used in the kit of the present invention include human anti-influenza A virus nucleoprotein antibodies and human anti-B influenza virus nucleoprotein antibodies described below.
- Examples of the label used in the kit of the present invention include the aforementioned label.
- Examples of the label measurement method in the kit of the present invention include the above-described label measurement method.
- kits of the present invention not only the above-mentioned aqueous medium but also additives such as the above-mentioned buffer, surfactant, preservative, salt, saccharide, metal ion, protein and the like may be included as necessary. .
- additives can be used as an additive-containing reagent separately from the solid phase and reagent constituting the kit of the present invention, but are contained in either or both of the solid phase and reagent constituting the kit of the present invention. It can also be used.
- the human anti-influenza virus nucleoprotein antibody of the present invention is an antibody that can be used in the device for detecting or quantifying the influenza virus of the present invention, the kit for detecting or quantifying the influenza virus, and the method for detecting or quantifying the influenza virus, Examples include human anti-influenza A virus nucleoprotein antibodies and human anti-influenza B virus nucleoprotein antibodies.
- the human anti-influenza virus nucleoprotein antibody in the present invention may be a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferred.
- the human anti-influenza A virus nucleoprotein monoclonal antibody of the present invention is preferably an antibody that specifically reacts with influenza A virus nucleoprotein but does not react with influenza B virus nucleoprotein.
- the human anti-influenza B virus nucleoprotein monoclonal antibody of the present invention is preferably an antibody that specifically reacts with influenza B virus nucleoprotein but does not react with influenza A virus nucleoprotein.
- human anti-influenza virus nucleoprotein monoclonal antibody is preferred as the human anti-influenza virus nucleoprotein antibody.
- Human anti-influenza virus nucleoprotein monoclonal antibodies can be prepared by known monoclonal antibody production methods [hybridoma method (see, for example, In Vitro Cell Dev Biol. (1985) 21 (10): 593-596), virus infection method (for example, J. gen. Virol. (1983), 64, 697-700), phage display method (see, for example, WO2001 / 062907 pamphlet), lymphocyte microarray method, etc.], but the lymphocyte microarray method is preferred.
- the lymphocyte microarray method is a technology related to a method for producing a monoclonal antibody jointly developed by the national university corporations Toyama University and SCI World, Inc. [JP 2004-173681; JP 2004-187676; JP 2005-261339; Anal. ; Chem. 77 (24), p.8050-8056 (2005); Cytometry A 71 (11), p.961-967 (2007); Cytometry A 71 (12), p.1003-1010 (2007) ] And includes the following steps.
- a step of preparing B lymphocytes from human blood [2] A step of selecting B lymphocytes that react with the measurement object; [3] A step of obtaining a gene related to an antibody that reacts with the measurement target from the selected B lymphocyte; [4] A step of expressing an antibody using the obtained gene; [5] A step of selecting an antibody having a desired property.
- Step of preparing B lymphocytes from human blood Human B lymphocytes can be prepared from human peripheral blood, especially influenza virus infection history or influenza vaccination history, and influenza virus nucleoprotein in serum. It can be prepared from human peripheral blood in which the presence of the antibody can be confirmed. It is desirable to prepare blood samples after a certain period of influenza vaccination, preferably after 1 to 30 days, more preferably after 5 to 10 days. Specific examples of the preparation method include, for example, a method using density gradient centrifugation, cell sorter, magnetic beads and the like.
- One antigen-specific B lymphocyte can be selected, for example, by a method using a microwell array chip.
- a microwell array chip for example, an antigen is added to each microwell of an antigen-specific B lymphocyte detection microwell array chip having a plurality of microwells containing one subject B lymphocyte,
- one antigen-specific B lymphocyte can be obtained by detecting B lymphocytes that have reacted with the antigen and removing the detected antigen-specific B lymphocytes from the microwell.
- this method will be described more specifically.
- the microwell array chip one having a plurality of microwells and each microwell containing one specimen B lymphocyte can be used.
- antigen-specific B lymphocytes can be identified at the cell level. That is, when this microwell array chip is used, since the subject B lymphocyte contained in the microwell is one, the subject B lymphocyte that reacts with the antigen can be identified as one cell, and as a result, Antigen-specific B lymphocytes can be detected as a single cell. Then, one detected antigen-specific B lymphocyte is taken out and the gene is cloned.
- the shape and dimensions of the microwell can be, for example, a cylindrical shape.
- a cylindrical shape a rectangular parallelepiped, an inverted cone, an inverted pyramid (an inverted triangular pyramid, An inverted quadrangular pyramid, an inverted pentagonal pyramid, an inverted hexagonal pyramid, an inverted polygonal pyramid with a heptagon or more can also be used, and a combination of two or more of these shapes can also be used.
- the bottom surface is the opening of the microwell, but a shape obtained by cutting a part from the top of the inverted cone or inverted pyramid (in this case, the bottom of the microwell is flat) Can also be).
- the bottom of the microwell is usually flat, but it can be curved (convex or concave).
- the bottom of the microwell can be a curved surface as well in the case of a shape obtained by cutting a part from the top of an inverted cone or inverted pyramid.
- the subject B lymphocyte is stored together with the culture solution. Examples of the culture solution include any of the following.
- Detection of cells that react with the antigen can be performed as follows. For example, when an antigen binds to an antigen receptor (immunoglobulin) of B lymphocytes, intracellular signal transduction occurs first, followed by cell proliferation and antibody production. Accordingly, by detecting intracellular signal transduction, cell proliferation, and antibody production by various methods, cells that react with the antigen can be detected. Detection of a cell that reacts with an antigen by detecting intracellular signal transduction can be performed, for example, by changing the concentration of intracellular Ca ions by using a Ca ion-dependent fluorescent dye. For changes in intracellular Ca ion concentration, Fura-2, Fluo-3 or Fluo-4 can be used as a fluorescent dye, and a fluorescence microscope or a microarray scanner can be used as a detection device.
- an antigen receptor immunoglobulin
- a step of obtaining a gene related to an antibody that reacts with the measurement target from the selected B lymphocyte is obtained.
- the extracted antigen-specific B lymphocyte is lysed using a cell lysing agent, and then PCR is used.
- the antigen-specific immunoglobulin (antibody) gene is cloned.
- the cell lysing agent known substances can be used as they are, and examples thereof include the following.
- the antigen receptor gene in B lymphocytes is the same as the antibody gene, and the protein is called immunoglobulin.
- Antigen receptors are present on the membrane surface of B lymphocytes (membrane-type immunoglobulin), and antibodies are normally produced as secretory proteins (secretory immunoglobulin). The difference is on the C-terminal side of the protein.
- Membrane immunoglobulin has a membrane domain buried in the cell membrane and a portion protruding to the cytoplasm side. Secreted immunoglobulin is also produced from the same gene. However, by alternative splicing, the C-terminal side of the protein does not have a membrane domain unlike membrane-type immunoglobulin. As a result, it is produced as a secreted protein, but the antigen-binding sites of these two proteins are the same. Therefore, in the case of B lymphocytes, antibody gene cloning and antigen receptor gene cloning are the same.
- cDNA is prepared using reverse transcriptase.
- tailing reaction to the 3 ′ end of the cDNA by DNA polymerase is performed, and any one of adenine, guanine, cytosine and thymine is tailed to the 3 ′ end of the cDNA.
- a desired antigen-specific immunoglobulin gene can be amplified by performing PCR twice using a primer mix for immunoglobulin genes.
- Preparation of cDNA using reverse transcriptase can be performed by a conventional method [eg, Sambrook J, Russell DWin Molecular Cloning: A Laboratory, Manual 3rd Ed (Cold Spring Harbor Laboratory, New York, 2001).
- RNA extracted and purified from cells it is preferable to perform a direct RT reaction using a cell lysate rather than a reverse transcription reaction using RNA extracted and purified from cells.
- the antibody molecule is composed of a combination of H chain and L chain, and the H chain of human antibody gene is composed of about 200 V region gene fragments, about 20 D fragments, and 6 J fragments in the germline.
- one antigen-binding site is formed by combining one V fragment, one D fragment, and one J fragment by gene rearrangement (V / D / J rearrangement). The same applies to the L chain.
- Each B lymphocyte expresses one type of antibody molecule on the cell surface.
- a primer corresponding to the sequence of each V fragment In order to amplify an antigen-specific antibody gene from antigen-specific B lymphocytes, it is necessary to use a primer corresponding to the sequence of each V fragment.
- cDNA encoding the heavy chain (H chain) and the light chain (L chain) of the antibody is obtained from the cell using genetic engineering techniques. Culturing the antibody-expressing cells obtained by constructing a recombinant vector in which the obtained cDNA is inserted downstream of the promoter of the antibody expression vector promoter and introducing it into a host cell, or in an animal It can be prepared by causing the cells to become ascites cancer and then separating and purifying the culture medium or ascites.
- Such an antibody expression vector is an expression vector for animal cells in which genes encoding a constant region heavy chain (CH) and a constant region light chain (CL), which are constant regions (C regions) of human antibodies, are incorporated. It is constructed by inserting genes encoding human antibodies CH and CL into expression vectors for animal cells, respectively.
- C ⁇ 1 and C ⁇ 4 can be used for human antibody H chains, and C ⁇ can be used for human antibody L chains.
- C ⁇ can be used for human antibody L chains.
- chromosomal DNA consisting of exons and introns can be used, and cDNA can also be used. Any expression vector for animal cells can be used as long as it can incorporate and express a gene encoding the antibody C region.
- promoters and enhancers used in expression vectors include SV40 early promoters and enhancers [J. Biochem, 101, 1307 (1987)], Moloney murine leukemia virus LTR promoter and enhancer [Biochen. Biophys. Res. Cons., , 149, 960 (1987)], and an immunoglobulin heavy chain promoter [Cell, 41, 479 (1985)], enhancer [Cell, 33, 717 (1983)] and the like.
- the expression vector can be of either the type in which the antibody H chain gene and the L chain gene are present on separate vectors or the type in which they are present on the same vector (tandem type).
- a transformed strain that stably produces an antibody can be obtained by introducing the expression vector into an appropriate host cell.
- a method for introducing an expression vector into a host cell include electroporation (Japanese Patent Laid-Open No. 2-257891, Cytotechnology, 3, 133 (1990)).
- Any host cell capable of expressing an antibody can be used as a host cell into which an expression vector is introduced.
- mouse SP2 / 0-Ag cells ATCC CRL1581
- mouse P3X63-Ag8.653 cells ATCC CRL1580
- DHFR gene CHO cells lacking a dihydrofolate reductase gene
- DHFR gene a dihydrofolate reductase gene
- the antibody obtained in the previous step can be purified from the culture supernatant of the transformant using a protein A column (Antibodies, Chapter 8). .
- other purification methods used for ordinary proteins such as gel filtration, ion exchange chromatography, and ultrafiltration can be used alone or in combination.
- the molecular weight of the purified recombinant antibody H chain, L chain, or whole antibody molecule can be determined by polyacrylamide electrophoresis (SDS-PAGE) [Nature, 227, 680 (1970)] or Western blotting (Antibodies Chapter 12). Measure with etc.
- the binding ability of antibodies in the culture supernatant to influenza virus nucleoprotein is measured by ELISA using a 96-well plate coated with influenza virus nucleoprotein. Measurement by ELISA can be performed as follows. That is, 50 ⁇ L of influenza virus nucleoprotein (10 ⁇ g / mL) diluted with PBS was dispensed into each well in a 96-well plate and allowed to stand overnight at 4 ° C. for adsorption. The wells are washed with PBS, and in order to remove non-specific binding, 400 ⁇ L of PBS containing 3% BSA and 0.05% Tween-20 is dispensed into each well and reacted for 2 hours at room temperature for blocking.
- a well blocked without antigen adsorption is also prepared.
- the wells are then washed with PBS containing 0.1% Tween-20 (PBS-T), and 50 ⁇ L of the above 293T cell culture supernatant is dispensed into each well and allowed to react at room temperature for 2 hours.
- the wells are washed with PBS-T, and 50 ⁇ L of alkaline phosphatase (ALP) -labeled anti-human immunoglobulin diluted 1000 times is dispensed into each well and allowed to react at room temperature for 2 hours.
- ALP alkaline phosphatase
- ALP substrate buffer ALP substrate
- the soluble influenza virus nucleoprotein and the culture supernatant are preincubated. I let you.
- the mixed solution is added to the well, and it is examined whether the binding of the antibody to the recombinant nucleoprotein adsorbed to the well is competitively inhibited by the soluble recombinant nucleoprotein.
- the amino acid sequence of the heavy chain variable region includes the amino acid sequence represented by any of SEQ ID NOs: 8 to 13, and the light chain variable region
- the human anti-influenza A virus nucleoprotein monoclonal antibody includes the amino acid sequence represented by any one of SEQ ID NOs: 22 to 27.
- a preferred embodiment includes a human anti-influenza A virus nucleoprotein monoclonal antibody comprising a heavy chain variable region and a light chain variable region comprising a combination of amino acid sequences selected from the group consisting of the following amino acid sequence combinations of SEQ ID NOs: It is done.
- amino acid sequence of the heavy chain variable region and the amino acid sequence of the light chain variable region are SEQ ID NO: 8 and SEQ ID NO: 22, SEQ ID NO: 9 and SEQ ID NO: 23, SEQ ID NO: 10 and SEQ ID NO: 24, respectively.
- a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28 are used.
- human anti-influenza B virus nucleoprotein monoclonal antibodies are used.
- a non-human anti-influenza virus nucleoprotein antibody can be used together with a human anti-influenza virus nucleoprotein antibody.
- non-human animals include mice, rats, rabbits and the like.
- the non-human anti-influenza virus nucleoprotein antibody may be a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferred.
- the non-human anti-influenza virus nucleoprotein monoclonal antibody can be produced, for example, by the aforementioned known monoclonal antibody production method.
- non-human anti-influenza virus nucleoprotein monoclonal antibody examples include mouse anti-influenza A virus nucleoprotein monoclonal antibody, mouse anti-B influenza virus nucleoprotein monoclonal antibody, rat anti-influenza A virus nucleoprotein monoclonal antibody, rat anti-B type Examples include influenza virus nucleoprotein monoclonal antibodies, rabbit anti-type A influenza virus nucleoprotein monoclonal antibodies, rabbit anti-type B influenza virus nucleoprotein monoclonal antibodies, and the like.
- non-human anti-influenza virus nucleoprotein monoclonal antibody not only the monoclonal antibody produced by the aforementioned known monoclonal antibody production method but also a commercially available product can be used.
- commercially available products include mouse anti-influenza A virus nucleoprotein monoclonal antibody products M322211, M2110169 (manufactured by Fitzgerald), ATCC ⁇ HB-65, etc. Examples thereof include M2110171 (manufactured by Fitzgerald) and 41027 (manufactured by Capricorn) which are commercially available protein monoclonal antibodies.
- Example 1 Device for detection or quantification of influenza virus, production of alkaline phosphatase-labeled antibody
- Two human anti-influenza virus nucleoprotein monoclonal antibodies [23G285 (type A), 23G327 (B Type)] and two mouse anti-influenza virus nucleoprotein monoclonal antibodies [M2110169 (type A: manufactured by Fitzgerald), M2110171 (type B: manufactured by Fitzgerald)] with Alkaline Phosphatase Labeling Kit (manufactured by Dojindo Laboratories)
- Alkaline Phosphatase Labeling Kit manufactured by Dojindo Laboratories
- influenza A virus solution H3N2: A / Panama / 2007/99
- a reaction solution containing Nonidet P-40 alkaline phosphatase labeled mouse anti-type A influenza
- the virus nucleoprotein monoclonal antibody M2110169 or alkaline phosphatase-labeled human anti-influenza A virus nucleoprotein monoclonal antibody 23G285 in Tris solution and BCIP / NBT (nitro-blue tetrazolium chloride) solution (manufactured by Sigma) were developed in this order. The results are shown in FIG.
- human anti-influenza A virus nucleoprotein antibody can be used for detection of influenza A virus by immunochromatography, and human anti-influenza A virus nucleoprotein antibody can detect influenza A virus with high sensitivity. It was found useful.
- this device was examined using a detection region in which the human anti-influenza A virus nucleoprotein antibody 23G268 was immobilized and immobilized, and a device having a specimen supply region below the filter paper (FIG. 2). -B).
- the sample was diluted with a reaction solution containing nonidet P-40, a type A influenza virus solution diluted with reaction solution containing nonidet P-40 (H3N2: A / Panama / 2007/99) Prepared influenza A virus solution (H1N1: A / Beijing / 262/95) and influenza B virus solution (B / Victoria / 504/00) diluted with reaction solution containing nonidet P-40, Next, a Tris solution of alkaline phosphatase-labeled antibody in which alkaline phosphatase is bound to human anti-influenza A virus nucleoprotein antibody 23G285 is developed in the specimen supply region of each device, and BCIP / NBT (nitro-blue tetrazolium chloride) The solution was developed from the developing solution supply area.
- H3N2 A / Panama / 2007/99
- H1N1 A / Beijing / 262/95
- influenza B virus solution B / Victoria / 504/00
- influenza A virus solution H3N2: A / Panama / 2007/99
- influenza A virus solution H1N1: A / Beijing / 262/95
- ⁇ Device for detection or quantification of influenza B virus using an alkaline phosphatase-labeled antibody A mouse anti-influenza B virus nucleoprotein monoclonal antibody M2110171 (Fitzgerald) in one area of filter paper (Millipore) cut to 5 cm x 0.5 cm PBS solution of 41027 (manufactured by Capricorn) was instilled, and the monoclonal antibody was immobilized and used as a detection region. From the bottom of this filter paper (sample supply area), the sample contains influenza A virus solution (H1N1: A / Beijing / 262/95) diluted with a reaction solution containing Nonidet P-40 and Nonidet P-40.
- H1N1 A / Beijing / 262/95
- Influenza B virus solution (B / Victoria / 504/00) diluted with the reaction solution to be prepared, and then, the alkaline phosphatase is added to the human anti-B influenza virus nucleoprotein antibody 23G327 in the specimen supply area of each device.
- a Tris solution of the bound alkaline phosphatase labeled antibody was supplied, and a BCIP / NBT (nitro-blue tetrazolium chloride) solution was further developed from the developing solution supply region. The results are shown in FIG. Only when the influenza B virus solution (B / Victoria / 504/00) was used, a blue spot was detected in the detection region.
- human anti-influenza B virus nucleoprotein antibody 23G327 is bound to the detection region and alkaline phosphatase is bound to mouse anti-influenza B virus nucleoprotein monoclonal antibody M2110171 (Fitzgerald) as the labeled antibody. Even when the alkaline phosphatase labeled antibody was used, a blue spot was detected in the detection region only when the influenza B virus solution (B / Victoria / 504/00) was used.
- mouse anti-influenza B virus nucleoprotein antibody 41027 (manufactured by Capricorn) is used as an antibody immobilized on the detection region
- mouse anti-influenza B virus nucleoprotein monoclonal antibody M2110171 (manufactured by Fitzgerald) is used as a labeled antibody. Even when an alkaline phosphatase-labeled antibody bound with alkaline phosphatase was used, a blue spot was detected in the detection region only when influenza B virus solution (B / Victoria / 504/00) was used. .
- human anti-B influenza virus nucleoprotein antibodies can be used for detection of influenza B virus by immunochromatography.
- Example 2 Production of colloidal gold-labeled antibody Two human anti-influenza A virus nucleoprotein monoclonal antibodies [23G272, 23G447] and one mouse anti-influenza A virus nucleoprotein monoclonal antibody [M322211 ( Fitzgerald) was used as a labeled antibody to produce a colloidal gold labeled antibody. That is, the PBS solution of the antibody was replaced with a Tris buffer to prepare a Tris solution of the antibody, and then mixed with 40 nm gold colloid (manufactured by BB International) and reacted for 15 minutes. After the reaction, after blocking with a Tris solution containing 1% BSA, unbound monoclonal antibodies were removed by centrifugation to obtain colloidal gold labeled antibodies.
- Influenza A virus detection device for detection or quantification of influenza A virus using colloidal gold-labeled antibody
- colloidal gold-labeled antibody In a region of filter paper (Millipore) cut into 5 cm x 0.5 cm, anti-influenza A as a solid phase antibody Virus nucleoprotein monoclonal antibodies [23G312, 23G494, M2110169 (Fitzgerald)] were immobilized and used as detection regions. That is, the PBS solution of the anti-influenza A virus nucleoprotein monoclonal antibody was instilled and immobilized to form a detection region.
- influenza A virus solution H1N1: A / Beijing / 262/95
- a reaction solution containing Nonidet P-40 and colloidal gold label produced above 0.1 mL of a solution in which an equal amount of the antibody Tris solution was mixed
- a remarkable red spot was confirmed in the detection region in any combination of antibodies (a combination of a solid phase antibody and a labeled antibody).
- the combination of the solid phase antibody and the labeled antibody using the human antibody was compared with the combination of the solid phase antibody and the labeled antibody using the mouse antibody. About twice as fast.
- influenza A virus solutions (H1N1: A / Beijing / 262 /) prepared at various concentrations using combinations of solid phase antibodies and labeled antibodies as shown in Tables 1 and 2 95, H3N2: A / Panama / 2007/99) and colloidal gold labeled antibodies were developed, and spot coloring was visually observed.
- human anti-influenza A virus nucleoprotein antibody can be used for detection of influenza A virus by immunochromatography, and moreover compared to the case of using mouse anti-influenza A virus antibody, human It was found that when an anti-influenza A virus nucleoprotein antibody was used, influenza A virus could be detected quickly and with high sensitivity.
- Example 3 Preparation of acridinium-labeled antibodies Seven human anti-influenza virus nucleoprotein monoclonal antibodies [23G268, 23G272, 23G285, 23G312, 23G447, 23G494 (above, type A); 23G327 (type B)] obtained in Example 4 described later Acridinium ester (manufactured by Dojindo Laboratories) is reacted with each of the mouse anti-influenza virus nucleoprotein monoclonal antibodies and commercially available mouse anti-influenza virus nucleoprotein, and unreacted acridinium ester is removed by gel filtration. Antibody was obtained.
- Influenza A virus detection using a monoclonal antibody A 96-well microtiter plate (manufactured by NUNK) was dispensed 50 ⁇ L of anti-influenza A virus nucleoprotein monoclonal antibody (10 ⁇ g / mL) diluted with PBS into each well. It was left overnight at 4 ° C. After removing the solution in the well, 100 ⁇ L of 1% BSA / PBS solution was dispensed and reacted at room temperature for 2 hours for blocking.
- both the solid phase antibody and the labeled antibody are commercially available mouse anti-influenza A virus nucleoprotein antibodies [M322211, M2110169 (Fitzgerald); Compared with the case of using ATCC HB-65], it was shown that H3N2 can be detected at the same level or higher, and H1N1 at least twice as sensitive.
- Influenza B virus detection using a monoclonal antibody 96-well microtiter plate 50 ⁇ L of influenza B virus nucleoprotein monoclonal antibody (10 ⁇ g / mL) diluted in PBS was dispensed into each well. Allowed to stand overnight at ° C. After removing the solution in the well, 100 ⁇ L of 1% BSA / PBS solution was dispensed and reacted at room temperature for 2 hours for blocking.
- H1N1 A / Beijing / 262/95
- H3N2 A / Panama / 2007/99
- B B / Victoria / 504/00
- 50 ⁇ L of an acridinium-labeled anti-influenza B virus nucleoprotein monoclonal antibody was added and allowed to react at room temperature for 1 hour.
- virus strain specificity As shown in FIGS. 7 and 8, in the detection of influenza virus using a monoclonal antibody, it was shown that the solid phase antibody and the labeled antibody can be detected as the same clone. Therefore, the virus strain specificity was evaluated using a solid phase antibody / labeled antibody of the same clone.
- influenza virus nucleoprotein monoclonal antibody 10 ⁇ g / mL
- PBS 1% BSA / PBS solution
- Various influenza virus strains [purchased from NIBSC (National Institute for Biological Standards and Control)] were diluted with PBS containing 0.5% nonidet P-40 and 0.5% BSA to 1 ⁇ g HA / mL, and then 50 ⁇ L was added to each well. And allowed to react at room temperature for 1 hour.
- the human anti-influenza A virus nucleoprotein antibody of the present invention does not react with a part of the equine influenza A virus strain H3N8 virus, but all other human influenza A virus strains and It was found that it reacts with the H5N1 virus, which is an avian influenza A virus strain, and does not react with the B virus strain. In contrast, commercially available mouse influenza A virus nucleoprotein antibodies were found to react with some of the B virus strains.
- human anti-B influenza virus nucleoprotein antibody (23G327) of the present invention does not react with all influenza A virus strains but specifically reacts with all influenza B strains.
- commercially available mouse influenza B virus nucleoprotein antibody 2/3 does not react with some influenza B strains.
- the human anti-influenza virus nucleoprotein antibody of the present invention exhibits a higher specificity for influenza virus strains than the commercially available mouse anti-influenza virus nucleoprotein antibody.
- goat anti-human IgG or goat anti-mouse IgG prepared at 10 ⁇ g / mL with Sodium Acetate Buffer, pH 5.0 was flowed for 7 minutes and immobilized on the surface of CM5.
- ethanolamine (1 mol / L, pH 8.5) was allowed to flow for 7 minutes in order to mask the remaining activated carboxylic acid.
- glycine-hydrochloric acid buffer (0.2 mol / L, pH 2.2) was washed for 1 minute. This operation was performed in the flow cell 2.
- the flow cell 1 was similarly activated with EDC and NHS, and masked with only ethanolamine without contacting IgG.
- the anti-influenza A virus nucleoprotein monoclonal antibody prepared at 10 ⁇ g / mL is flowed to the sensor chip prepared above for 10 minutes at a flow rate of 5 ⁇ L / min to capture the antibody on the sensor chip. It was. At this time, a value obtained by subtracting the signal (RU) obtained in the flow cell 1 from the signal (RU) obtained in the flow cell 2 was calculated as the antibody binding amount.
- H1N1 A / Beijing / 262/95
- H3N2 A / Panama / 2007/99
- B B / Victoria / 504/00
- solutions prepared to 10 ⁇ g / mL with HBS-EP were reacted for 5 minutes at a flow rate of 5 ⁇ L / min
- further HBS-EP (0.01 mol / L HEPES, 0.15 mol / L NaCl, 3 mmol / L EDTA, 0.005% surfactant P20) buffer was flowed for 5 minutes at a flow rate of 5 ⁇ L / min. .
- the sensor chip was regenerated by flowing glycine-hydrochloric acid buffer (0.2 mol / L, pH 1.7) for 1 minute.
- glycine-hydrochloric acid buffer 0.2 mol / L, pH 1.7
- H1N1 A / Beijing / 262/95
- H3N2 A / Panama / 2007/99
- B B / Victoria / 504/00
- the sensorgram representing the interaction with the solution is shown in FIG. 9-a.
- the human anti-influenza A virus nucleoprotein antibody obtained in the present invention and three influenza viruses (H1N1: A / Beijing / 262/95) , H3N2: A / Panama / 2007/99, B: B / Victoria / 504/00)
- a sensorgram representing the interaction with the solution is shown in FIG. 9-b.
- 23G272 which is the human antibody of the present invention, no antigen dissociation reaction was observed, and it was revealed that the antibody is an antibody with strong affinity.
- the 23G268 antibody, 23G272 antibody, 23G285 antibody, 23G312 antibody, 23G447 antibody, and 23G494 antibody have the same or higher reactivity than the commercially available mouse anti-influenza A virus nucleoprotein antibody.
- the 23G272 antibody was more than twice as reactive as the commercially available mouse anti-influenza A virus nucleoprotein antibody, indicating that the binding reaction rate is very fast.
- the human anti-influenza B virus nucleoprotein antibody (23G327) is approximately 0.8 times more reactive than one of the commercially available mouse anti-influenza B virus nucleoprotein antibodies (2/3), compared to antibody 2/3 The reactivity was slightly inferior.
- 23G272 was immobilized on the flow cell 2
- M2110169 was immobilized on the flow cell 3
- HB-65 was immobilized on the flow cell 4 in the same manner as the above sensor chip production method.
- the immobilization amounts were 1519 RU for 23G272, 1773 RU for M2110169, and 1734 RU for HB-65, respectively.
- the prepared chip was reacted with A / Beijing / 262/95 adjusted to 10 ⁇ g / mL at a flow rate of 5 ⁇ L / min for 2 minutes.
- the results are shown in FIG. 23G272 was found to have about 10 times the amount of antigen binding compared to HB-65.
- M2110169 was inactivated by glycine-hydrochloric acid buffer and showed no reaction with virus.
- influenza virus nucleoprotein gene was synthesized based on the publicly available sequence information (A / Puerto Rico / 8/34, B / Ann Arbor / 1/86) and incorporated into the pSUPEX vector. Using this vector, E. coli (NY49 strain) was transformed, and influenza virus nucleoprotein was expressed in the transformant. The obtained influenza virus nucleoprotein was purified by affinity chromatography using an antibody against the influenza virus nucleoprotein.
- lymphocytes were prepared from peripheral blood 9 days or 1 month after immunization. That is, human lymphocytes are centrifuged from peripheral blood using Lymphosepar I solution (manufactured by Immunobiological Research Laboratories), and further, AutoMACS (Miltenyi Biotec, Bergisch Gladbac, Germany) is used to fractionate lymphocytes from non-B lymphocytes. Cells were removed and the B lymphocyte fraction was separated and purified.
- Lymphosepar I solution manufactured by Immunobiological Research Laboratories
- AutoMACS Miltenyi Biotec, Bergisch Gladbac, Germany
- Fluo 4-AM is a fluorescent dye whose fluorescence intensity increases when calcium binds, and is used to detect activation of B lymphocytes by an antigen.
- the microwell array chip is manufactured using silicon (manufactured by Toyama Prefectural Industrial Technology Center), and microwells with a diameter of 10 ⁇ m and a depth of 14 ⁇ m are arranged vertically and horizontally at a pitch of 25 ⁇ m (distance between well centers). .
- the microwells were 30 x 30 (900 wells), forming one cluster, and 10 clusters vertically and 5 clusters horizontally were used.
- the microwell array chip was immersed in Hank's buffer (HBSS) and degassed under reduced pressure to remove air in the well, thereby filling the microwell with the solution. Block the surface of the microwell chip with 0.2% LIPIDURE-BL-B03 (manufactured by NOF Corporation) for 10 to 15 minutes, remove excess buffer, add the cell suspension to it, and leave it for 10 minutes did. Cells that did not enter the microwells on the chip were washed away with HBSS.
- the diameter of the lymphocyte is about 8 ⁇ m, and since the diameter of the microwell used is 10 ⁇ m, one lymphocyte is contained in one microwell.
- a cover glass was placed on the seal and HBSS was filled between the chip and the cover glass.
- a microwell array chip seeded with B lymphocytes was placed on a microchip CCD imager (Micro Chip VIEW 1100; manufactured by Nanosystem Solutions), and the time course of fluorescence intensity of Fluo 4 before antigen stimulation was 100 seconds at 10 second intervals. It was measured. The measurement was temporarily stopped, the chip was pulled out of the scanner, HBSS between the chip and the cover glass was removed, and recombinant nucleoprotein (10-100 ⁇ g / mL) dissolved in HBSS was added thereto.
- the microwell array chip was returned to the measurement section of the microchip CCD imager, and the time-dependent change in fluorescence intensity of Fluo 4 in the cells after addition of the antigen was scanned at 10 second intervals, and the data was stored. Analyze using software how the time course of individual intracellular calcium concentration correlates with the typical pattern of time course of intracellular calcium concentration observed when antigens bind to specific antibody-expressing cells, Cells showing an antigen-specific intracellular calcium response were identified.
- B lymphocytes with increased intracellular calcium in response to the detected recombinant nucleoprotein were collected using a micromanipulator under a fluorescence microscope. That is, first, the HBSS between the cover glass and the chip was removed, and the cover glass was removed by putting air between the cover glass and the chip. The RPMI1640 / 10% FCS solution was added to the chip so that the chip did not dry, and the target cells were collected with a micromanipulator while observing the fluorescence of Fluo 4 of the cells under a fluorescence microscope.
- the parentheses () indicate that any one of the bases in the parentheses is selected.
- the PCR product was analyzed and purified using an agarose gel, inserted into pGEM-T Easy vector (Promega), the nucleotide sequence of the antibody gene was determined, and it was confirmed that it was translated into protein.
- the nucleotide sequences of the H chain V region cDNA are shown in SEQ ID NOs: 1 to 7, and the amino acid sequences are shown in SEQ ID NOs: 8 to 14, respectively.
- the base sequence of the cDNA of the L chain V region is shown in SEQ ID NOs: 15 to 21, and the amino acid sequence is shown in SEQ ID NOs: 22 to 28.
- the antibody H chain and L chain variable region genes amplified by RT-PCR were incorporated into antibody protein expression vectors (FIGS. 4-a, 4-b, 4-c). That is, the gene fragment of the heavy chain variable region of the amplified antibody is placed in the V ⁇ portion of the pMXv6-hIg ⁇ vector, and the gene fragment of the L chain variable region is placed in the V ⁇ portion of the pMXv6-hIg ⁇ or the V ⁇ portion of the pMXv6-hIg ⁇ vector. Inserted. In order to express antibody proteins from the prepared H chain and L chain expression vectors, both expression vectors were simultaneously introduced into human fetal kidney-derived cell 293T cells.
- Gene transfer was performed according to a conventional method using Lipofectamine 2000 (Invitrogen). Cell supernatants were collected after 5-7 days. The binding ability of the antibody in this culture supernatant to the recombinant nucleoprotein was measured by ELISA using a 96-well plate coated with the recombinant nucleoprotein. Measurement by ELISA was performed as follows. Specifically, 50 ⁇ L of recombinant nucleoprotein (10 ⁇ g / mL) diluted in PBS was dispensed into each well in a 96-well plate, and allowed to stand overnight at 4 ° C. for adsorption.
- the soluble recombinant nucleoprotein and the culture supernatant are preincubated. It was. The mixed solution was added to the well, and it was examined whether the binding of the antibody to the recombinant nucleoprotein adsorbed to the well was competitively inhibited by the soluble recombinant nucleoprotein. The results are shown in FIGS. 5-a and 5-b.
- the prepared 23G268 antibody, 23G272 antibody, 23G285 antibody, 23G312 antibody, 23G447 antibody and 23G494 antibody bind specifically to the recombinant influenza A virus nucleoprotein and It did not bind to influenza B virus nucleoprotein. Furthermore, in order to examine the specificity of binding, a mixture of the above six types of antibodies and soluble recombinant influenza A virus nucleoprotein was added to wells to which recombinant influenza A virus nucleoprotein was bound, and ELISA was similarly performed. went.
- the prepared 23G327 antibody specifically bound to the recombinant influenza B virus nucleoprotein and did not bind to the recombinant influenza A virus nucleoprotein. Further, in order to examine the specificity of binding, a mixture of 23G327 antibody and soluble recombinant influenza B virus nucleoprotein was added to wells to which recombinant influenza B virus nucleoprotein was immobilized, and ELISA was performed in the same manner. As a result, it was observed that the binding of the 23G327 antibody to the recombinant influenza B virus nucleoprotein immobilized on the well was dose-dependently and competitively inhibited by the soluble recombinant influenza B virus nucleoprotein. From these results, it was shown that the 23G327 antibody specifically binds to the recombinant influenza B virus nucleoprotein.
- virus specificity by direct ELISA 50 ⁇ L of various influenza virus nucleoprotein antigens diluted with PBS (10 ⁇ g / mL) were dispensed into each well in a 96-well microtiter plate (manufactured by NUNK) at 4 ° C. I left still overnight. After removing the solution in the well, 100 ⁇ L of 1% BSA / PBS solution was dispensed and reacted at room temperature for 2 hours for blocking.
- the culture supernatant or human IgG1 obtained above was diluted with 0.1% BSA / PBS solution, added 50 ⁇ L per well, and allowed to react at room temperature for 1 hour. After washing with PBS containing 0.05% Tween 20, HRP-labeled-rabbit anti-human IgG antibody was added and reacted at room temperature for 1 hour. After washing with PBS containing 0.05% Tween 20, 50 ⁇ L of TMB coloring solution was added and reacted at room temperature for 30 minutes. Finally, 50 ⁇ L of a reaction stop solution (1 mol / L H 2 SO 4 ) was added, and the absorbance at 450 nm was measured with a microplate reader. The results are shown in FIG.
- devices and kits for diagnosing influenza virus infection and human anti-influenza virus nucleoprotein antibodies used therefor are provided.
Abstract
Description
〔2〕 抗インフルエンザウィルス核タンパク質抗体(抗体1)が支持体上に不動化された検出領域、検体供給領域及び検体移動領域を具備し、抗インフルエンザウィルス核タンパク質抗体(抗体2)に標識が結合した標識化抗体が検体供給領域より供給され、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする検体中のインフルエンザウィルス検出又は定量用デバイス。
〔3〕 抗インフルエンザウィルス核タンパク質抗体(抗体1)が支持体上に不動化された検出領域、抗インフルエンザウィルス核タンパク質抗体(抗体2)に標識が結合した標識化抗体が移動可能なように支持体上に保持されている標識試薬領域、検体供給領域及び検体移動領域を具備し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、検体中のインフルエンザウィルス検出又は定量用デバイス。
〔4〕 さらに、展開液供給領域、余剰液吸収領域及び検体供給確認領域からなる群より選ばれる領域を少なくとも1つを具備する、〔2〕又は〔3〕記載のデバイス。
〔5〕 ヒト抗インフルエンザウィルス核タンパク質抗体が、ヒト抗A型インフルエンザウィルス核タンパク質抗体である、〔1〕~〔4〕のいずれかに記載のデバイス。
〔6〕 ヒト抗A型インフルエンザウィルス核タンパク質抗体が、A型インフルエンザウィルス核タンパク質と特異的に反応し、B型インフルエンザウィルスの核タンパク質とは反応しないヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体である、〔5〕記載のデバイス。
〔7〕 ヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体の重鎖可変領域のアミノ酸配列が、配列番号:8~13のいずれかで表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:22~27のいずれかで表されるアミノ酸配列を含む〔6〕記載のデバイス。
〔8〕 ヒト抗インフルエンザウィルス核タンパク質抗体が、ヒト抗B型インフルエンザウィルス核タンパク質抗体である、〔1〕~〔4〕のいずれかに記載のデバイス。
〔9〕 ヒト抗B型インフルエンザウィルス核タンパク質抗体が、B型インフルエンザウィルス核タンパク質と特異的に反応し、A型インフルエンザウィルスの核タンパク質とは反応しないヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体である〔8〕記載のデバイス。
〔10〕 ヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体の重鎖可変領域のアミノ酸配列が、配列番号:14で表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:28で表されるアミノ酸配列を含む〔9〕記載のデバイス。
〔11〕 ヒト抗インフルエンザウィルス核タンパク質抗体が担体に固定化されてなる固相を含有することを特徴とする、インフルエンザウィルス検出又は定量用キット。
〔12〕 抗インフルエンザウィルス核タンパク質抗体(抗体1)が担体に固定化されてなる固相と、抗インフルエンザウィルス核タンパク質抗体(抗体2)を含有する試薬とを含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルス検出又は定量用キット。
〔13〕 抗インフルエンザウィルス核タンパク質抗体(抗体1)が担体に固定化されてなる固相と、抗インフルエンザウィルス核タンパク質抗体(抗体2)に標識が結合した標識化抗体を含有する試薬とを含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルス検出又は定量用キット。
〔14〕 ヒト抗インフルエンザウィルス核タンパク質抗体が担体に固定化されてなる固相と、インフルエンザウィルス核タンパク質抗原類似物に標識が結合した標識化抗原類似物を含有する試薬とを含有することを特徴とする、インフルエンザウィルス検出又は定量用キット。
〔15〕 インフルエンザウィルス核タンパク質抗原類似物が担体に固定化されてなる固相と、ヒト抗インフルエンザウィルス核タンパク質抗体に標識が結合した標識化抗体を含有する試薬とを含有することを特徴とする、インフルエンザウィルス検出又は定量用キット。
〔16〕 ヒト抗インフルエンザウィルス核タンパク質抗体が、ヒト抗A型インフルエンザウィルス核タンパク質抗体である、〔11〕~〔15〕のいずれかに記載のキット。
〔17〕 ヒト抗A型インフルエンザウィルス核タンパク質抗体が、A型インフルエンザウィルス核タンパク質と特異的に反応し、B型インフルエンザウィルスの核タンパク質とは反応しないヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体である、〔16〕記載のキット。
〔18〕 ヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体の重鎖可変領域のアミノ酸配列が、配列番号:8~13のいずれかで表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:22~27のいずれかで表されるアミノ酸配列を含む〔17〕記載のキット。
〔19〕 ヒト抗インフルエンザウィルス核タンパク質抗体が、ヒト抗B型インフルエンザウィルス核タンパク質抗体である、〔11〕~〔15〕のいずれかに記載のキット。
〔20〕 ヒト抗B型インフルエンザウィルス核タンパク質抗体が、B型インフルエンザウィルス核タンパク質と特異的に反応し、A型インフルエンザウィルスの核タンパク質とは反応しないヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体である〔19〕記載のキット。
〔21〕 ヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体の重鎖可変領域のアミノ酸配列が、配列番号:14で表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:28で表されるアミノ酸配列を含む〔20〕記載のキット。
〔22〕 (1)検体と、担体上に固定化されているヒト抗インフルエンザウィルス核タンパク質抗体とを反応させる工程;及び、
(2)工程(1)で生じた物理的変化を測定する工程
を含有することを特徴とする、インフルエンザウィルスの検出又は定量方法。
〔23〕 (1)検体と、担体上に固定化されている抗インフルエンザウィルス核タンパク質抗体(抗体1)とを反応させて、担体上に抗体1-インフルエンザウィルス核タンパク質の複合体を形成させる工程;
(2)工程(1)において、担体上に形成した該複合体と、抗インフルエンザウィルス核タンパク質抗体(抗体2)とを反応させる工程;及び、
(3)工程(2)で生じた物理的変化を測定する工程
を含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルスの検出又は定量方法。
〔24〕 物理的変化が、濁度変化又は質量変化である〔22〕又は〔23〕記載の方法。
〔25〕 (1)検体と、担体上に固定化されている抗インフルエンザウィルス核タンパク質抗体(抗体1)とを反応させて、担体上に抗体1-インフルエンザウィルス核タンパク質の複合体を形成させる工程;
(2)工程(1)において、担体上に形成した該複合体と、抗インフルエンザウィルス核タンパク質抗体(抗体2)に標識が結合した標識化抗体(標識化抗体2)とを反応させて、担体上に抗体1-インフルエンザウィルス核タンパク質-標識化抗体2の複合体を形成させる工程;
(3)工程(2)後の担体を洗浄し、担体上に結合していない物質を除去する工程;
(4)工程(3)後の担体上の抗体1-インフルエンザウィルス核タンパク質-標識化抗体2の複合体の中の標識を測定する工程
を含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルスの検出又は定量方法。
〔26〕 (1)検体と、抗インフルエンザウィルス核タンパク質抗体(抗体2)に標識が結合した標識化抗体(標識化抗体2)とを反応させて、標識化抗体2-インフルエンザウィルス核タンパク質の複合体を形成させる工程;
(2)工程(1)において形成した複合体と、担体上に固定化されている抗インフルエンザウィルス核タンパク質抗体(抗体1)とを反応させて、担体上に抗体1-インフルエンザウィルス核タンパク質-標識化抗体2の複合体を形成させる工程;
(3)工程(2)後の担体を洗浄し、担体上に結合していない物質を除去する工程;
(4)工程(3)後の担体上の抗体1-インフルエンザウィルス核タンパク質-標識化抗体2の複合体の中の標識を測定する工程
を含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルスの検出又は定量方法。
〔27〕 (1)検体と、担体上に固定化されているヒト抗インフルエンザウィルス核タンパク質抗体とを、インフルエンザウィルス核タンパク質抗原類似物に標識が結合した標識化抗原類似物の共存下に反応させて、担体上に、ヒト抗インフルエンザウィルス核タンパク質抗体-インフルエンザウィルス核タンパク質の複合体、及び、ヒト抗インフルエンザウィルス核タンパク質抗体-標識化抗原類似物の複合体を形成させる工程;
(2)工程(1)後の担体を洗浄し、担体上に結合していない物質を除去する工程;
(3)工程(2)後の担体上の複合体中の標識を測定する工程
を含有することを特徴とする、インフルエンザウィルスの検出又は定量方法。
〔28〕 (1)検体と、担体上に固定化されているインフルエンザウィルス核タンパク質抗原類似物とを、ヒト抗インフルエンザウィルス核タンパク質抗体に標識が結合した標識化抗体の共存下に反応させて、担体上に、インフルエンザウィルス核タンパク質抗原類似物-標識化抗体の複合体を形成させる工程;
(2)工程(1)後の担体を洗浄し、担体上に結合していない物質を除去する工程;
(3)工程(2)後の担体上の複合体中の標識を測定する工程
を含有することを特徴とする、インフルエンザウィルスの検出又は定量方法。
〔29〕 ヒト抗インフルエンザウィルス核タンパク質抗体が、ヒト抗A型インフルエンザウィルス核タンパク質抗体である〔22〕~〔28〕のいずれかに記載の方法。
〔30〕 ヒト抗A型インフルエンザウィルス核タンパク質抗体が、A型インフルエンザウィルス核タンパク質と特異的に反応し、B型インフルエンザウィルスの核タンパク質とは反応しないヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体である〔29〕記載の方法。
〔31〕 ヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体の重鎖可変領域のアミノ酸配列が、配列番号:8~13のいずれかで表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:22~27のいずれかで表されるアミノ酸配列を含む〔30〕記載の方法。
〔32〕 ヒト抗インフルエンザウィルス核タンパク質抗体が、ヒト抗B型インフルエンザウィルス核タンパク質抗体である〔22〕~〔28〕のいずれかに記載の方法。
〔33〕 ヒト抗B型インフルエンザウィルス核タンパク質抗体が、B型インフルエンザウィルス核タンパク質と特異的に反応し、A型インフルエンザウィルスの核タンパク質とは反応しないヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体である〔32〕記載の方法。
〔34〕 ヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体の重鎖可変領域のアミノ酸配列が、配列番号:14で表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:28で表されるアミノ酸配列を含む〔33〕記載の方法。
〔35〕 重鎖可変領域のアミノ酸配列が、配列番号:8~13のいずれかで表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:22~27のいずれかで表されるアミノ酸配列を含む、A型インフルエンザウィルス核タンパク質と特異的に反応し、B型インフルエンザウィルスの核タンパク質と反応しないヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体。
〔36〕 B型インフルエンザウィルス核タンパク質と特異的に反応し、A型インフルエンザウィルスの核タンパク質と反応しないヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体。
〔37〕 重鎖可変領域のアミノ酸配列が、配列番号:14で表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:28で表されるアミノ酸配列を含む、B型インフルエンザウィルス核タンパク質と特異的に反応し、A型インフルエンザウィルスの核タンパク質と反応しないヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体。
本発明のインフルエンザウィルス検出又は定量用デバイスは、インフルエンザウィルスの検出又は定量に使用され得るデバイスであり、具体的にはイムノクロマトグラフィー等に適したデバイスである。本発明のデバイスは、フロースルー型、ラテラルフロー型のどちらでもよい。
本発明のデバイスにおける支持体は、検体移動領域で、液体が展開可能な材料であれば特に制限はなく、例えばガラス繊維、セルロース、ナイロン、架橋デキストラン、各種のクロマトグラフィー用紙、ニトロセルロース、金等の金属等が挙げられる。この支持体の大きさに制限はないが、幅3mm~10mm程度、長さ30mm~100mm程度のストリップ状のものが好ましい。支持体の厚さは100μm~1mmのものを用いることができる。また支持体は、その一部又は全体を測定時に検体由来のタンパク質の支持体への非特異吸着防止のために、例えば牛血清アルブミン(BSA)、カゼイン等の動物血清、カゼイン、スクロース等でブロッキングして用いることができる。
本発明のデバイスにおける検出領域には、抗インフルエンザウィルス核タンパク質抗体が不動化されている。検出領域は支持体とは別個に形成されてもよいが、支持体上に形成されることが好ましい。検出領域に不動化される抗インフルエンザウィルス核タンパク質抗体はモノクローナル抗体でもポリクローナル抗体でもよいが、モノクローナル抗体であることが好ましい。検出領域に不動化される抗インフルエンザウィルス核タンパク質抗体、及び、標識試薬領域で移動可能なように保持されている標識化抗インフルエンザウィルス核タンパク質抗体を形成する抗インフルエンザウィルス核タンパク質抗体の少なくとも1つはヒト抗体であり、両抗体ともモノクローナル抗体であることが好ましい。検出領域に不動化される抗インフルエンザウィルス核タンパク質抗体としては、例えば後述のヒト抗A型インフルエンザウィルス核タンパク質抗体、ヒト抗B型インフルエンザウィルス核タンパク質抗体、これらのヒト抗体のフラグメント[Fab、F(ab’)2、F(ab’)]等が挙げられる。
本発明のデバイスにおける標識試薬領域には、抗インフルエンザウィルス核タンパク質抗体に標識が結合した標識化抗体が移動可能なように保持されている。標識試薬領域は支持体とは別個に形成されてもよいが、支持体上に形成されることが好ましい。標識試薬領域に移動可能なように保持される標識化抗インフルエンザウィルス核タンパク質抗体を形成する抗インフルエンザウィルス核タンパク質抗体はモノクローナル抗体でもポリクローナル抗体でもよいが、モノクローナル抗体であることが好ましい。標識試薬領域に移動可能なように保持される抗インフルエンザウィルス核タンパク質抗体としては、例えば後述のヒト抗A型インフルエンザウィルス核タンパク質抗体、ヒト抗B型インフルエンザウィルス核タンパク質抗体、これらのヒト抗体のフラグメント[Fab、F(ab’)2、F(ab’)]等が挙げられる。標識試薬領域の構築方法としては、例えば支持体に標識化抗体含有試薬を支持体に点着する方法、標識化抗体含有試薬を含ませた吸水性パッドを支持体上に積層する方法等が挙げられる。吸水性パッドとしては、例えば後述の検体供給領域で使用される吸水性パッド等が挙げられる。
本発明のデバイスにおける検体供給領域は、検出領域の上流に位置し、本領域を通じて検体が供給される。検体供給領域は、支持体上に形成されるが、支持体上に、吸水性パッドを積層させて形成してもよい。検体供給領域を形成する支持体及び吸水性パッドには、適宜、塩類、界面活性剤等が含有されてもよい。塩類、界面活性剤としては、例えば前述の塩類、界面活性剤等が挙げられる。吸水性パッドとしては、例えばガラス繊維、セルロース、不織布、ポリビニルアルコール等が挙げられる。
本発明のデバイスにおける検体移動領域は、支持体全般に渡って存在し、検体、標識化抗体、後述の展開液が移動する領域である。検体供給領域に供給された検体は、検体移動領域を通じて標識化抗体と共に検出領域に運ばれる。また、検体は、後述の展開液供給領域から供給された展開液によっても、検体移動領域を通じて検出領域に運ばれ得る。
本発明のデバイスにおける展開液供給領域は、支持体の一端に設けられ、展開液が供給される領域である。検体供給領域に検体が供給されたデバイスの展開液供給領域を展開液に浸すことにより、検体は展開液により支持体を移動し、検体移動領域を通じて検出領域に運ばれ得る。この過程で、検体中のインフルエンザウィルス核タンパク質は、標識試薬領域で標識化抗体と反応し、生成した移動可能な、標識化抗体-インフルエンザウィルス核タンパク質の複合体はさらに検出領域に運ばれ、検出領域において、抗インフルエンザウィルス核タンパク質抗体-インフルエンザウィルス核タンパク質-標識化抗体の複合体を生成する。展開液を用いずとも検体を支持体上で展開させることもできるが、展開液を用いることにより効率的に検体を展開させることができる。展開液は、検体を支持体上で展開させる水性媒体であるが、この水性媒体には必要に応じて、前述の緩衝剤、界面活性剤、防腐剤、塩類、糖類、金属イオン、蛋白等の添加物を含んでいてもよい。
本発明のデバイスにおける検体供給確認領域は、余剰液吸収領域の上流に設けられ、検体が確実にデバイスの検体供給領域に添加されたことを確認するための領域である。
本発明のデバイスにおける余剰液吸収領域は、支持体の最下流に設けられ、支持体上を要すれば展開液と共に展開してきた検体及び標識化抗体が検出領域で捕捉されて抗インフルエンザ核タンパク質抗体-インフルエンザ核タンパク質-標識化抗体の複合体を生成し、該複合体を生成した後の余剰液を吸収するための領域である。余剰液吸収領域は、支持体から形成することもできるが、支持体に吸水性物質を付随させて形成することもでき、また、支持体上に吸水性物質を積層させて形成することもできる。吸水性物質としては、例えば吸水性の高いろ紙、スポンジ等が挙げられる。
本発明のデバイスを用いたインフルエンザウィルスの検出と定量は、例えば次のように行うことができる。
検体供給領域に供給された検体は検出移動領域を展開し、検出領域に到達する。検出領域において、検体中のインフルエンザウィルス核タンパク質は、ヒト抗インフルエンザウィルス核タンパク質抗体と反応し、インフルエンザウィルス核タンパク質-ヒト抗インフルエンザウィルス核タンパク質抗体の複合体を生成する。この複合体形成に伴う物理的変化を測定することにより、インフルエンザウィルスを検出又は定量することができる。ここでの物理的変化としては、例えば質量変化等が挙げられる。
検体供給領域に供給された検体及び標識化抗体(標識化抗インフルエンザウィルス核タンパク質抗体)は検出移動領域を展開し、検出領域に到達する。供給時又は展開時に生成したインフルエンザウィルス核タンパク質-標識化抗体の複合体は、検出領域において、抗インフルエンザウィルス核タンパク質抗体と反応し、抗インフルエンザウィルス核タンパク質抗体-インフルエンザウィルス核タンパク質-標識化抗体の複合体を生成する。検出領域において生成したこの抗インフルエンザウィルス核タンパク質抗体-インフルエンザウィルス核タンパク質-標識化抗体の複合体中の標識を測定することにより、インフルエンザウィルスを検出又は定量することができる。
検体供給領域に供給された検体は標識試薬領域において、標識化抗インフルエンザウィルス核タンパク質抗体(標識化抗体2)と反応し、インフルエンザウィルス核タンパク質-標識化抗体2の複合体(複合体1)が生成し、次いで、検出領域に検体移動領域を通じて展開してきた複合体1は検出領域において、抗インフルエンザウィルス核タンパク質抗体(抗体1)と反応し、抗体1-抗インフルエンザウィルス核タンパク質-標識化抗体2の複合体(複合体2)が検出領域において不動化されて生成し、検出領域内に生成した複合体2の標識を測定することにより、インフルエンザウィルスを検出又は定量することができる。ここで、標識化抗インフルエンザウィルス核タンパク質抗体(標識化抗体2)を構成する抗インフルエンザウィルス核タンパク質抗体(抗体2)と、抗体1の少なくとも1つはヒト抗体である。検出供給領域に供給された検体の展開、標識試薬領域で生成した複合体1の検出領域への展開には、展開液を用いることもできる。
検体供給領域に供給された検体及び標識化抗原類似物(標識化インフルエンザウィルス核タンパク質抗原類似物)は検出移動領域を展開し、検出領域に到達する。検出領域において、検体中のインフルエンザウィルス核タンパク質抗原と標識化抗原類似物とは、ヒト抗インフルエンザウィルス核タンパク質抗体に対して競合的に反応し、ヒト抗インフルエンザウィルス核タンパク質抗体-インフルエンザウィルス核タンパク質抗原の複合体、及び、ヒト抗インフルエンザウィルス核タンパク質抗体-標識化抗原類似物の複合体を精製する。この検出領域に生成した複合体中の標識を測定することにより、インフルエンザウィルスを検出又は定量することができる。
検体供給領域に供給された検体は、検体移動領域を展開し、標識試薬領域に到達する。標識試薬領域に到達した検体は、標識化抗原類似物(標識化インフルエンザウィルス核タンパク質抗原類似物)と共に検体移動領域を更に展開し、検出領域に到達する。検出領域において、検体中のインフルエンザウィルス核タンパク質抗原と標識化抗原類似物とは、ヒト抗インフルエンザウィルス核タンパク質抗体に対して競合的に反応し、ヒト抗インフルエンザウィルス核タンパク質抗体-インフルエンザウィルス核タンパク質抗原の複合体、及び、ヒト抗インフルエンザウィルス核タンパク質抗体-標識化抗原類似物の複合体を精製する。この検出領域に生成した複合体中の標識を測定することにより、インフルエンザウィルスを検出又は定量することができる。
検体供給領域に供給された検体及び標識化抗体(標識化ヒト抗インフルエンザウィルス核タンパク質抗体)は検出移動領域を展開する。供給時又は展開時に生成したインフルエンザウィルス核タンパク質-標識化抗体の複合体は、未反応の標識化抗体と共に検出領域に到達する。検出領域において、未反応の標識化抗体は、インフルエンザウィルス核タンパク質抗原類似物と反応し、インフルエンザウィルス核タンパク質抗原類似物-標識化抗体の複合体を生成する。検出領域において生成したこのインフルエンザウィルス核タンパク質抗原類似物-標識化抗体の複合体中の標識を測定することにより、インフルエンザウィルスを検出又は定量することができる。
検体供給領域に供給された検体は、検体移動領域を展開し、標識試薬領域に到達する。標識試薬領域において、インフルエンザウィルス核タンパク質は標識化抗体(標識化ヒト抗インフルエンザウィルス核タンパク質抗体)と反応し、インフルエンザウィルス核タンパク質-標識化抗体の複合体を生成する。生成した複合体は、未反応の標識化抗体と共にさらに検体移動領域を展開し、検出領域に到達する。検出領域において、未反応の標識化抗体は、インフルエンザウィルス核タンパク質抗原類似物と反応し、インフルエンザウィルス核タンパク質抗原類似物-標識化抗体の複合体を生成する。検出領域において生成したこのインフルエンザウィルス核タンパク質抗原類似物-標識化抗体の複合体中の標識を測定することにより、インフルエンザウィルスを検出又は定量することができる。
本発明のインフルエンザウィルス検出又は定量方法は、ヒト抗インフルエンザウィルス核タンパク質抗体を少なくとも1つ以上用いることを特徴とする方法である。後述の、本発明のインフルエンザウィルス検出又は定量用キットを用いて、インフルエンザウィルスを検出又は定量することができる。
(1)検体と、担体上に固定化されているヒト抗インフルエンザウィルス核タンパク質抗体とを反応させる工程;及び、
(2)工程(1)で生じた物理的変化を測定する工程
を含有することを特徴とする、インフルエンザウィルスの検出又は定量方法。
(1)検体と、担体上に固定化されている抗インフルエンザウィルス核タンパク質抗体(抗体1)とを反応させて、担体上に抗体1-インフルエンザウィルスの複合体を形成させる工程;
(2)工程(1)において、担体上に形成した該複合体と、抗インフルエンザウィルス核タンパク質抗体(抗体2)とを反応させる工程;及び、
(3)工程(2)で生じた物理的変化量を測定する工程
を含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルス検出又は定量方法。
(1)検体と、担体上に固定化されている抗インフルエンザウィルス核タンパク質抗体(抗体1)とを反応させて、担体上に抗体1-インフルエンザウィルス核タンパク質の複合体を形成させる工程;
(2)工程(1)において、担体上に形成した該複合体と、抗インフルエンザウィルス核タンパク質抗体(抗体2)に標識が結合した標識化抗体(標識化抗体2)とを反応させて、担体上に抗体1-インフルエンザウィルス核タンパク質-標識化抗体2の複合体を形成させる工程;
(3)工程(2)後の担体を洗浄し、担体上に結合していない物質を除去する工程;
(4)工程(3)後の担体上の抗体1-インフルエンザウィルス核タンパク質-標識化抗体2の複合体の中の標識を測定する工程
を含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルス検出又は定量方法。
(1)検体と、抗インフルエンザウィルス核タンパク質抗体(抗体2)に標識が結合した標識化抗体(標識化抗体2)とを反応させて、標識化抗体2-インフルエンザウィルス核タンパク質の複合体を形成させる工程;
(2)工程(1)において形成した複合体と、担体上に固定化されている抗インフルエンザウィルス核タンパク質抗体(抗体1)とを反応させて、担体上に抗体1-インフルエンザウィルス核タンパク質-標識化抗体2の複合体を形成させる工程;
(3)工程(2)後の担体を洗浄し、担体上に結合していない物質を除去する工程;
(4)工程(3)後の担体上の抗体1-インフルエンザウィルス核タンパク質-標識化抗体2の複合体の中の標識を測定する工程
を含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルス検出又は定量方法。
(1)検体と、担体上に固定化されているヒト抗インフルエンザウィルス核タンパク質抗体とを、インフルエンザウィルス核タンパク質抗原類似物に標識が結合した標識化抗原類似物の共存下に反応させて、担体上に、ヒト抗インフルエンザウィルス核タンパク質抗体-インフルエンザウィルス核タンパク質の複合体、及び、ヒト抗インフルエンザウィルス核タンパク質抗体-標識化抗原類似物の複合体を形成させる工程;
(2)工程(1)後の担体を洗浄し、担体上に結合していない物質を除去する工程;
(3)工程(2)後の担体上の複合体中の標識を測定する工程
を含有することを特徴とする、インフルエンザウィルスの検出又は定量方法。
(1)検体と、担体上に固定化されているインフルエンザウィルス核タンパク質抗原類似物とを、ヒト抗インフルエンザウィルス核タンパク質抗体に標識が結合した標識化抗体の共存下に反応させて、担体上に、インフルエンザウィルス核タンパク質抗原類似物-標識化抗体の複合体を形成させる工程;
(2)工程(1)後の担体を洗浄し、担体上に結合していない物質を除去する工程;
(3)工程(2)後の担体上の複合体中の標識を測定する工程
を含有することを特徴とする、インフルエンザウィルスの検出又は定量方法。
本発明のインフルエンザウィルス検出又は定量用キットは、本発明のインフルエンザウィルス検出又は定量方法に用いられるキットである。
本発明のインフルエンザウィルス検出又は定量用キットとしては、例えば以下に示すキットを挙げることができる。
ヒト抗インフルエンザウィルス核タンパク質抗体が担体に固定化されてなる固相を含有することを特徴とする、インフルエンザウィルス検出又は定量用キット。
本キットは比濁免疫測定法、表面プラズモン共鳴(SPR)法等の方法に適したキットである。
抗インフルエンザウィルス核タンパク質抗体(抗体1)が担体に固定化されてなる固相と、
抗インフルエンザウィルス核タンパク質抗体(抗体2)を含有する試薬
とを含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルス検出又は定量用キット。
本キットは比濁免疫測定法、表面プラズモン共鳴(SPR)法等の方法に適したキットである。
抗インフルエンザウィルス核タンパク質抗体(抗体1)が担体に固定化されてなる固相と、
抗インフルエンザウィルス核タンパク質抗体(抗体2)に標識が結合した標識化抗体を含有する試薬
とを含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルス検出又は定量用キット。
本キットはサンドイッチ法に基づく免疫学的測定法に適したキットである。
ヒト抗インフルエンザウィルス核タンパク質抗体が担体に固定化されてなる固相と、
インフルエンザウィルス核タンパク質抗原類似物に標識が結合した標識化抗原類似物を含有する試薬
とを含有することを特徴とする、インフルエンザウィルス検出又は定量用キット。
インフルエンザウィルス核タンパク質抗原類似物が担体に固定化されてなる固相と、
ヒト抗インフルエンザウィルス核タンパク質抗体に標識が結合した標識化抗体を含有する試薬
とを含有することを特徴とする、インフルエンザウィルス検出又は定量用キット。
本発明のヒト抗インフルエンザウィルス核タンパク質抗体は、本発明のインフルエンザウィルスの検出又は定量用デバイス、インフルエンザウィルスの検出又は定量用キット、並びに、インフルエンザウィルスの検出又は定量方法に使用され得る抗体であり、ヒト抗A型インフルエンザウィルス核タンパク質抗体、ヒト抗B型インフルエンザウィルス核タンパク質抗体等が挙げられる。また、本発明におけるヒト抗インフルエンザウィルス核タンパク質抗体は、モノクローナル抗体でもポリクローナル抗体でもよいが、モノクローナル抗体が好ましい。
上述の通り、本発明のヒト抗インフルエンザウィルス核タンパク質抗体としては、ヒト抗インフルエンザウィルス核タンパク質モノクローナル抗体が好ましい。ヒト抗インフルエンザウィルス核タンパク質モノクローナル抗体は、公知のモノクローナル抗体製造方法[ハイブリドーマ法(例えば、In Vitro Cell Dev Biol. (1985)21(10):593-596参照)、ウィルス感染法(例えば、J. gen. Virol. (1983), 64, 697-700参照)、ファージディスプレイ法(例えば、WO2001/062907パンフレット参照)、リンパ球マイクロアレイ法等]で製造することができるが、リンパ球マイクロアレイ法が好ましい。
[2]測定対象物に反応するBリンパ球を選択する工程;
[3]選択されたBリンパ球から、測定対象物と反応する抗体に係る遺伝子を取得する工程;
[4]取得した遺伝子を用いて抗体を発現させる工程;
[5]目的の性質を有する抗体を選択する工程。
ヒトBリンパ球は、ヒト末梢血より調製することができ、特に、インフルエンザウィルス感染歴あるいはインフルエンザワクチン接種歴があり、血清中にインフルエンザウィルス核タンパク質抗体の存在が確認できるヒト末梢血から調製することができる。インフルエンザワクチン接種一定期間後、好ましくは1~30日後、より好ましくは5~10日後に採血を行い、調製することが望ましい。調製方法の具体例としては、例えば密度勾配遠心、セルソーター、磁気ビーズ等を用いる方法等が挙げられる。
1個の抗原特異的Bリンパ球の選択は、例えば、マイクロウェルアレイチップを用いた方法により行うことができる。マイクロウェルアレイチップを用いた方法では、例えば、1個の被検体Bリンパ球が含まれるマイクロウェルを複数有する抗原特異的Bリンパ球検出用マイクロウェルアレイチップの各マイクロウェルに抗原を添加し、次いで、抗原に反応したBリンパ球を検出し、検出された抗原特異的Bリンパ球をマイクロウェルから取り出すことにより、1個の抗原特異的Bリンパ球を得ることができる。以下、この方法を、より具体的に説明する。
2. 10% FCS(牛胎仔血清)含有RPMI1640培地
3. 1 mg/mL BSA含有RPMI1640培地
4. 10% FCS(牛胎仔血清)含有Dulbecco's MEM培地
5. 1 mg/mL BSA含有Dulbecco's MEM培地
例えば、Bリンパ球の抗原受容体(免疫グロブリン)に抗原が結合するとまず細胞内シグナル伝達が起こり、それに続いて細胞増殖、抗体産生が起こる。従って、細胞内シグナル伝達、細胞増殖、抗体産生を種々の方法により検知することにより、抗原に反応する細胞を検出することができる。細胞内シグナル伝達を検出することによる、抗原に反応する細胞の検出は、例えば、細胞内Caイオンの濃度変化をCaイオン依存性の蛍光色素を用いることにより行うことができる。細胞内Caイオン濃度変化は、蛍光色素としてFura-2、Fluo-3あるいはFluo-4を用い、検出装置として蛍光顕微鏡あるいはマイクロアレイスキャナーを用いることができる。
取り出された抗原特異的Bリンパ球は、細胞溶解剤を用いて溶解した後、PCRを用いて、抗原特異的免疫グロブリン(抗体)遺伝子がクローニングされる。細胞溶解剤としては、公知の物をそのまま使用でき、例えば、以下のものを挙げることができる。
PCR法による抗体遺伝子の増幅では、PCR反応を2回実施することにより抗体遺伝子のV領域遺伝子を増幅することが好ましい。
上述の通り、細胞から遺伝子工学的手法を用いて該抗体の重鎖(H鎖)および軽鎖(L鎖)をコードするcDNAを取得し、取得したcDNAを抗体発現用ベクターのプロモーターの下流に挿入した組換えベクターを造成し、それを宿主細胞に導入することにより得られた該抗体発現細胞を適当な培地中で培養するか、動物に投与して該細胞を腹水癌化させ、該培養液または腹水を分離精製することにより調製することができる。このような抗体発現用ベクターは、ヒト抗体の定常領域(C領域)である定常領域重鎖(CH)および定常領域軽鎖(CL)をコードする遺伝子が組み込まれた動物細胞用発現ベクターであり、動物細胞用発現ベクターにヒト抗体のCHおよびCLをコードする遺伝子をそれぞれ挿入することにより構築される。
前工程で得られた抗体は、形質転換株の培養上清よりプロテインAカラムを用いて精製することができる(アンチボディズ、第8章)。また、その他に通常の蛋白質に用いられる精製方法、例えば、ゲル濾過、イオン交換クロマトグラフィーおよび限外濾過等を単独又は組み合わせて使用することができる。精製した組換え抗体のH鎖、L鎖あるいは抗体分子全体の分子量は、ポリアクリルアミド電気泳動(SDS-PAGE)[Nature,227,680(1970)]やウェスタンブロッティング法(アンチボディズ 第12章)等で測定する。
以下、実施例により本発明をより詳細に説明するが、これらは本発明の範囲を何ら限定するものではない。
・アルカリホスファターゼ標識化抗体の作製
後述の実施例4において取得した2種のヒト抗インフルエンザウィルス核タンパク質モノクローナル抗体[23G285(A型), 23G327(B型)]および2種のマウス抗インフルエンザウィルス核タンパク質モノクローナル抗体[M2110169(A型:Fitzgerald社製)、M2110171(B型:Fitzgerald社製)]をAlkaline Phosphatase Labeling Kit(同仁化学研究所社製)を用いてアルカリホスファターゼ標識化抗体を作製した。
5 cm×0.5 cmに切断したろ紙(ミリポア社製)の一領域にマウス抗A型インフルエンザウィルス核タンパク質モノクローナル抗体M2110169, M322211(Fitzgerald社製)及びヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体23G268のPBS溶液をそれぞれ点滴し、当該モノクローナル抗体を固定化し、検出領域とした。このろ紙の下方(検体供給領域)より、ノニデットP-40を含有する反応用溶液で希釈したA型インフルエンザウィルス液(H3N2:A/Panama/2007/99)、アルカリホスファターゼ標識化マウス抗A型インフルエンザウィルス核タンパク質モノクローナル抗体M2110169又はアルカリホスファターゼ標識化ヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体23G285のTris溶液、BCIP/NBT(nitro-blue tetrazolium chloride)溶液(シグマ社製)の順に展開させた。結果を図2-aに示す。特に、ヒト抗インフルエンザウィルス核タンパク質抗体を用いる固相抗体(検出領域で不動化される抗体)と標識抗体(標識化に使用される抗体)との組み合わせにおいて、検出領域において、顕著な青いスポットが確認された。このことから、イムノクロマトグラフィーによるA型インフルエンザウィルスの検出に、ヒト抗A型インフルエンザウィルス核タンパク質抗体が利用可能であると共に、ヒト抗A型インフルエンザウィルス核タンパク質抗体が高感度のA型インフルエンザウィルスの検出に有用であることが判明した。
5 cm×0.5 cmに切断したろ紙(ミリポア社製)の一領域にマウス抗B型インフルエンザウィルス核タンパク質モノクローナル抗体M2110171(Fitzgerald社製)及び41027(Capricorn社製)のPBS溶液をそれぞれ点滴し、当該モノクローナル抗体を固定化し、検出領域とした。このろ紙の下方(検体供給領域)より、検体として、ノニデットP-40を含有する反応用溶液で希釈したA型インフルエンザウィルス液(H1N1:A/Beijing/262/95)及びノニデットP-40を含有する反応用溶液で希釈したB型インフルエンザウィルス液(B/Victoria/504/00)をそれぞれ供給し、次いで、各デバイスの検体供給領域に、ヒト抗B型インフルエンザウィルス核タンパク質抗体23G327にアルカリホスファターゼが結合したアルカリホスファターゼ標識化抗体のTris溶液を供給し、さらに、BCIP/NBT(nitro-blue tetrazolium chloride)溶液を展開液供給領域から展開させた。結果を図3に示す。B型インフルエンザウィルス液(B/Victoria/504/00)を用いた場合にのみ、検出領域に青いスポットが検出された。
金コロイド標識化抗体の作製
後述の実施例4において取得した2種のヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体[23G272、23G447]および1種のマウス抗A型インフルエンザウィルス核タンパク質モノクローナル抗体[M322211(Fitzgerald社製)]を標識抗体として用いて金コロイド標識化抗体を作製した。すなわち、当該抗体のPBS溶液をそれぞれTris緩衝液で置換して当該抗体のTris溶液を調製し、次いで、40 nm金コロイド(BBInternational社製)と混合し15分間反応させた。反応後、1%BSAを含むTris溶液によるブロッキングの後、未結合のモノクローナル抗体を遠心分離により除去し、金コロイド標識化抗体とした。
金コロイド標識化抗体を用いたA型インフルエンザウィルスの検出又は定量用デバイス5 cm×0.5 cmに切断したろ紙(ミリポア社製)の一領域に、固相抗体として抗A型インフルエンザウィルス核タンパク質モノクローナル抗体[23G312、23G494、M2110169(Fitzgerald社製)]を固定化し、検出領域とした。すなわち、当該抗A型インフルエンザウィルス核タンパク質モノクローナル抗体のPBS溶液をそれぞれ点滴・固定化し、検出領域とした。このろ紙の下方(検体供給領域)より、ノニデットP-40を含有する反応用溶液で希釈したA型インフルエンザウィルス液(H1N1:A/Beijing/262/95)と上記で作製された金コロイド標識化抗体のTris溶液とを等量混合した溶液0.1 mLを展開させた。その結果、いずれの抗体の組み合わせ(固相抗体と標識抗体との組み合わせ)においても検出領域に顕著な赤色のスポットが確認された。スポットが目視で確認できる時間を比較したところ、固相抗体と標識抗体にヒト抗体を用いた組み合わせにおいては、いずれの組み合わせにおいても、固相抗体と標識抗体にマウス抗体を用いた組み合わせに比較して、約2倍速いことが認められた。
アクリジニウム標識化抗体の作製
後述の実施例4において取得した7種のヒト抗インフルエンザウィルス核タンパク質モノクローナル抗体[23G268, 23G272, 23G285, 23G312, 23G447, 23G494(以上、A型);23G327(B型)]および市販マウス抗インフルエンザウィルス核タンパク質モノクローナル抗体それぞれにアクリジニウムエステル(同仁化学研究所社製)を反応させ、ゲルろ過により未反応のアクリジニウムエステルを除去し、アクリジニウム標識化抗インフルエンザウィルス核タンパク質抗体を得た。
96ウェルマイクロタイタープレート(ヌンク社製)に、PBSで希釈した抗A型インフルエンザウィルス核タンパク質モノクローナル抗体(10μg/mL)を各ウェルに50μLずつ分注し、4℃で一晩静置した。ウェル内の溶液を除去後、1% BSA/PBS溶液を100μLずつ分注し、室温で2時間反応させてブロッキングした。0.5%ノニデットP-40、0.5% BSAを含有するPBSで希釈した3種のインフルエンザウィルス液(H1N1:A/Beijing/262/95、H3N2:A/Panama/2007/99、B:B/Victoria/504/00)を各ウェルに50μL加え、室温で1時間反応させた。0.05% Tween20を含むPBSで洗浄した後、アクリジニウム標識化抗A型インフルエンザウィルス核タンパク質モノクローナル抗体を50μL加え、室温で1時間反応させた。0.05% Tween20を含むPBSで洗浄した後、アクリジニウム由来の発光量を測定し、ブランクの発光量とのS/N比を求めた。結果を図7に示す。この結果より固相抗体として23G268抗体を、標識抗体として23G285抗体を用いる組み合わせが最も高感度であることが示された。
96ウェルマイクロタイタープレート(ヌンク社製)に、PBSで希釈したインフルエンザB型ウィルス核タンパク質モノクローナル抗体(10μg/mL)を各ウェルに50μLずつ分注し、4℃で一晩静置した。ウェル内の溶液を除去後、1% BSA/PBS溶液を100μLずつ分注し、室温で2時間反応させてブロッキングした。0.5%ノニデットP-40、0.5% BSAを含有するPBSで希釈した3種のインフルエンザウィルス液(H1N1:A/Beijing/262/95、H3N2:A/Panama/2007/99、B:B/Victoria/504/00)を各ウェルに50μL加え、室温で1時間反応させた。0.05% Tween20を含むPBSで洗浄した後、アクリジニウム標識抗B型インフルエンザウィルス核タンパク質モノクローナル抗体を50μL加え、室温で1時間反応させた。0.05% Tween20を含むPBSで洗浄した後、アクリジニウム由来の発光量を測定し、ブランクの発光量とのS/N比を求めた。結果を図8に示す。固相抗体として23G237を、標識抗体としてM2110171(Fitzgerald社製)を用いることで最も高感度で検出できることが判明した。
図7、8に示す様に、モノクローナル抗体を用いたインフルエンザウィルス検出評価において、固相抗体と標識抗体を同一クローンとしても検出可能であることが示された。そこで同一クローンの固相抗体・標識抗体を用いてウィルス株特異性評価を行った。
センサーチップの作製
センサーチップCM5をBiacore2000にセットし、Running BufferとしてHBS-EP(GEヘルスケアバイオサイエンス社製)を10μL/分の流速で流した。活性化液(0.4 mol/LのEDCと0.1 mol/LのNHSの等量混合溶液:各GEヘルスケアバイオサイエンス社製)を7分間流して接触させた。続けて、Sodium Acetate Buffer, pH5.0で10μg/mLに調製したヤギ抗ヒトIgGもしくはヤギ抗マウスIgG(Thermo Scientific社製)を7分間流してCM5表面に固定化した。続いて、活性化されている残りのカルボン酸をマスクするために、エタノールアミン(1 mol/L, pH8.5)を7分間流した。続いて、グリシン-塩酸バッファー(0.2 mol/L, pH2.2)を1分間流して洗浄した。本操作をフローセル2において行い、フローセル1に対しては同様にEDC、NHSで活性化を行い、IgGの接触を行わずにエタノールアミンのみでマスクした。
10μg/mLに調製した抗A型インフルエンザウィルス核タンパク質モノクローナル抗体を上記で作製したセンサーチップに流速5μL/分で10分間流すことにより、抗体をセンサーチップ上に捕捉させた。この時、フローセル2において得られた信号(RU)からフローセル1において得られた信号(RU)を差し引いた値を抗体結合量として算出した。その後、HBS-EPにて10μg/mLに調製した3種のインフルエンザウィルス(H1N1:A/Beijing/262/95、H3N2:A/Panama/2007/99、B:B/Victoria/504/00)溶液を流速5μL/分で5分間反応させ、さらにHBS-EP(0.01 mol/L HEPES, 0.15 mol/L NaCl, 3 mmol/L EDTA, 0.005% surfactant P20)バッファーを流速5μL/分で5分間流した。この時、フローセル2において得られた信号(RU)からフローセル1において得られた信号(RU)を差し引いた値から先述の抗体結合量を差し引いた値を抗原結合量として算出した。反応後、グリシン-塩酸バッファー(0.2 mol/L, pH1.7)を1分間流してセンサーチップを再生させた。市販マウス抗A型インフルエンザウィルス核タンパク質抗体(M322211)と3種のインフルエンザウィルス(H1N1:A/Beijing/262/95、H3N2:A/Panama/2007/99、B:B/Victoria/504/00)溶液との相互作用を表すセンサーグラムを図9-aに、本発明で得られたヒト抗A型インフルエンザウィルス核タンパク質抗体(23G272)と3種のインフルエンザウィルス(H1N1:A/Beijing/262/95、H3N2:A/Panama/2007/99、B:B/Victoria/504/00)溶液との相互作用を表すセンサーグラムを図9-bに示す。本発明のヒト抗体である23G272においては、抗原解離反応が認められず、当該抗体が親和性の強い抗体であることが明らかとなった。
[インフルエンザウィルス核タンパク質の調製]
インフルエンザウィルス核タンパク質遺伝子は公開されている配列情報(A/Puerto Rico/8/34、B/Ann Arbor/1/86)を基に合成し、pSUPEXベクターへ組み込んだ。このベクターを用いて大腸菌(NY49株)を形質転換させ、該形質転換体にインフルエンザウィルス核タンパク質を発現させた。得られたインフルエンザウィルス核タンパク質は、インフルエンザウィルス核タンパク質に対する抗体を用いたアフィニティークロマトグラフィーにより精製した。
市販のインフルエンザHAワクチンをヒトボランティアに免疫し、免疫後9日目或いは1ヶ月後に末梢血よりリンパ球を調製した。すなわち、末梢血からヒトリンパ球をリンホセパールI液(免疫生物研究所社製)を用いて遠心分離し、さらにAutoMACS(Miltenyi Biotec, Bergisch Gladbac, Germany)を用いてリンパ球画分から非Bリンパ球分画細胞を除去しBリンパ球画分を分離精製した。
調製した2x106個Bリンパ球を1μmol/L Fluo 4-AM(カルシウム依存性蛍光色素、Invitrogen社)/BSA含有RPMIバッファー(loading buffer)[RPMI1640, 3 mg/mL BSA,25 mmol/L HEPES(pH7.4)]に懸濁し、ゆっくり震盪しながら室温で40~60分インキュベーションした。Fluo 4-AMは細胞膜透過性であるが、一旦細胞内に取り込まれてエステラーゼでAM基がはずされると、Fluo 4-AMは細胞質に留まる。細胞をLoading Bufferで洗浄し、細胞内に導入されなかった余分なFluo 4-AMを除いた後、RPMI1640/10% FCS溶液に懸濁した。なお、以下の全ての実験においてはPhenol Redを含まないRPMI1640を用いた。Fluo 4-AMはカルシウムが結合すると蛍光強度が増加する蛍光色素であり、Bリンパ球の抗原による活性化の検出に用いる。
マイクロウェルアレイチップはシリコンを用いて作製されており(富山県工業技術センター製造)、直径10μm、深さ14μmのマイクロウェルが25μmのピッチ(ウェルの中心間の距離)で縦横に配列されている。マイクロウェルは30 x 30(900ウェル)でひとつのクラスターを形成しており、それが縦に10クラスター、横5クラスター並んでいるものを用いた。
マイクロウェルアレイチップをハンクス緩衝液(HBSS)に浸し、減圧脱泡することでウェル中の空気を除くことにより、マイクロウェルへ溶液を満たした。0.2% LIPIDURE-BL-B03(日油株式会社製)でマイクロウェルチップ表面を10~15分間ブロッキング処理した後、余分なバッファーを除き、そこへ上記細胞懸濁液を添加し、10分間静置した。チップ上のマイクロウェルに入らなかった細胞をHBSSで洗い流した。リンパ球の直径は約8μmであり、使用するマイクロウェルの直径が10μmであるために一つのマイクロウェルにはリンパ球が1個入る。カバーグラスを上記シール上に置き、チップとカバーグラスの間にHBSSを満たした。
Bリンパ球を播種したマイクロウェルアレイチップをマイクロチップCCDイメージャー(Micro Chip VIEW 1100;ナノシステムソリューションズ社製)に載せ、抗原刺激前のFluo 4の蛍光強度の経時変化を10秒間隔で100秒測定した。測定を一旦停止して、チップをスキャナーから引き出し、チップとカバーグラスの間のHBSSを除き、そこへHBSSに溶解させたリコンビナント核タンパク質(10~100μg/mL)を加えた。直ちにマイクロウェルアレイチップをマイクロチップCCDイメージャーの測定部へ戻し、抗原添加後の細胞内のFluo 4の蛍光強度の経時変化を10秒間隔でスキャンし、データを保存した。個々の細胞内カルシウム濃度の経時変化が、抗原が特異抗体発現細胞に結合した時に観察される細胞内カルシウム濃度経時変化の典型的パターンとどの程度相関しているかについて、ソフトウェアを用いて解析し、抗原特異的な細胞内カルシウム応答を示す細胞を特定した。
検出したリコンビナント核タンパク質に反応し細胞内カルシウムが上昇したBリンパ球は蛍光顕微鏡下でマイクロマニピュレータを用いて回収した。すなわち、まず、カバーグラスとチップの間のHBSSを取り除き、カバーグラスとチップの間に空気を入れることでカバーグラスを取り外した。チップが乾燥しないようにチップにRPMI1640/10% FCS溶液を加え、蛍光顕微鏡下で細胞のFluo 4の蛍光を観察しながら、マイクロマニピュレータで目的の細胞を回収した。
回収したBリンパ球を予め2.5μLの逆転写反応溶液[1x 1st strand buffer(Invitrogen, SuperScript IIIに添付されている),0.25 pmol each GSP RTプライマー(Cg-RT, Cl-RT及びCk-RT), 0.1 mmol/L each dNTP,10 mmol/L DTT,1 mg/mL Bovine Serum Albumine (BSA),0.25% NP-40,1.6 U RNaseOUT(Invitrogen), 0.2x Ampdirect Plus(株式会社島津製作所), 16 U SuperScript III(Invitrogen)]の入ったPCR用チューブに移す。25℃,10分;30℃, 5秒;35℃, 5秒;40℃, 5秒;45℃, 5秒;50℃, 5秒;55℃, 1時間反応させmRNAよりcDNAを合成した。
合成したcDNA溶液2.5μLに、Terminal Deoxynucleotidyl Transferase(TdT)酵素を7.5 U添加し、10 mmol/L Tris-HCl(pH7.5), 10 mmol/L MgCl2, 1 mM DTT, 2 mmol/L dGTP存在下、37℃、1時間反応させた。
Gテーリング反応が終了したcDNA溶液10μLに、ヘラクレスII fusion DNAポリメラーゼ酵素を0.25μL添加し、1x ヘラクレスII fusion DNAポリメラーゼ・バッファー、0.25 mmol/L each dNTP、25 pmol Oligo(dC) adaptor、8% DMSO存在下、98℃, 2分間;(98℃, 20秒;60℃, 20秒;72℃, 30秒)x 23サイクル;72℃, 3分間反応させる。このPCRの反応により抗体遺伝子のOligo(dC) adaptor配列から定常部領域までのDNAが増幅される。
続いて2回目のPCR反応を行い、取得した細胞のH鎖及びL鎖のV領域のcDNAを別個のチューブを用いて増幅した。具体的には、1回目に得られたPCR反応溶液3.3μLを鋳型に、ヘラクレスII fusion DNAポリメラーゼ酵素を0.05μL添加し、1x ヘラクレスII fusion DNAポリメラーゼ・バッファー、0.25 mmol/L each dNTP、2.5 pmol AP1プライマー、2.5 pmol Cg-1stプライマー或いはCl-1st & Ck-1stプライマー、8% DMSO存在下、98℃, 2分間;(98℃, 20秒;55℃, 20秒;72℃, 30秒)x 30サイクル;72℃,3分間反応させる。
2回のPCRではcDNAが十分増幅されないため、3回目の増幅を行った。2回目に得られたPCR反応溶液0.02μLを鋳型に、TaKaRa LA Taqポリメラーゼ酵素を0.05μL添加し、1x GCバッファー、0.2 mmol/L each dNTP、3 pmol AP2プライマー、3 pmol Cg-nestプライマー或いはCl-nest & Ck-nestプライマー存在下、94℃, 3分間;(94℃, 20秒;55℃, 20秒;72℃, 90秒)x 30サイクル;72℃, 3分間反応させる。3回目のPCRにより、抗体遺伝子の可変部領域から定常部領域までのcDNA配列が増幅される。使用したプライマーの配列を表7(単一細胞5'-RACEに用いたプライマー)に示す。
PCR産物を、アガロースゲルを用いて解析、精製し、pGEM-T Easy vector(Promega)に挿入し、抗体遺伝子の塩基配列を決定し、タンパクに翻訳されることを確認した。H鎖のV領域のcDNAの塩基配列を配列番号:1~7に、アミノ酸配列を配列番号:8~14に示す。さらに、L鎖のV領域のcDNAの塩基配列を配列番号:15~21に、アミノ酸配列を配列番号:22~28に示す。
RT-PCR法にて増幅した抗体のH鎖およびL鎖の可変領域遺伝子を抗体タンパク発現用ベクター(図4-a、4-b、4-c)に組込んだ。すなわち、増幅した抗体のH鎖可変領域の遺伝子断片はpMXv6-hIgγベクターのVγの部分に、L鎖可変領域の遺伝子断片はpMXv6-hIgκのVκ又はpMXv6-hIgλベクターのVλの部分に制限酵素を用いて挿入した。作製したH鎖およびL鎖の発現ベクターから抗体タンパクを発現させるために、両方の発現ベクターを同時にヒト胎児腎由来細胞293T細胞に遺伝子導入した。遺伝子導入はLipofectamine 2000 (Invitrogen社)を用いて常法に従い行った。5~7日後に細胞上清を回収した。この培養上清中の抗体のリコンビナント核タンパク質への結合能を、リコンビナント核タンパク質をコートした96ウェルプレートを用いたELISA法により測定した。ELISAによる測定は以下のように行った。すなわち、96穴プレートにPBSで希釈したリコンビナント核タンパク質(10μg/mL)を各ウェルに50μLずつ分注し、4℃で一晩静置し吸着させた。非特異的結合を除去する為に3%BSA添加PBSを各ウェルに150μLずつ分注し、室温で1時間反応させブロッキングした。ネガティブコントロールのウェルとして、抗原を吸着させず、ブロッキングしたウェルも作成した。その後、上記293T細胞の培養上清を各ウェルに50μLずつ分注し、室温で2時間反応させた。ウェルをTBS-Tで洗浄し、1/2,000倍希釈したhorseradish peroxidase (HRP)標識抗ヒト免疫グロブリンを各ウェルに50μLずつ分注し、室温で1.5時間反応させた。ウェルをTBS-Tで洗浄し、クエン酸-リン酸緩衝液(12 mmol/Lクエン酸、26 mmol/L Na2HPO4,pH5.0)に溶解した0.4 mg/mL o-phenylenediamine(OPD; HRP基質)を各ウェルに50μLずつ分注し、室温で15分反応後にマイクロプレートリーダーにて、492 nmにおける吸光度を測定した。
96ウェルマイクロタイタープレート(ヌンク社製)に、PBSで希釈した種々のインフルエンザウィルス核タンパク質抗原(10μg/mL)を各ウェルに50μLずつ分注し、4℃で一晩静置した。ウェル内の溶液を除去後、1% BSA/PBS溶液を100μLずつ分注し、室温で2時間反応させてブロッキングした。
Claims (37)
- ヒト抗インフルエンザウィルス核タンパク質抗体が支持体上に不動化された検出領域、検体供給領域及び検体移動領域を具備することを特徴とする、検体中のインフルエンザウィルス検出又は定量用デバイス。
- 抗インフルエンザウィルス核タンパク質抗体(抗体1)が支持体上に不動化された検出領域、検体供給領域及び検体移動領域を具備し、抗インフルエンザウィルス核タンパク質抗体(抗体2)に標識が結合した標識化抗体が検体供給領域より供給され、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする検体中のインフルエンザウィルス検出又は定量用デバイス。
- 抗インフルエンザウィルス核タンパク質抗体(抗体1)が支持体上に不動化された検出領域、抗インフルエンザウィルス核タンパク質抗体(抗体2)に標識が結合した標識化抗体が移動可能なように支持体上に保持されている標識試薬領域、検体供給領域及び検体移動領域を具備し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、検体中のインフルエンザウィルス検出又は定量用デバイス。
- さらに、展開液供給領域、余剰液吸収領域及び検体供給確認領域からなる群より選ばれる領域を少なくとも1つを具備する、請求項2又は3記載のデバイス。
- ヒト抗インフルエンザウィルス核タンパク質抗体が、ヒト抗A型インフルエンザウィルス核タンパク質抗体である、請求項1~4のいずれかに記載のデバイス。
- ヒト抗A型インフルエンザウィルス核タンパク質抗体が、A型インフルエンザウィルス核タンパク質と特異的に反応し、B型インフルエンザウィルスの核タンパク質とは反応しないヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体である、請求項5記載のデバイス。
- ヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体の重鎖可変領域のアミノ酸配列が、配列番号:8~13のいずれかで表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:22~27のいずれかで表されるアミノ酸配列を含む請求項6記載のデバイス。
- ヒト抗インフルエンザウィルス核タンパク質抗体が、ヒト抗B型インフルエンザウィルス核タンパク質抗体である、請求項1~4のいずれかに記載のデバイス。
- ヒト抗B型インフルエンザウィルス核タンパク質抗体が、B型インフルエンザウィルス核タンパク質と特異的に反応し、A型インフルエンザウィルスの核タンパク質とは反応しないヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体である請求項8記載のデバイス。
- ヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体の重鎖可変領域のアミノ酸配列が、配列番号:14で表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:28で表されるアミノ酸配列を含む請求項9記載のデバイス。
- ヒト抗インフルエンザウィルス核タンパク質抗体が担体に固定化されてなる固相を含有することを特徴とする、インフルエンザウィルス検出又は定量用キット。
- 抗インフルエンザウィルス核タンパク質抗体(抗体1)が担体に固定化されてなる固相と、抗インフルエンザウィルス核タンパク質抗体(抗体2)を含有する試薬とを含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルス検出又は定量用キット。
- 抗インフルエンザウィルス核タンパク質抗体(抗体1)が担体に固定化されてなる固相と、抗インフルエンザウィルス核タンパク質抗体(抗体2)に標識が結合した標識化抗体を含有する試薬とを含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルス検出又は定量用キット。
- ヒト抗インフルエンザウィルス核タンパク質抗体が担体に固定化されてなる固相と、インフルエンザウィルス核タンパク質抗原類似物に標識が結合した標識化抗原類似物を含有する試薬とを含有することを特徴とする、インフルエンザウィルス検出又は定量用キット。
- インフルエンザウィルス核タンパク質抗原類似物が担体に固定化されてなる固相と、ヒト抗インフルエンザウィルス核タンパク質抗体に標識が結合した標識化抗体を含有する試薬とを含有することを特徴とする、インフルエンザウィルス検出又は定量用キット。
- ヒト抗インフルエンザウィルス核タンパク質抗体が、ヒト抗A型インフルエンザウィルス核タンパク質抗体である、請求項11~15のいずれかに記載のキット。
- ヒト抗A型インフルエンザウィルス核タンパク質抗体が、A型インフルエンザウィルス核タンパク質と特異的に反応し、B型インフルエンザウィルスの核タンパク質とは反応しないヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体である、請求項16記載のキット。
- ヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体の重鎖可変領域のアミノ酸配列が、配列番号:8~13のいずれかで表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:22~27のいずれかで表されるアミノ酸配列を含む請求項17記載のキット。
- ヒト抗インフルエンザウィルス核タンパク質抗体が、ヒト抗B型インフルエンザウィルス核タンパク質抗体である、請求項11~15のいずれかに記載のキット。
- ヒト抗B型インフルエンザウィルス核タンパク質抗体が、B型インフルエンザウィルス核タンパク質と特異的に反応し、A型インフルエンザウィルスの核タンパク質とは反応しないヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体である請求項19記載のキット。
- ヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体の重鎖可変領域のアミノ酸配列が、配列番号:14で表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:28で表されるアミノ酸配列を含む請求項20記載のキット。
- (1)検体と、担体上に固定化されているヒト抗インフルエンザウィルス核タンパク質抗体とを反応させる工程;及び、
(2)工程(1)で生じた物理的変化を測定する工程
を含有することを特徴とする、インフルエンザウィルスの検出又は定量方法。 - (1)検体と、担体上に固定化されている抗インフルエンザウィルス核タンパク質抗体(抗体1)とを反応させて、担体上に抗体1-インフルエンザウィルス核タンパク質の複合体を形成させる工程;
(2)工程(1)において、担体上に形成した該複合体と、抗インフルエンザウィルス核タンパク質抗体(抗体2)とを反応させる工程;及び、
(3)工程(2)で生じた物理的変化を測定する工程
を含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルスの検出又は定量方法。 - 物理的変化が、濁度変化又は質量変化である請求項22又は23記載の方法。
- (1)検体と、担体上に固定化されている抗インフルエンザウィルス核タンパク質抗体(抗体1)とを反応させて、担体上に抗体1-インフルエンザウィルス核タンパク質の複合体を形成させる工程;
(2)工程(1)において、担体上に形成した該複合体と、抗インフルエンザウィルス核タンパク質抗体(抗体2)に標識が結合した標識化抗体(標識化抗体2)とを反応させて、担体上に抗体1-インフルエンザウィルス核タンパク質-標識化抗体2の複合体を形成させる工程;
(3)工程(2)後の担体を洗浄し、担体上に結合していない物質を除去する工程;
(4)工程(3)後の担体上の抗体1-インフルエンザウィルス核タンパク質-標識化抗体2の複合体の中の標識を測定する工程
を含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルスの検出又は定量方法。 - (1)検体と、抗インフルエンザウィルス核タンパク質抗体(抗体2)に標識が結合した標識化抗体(標識化抗体2)とを反応させて、標識化抗体2-インフルエンザウィルス核タンパク質の複合体を形成させる工程;
(2)工程(1)において形成した複合体と、担体上に固定化されている抗インフルエンザウィルス核タンパク質抗体(抗体1)とを反応させて、担体上に抗体1-インフルエンザウィルス核タンパク質-標識化抗体2の複合体を形成させる工程;
(3)工程(2)後の担体を洗浄し、担体上に結合していない物質を除去する工程;
(4)工程(3)後の担体上の抗体1-インフルエンザウィルス核タンパク質-標識化抗体2の複合体の中の標識を測定する工程
を含有し、抗体1と抗体2の少なくとも1つは、ヒト抗インフルエンザウィルス核タンパク質抗体であることを特徴とする、インフルエンザウィルスの検出又は定量方法。 - (1)検体と、担体上に固定化されているヒト抗インフルエンザウィルス核タンパク質抗体とを、インフルエンザウィルス核タンパク質抗原類似物に標識が結合した標識化抗原類似物の共存下に反応させて、担体上に、ヒト抗インフルエンザウィルス核タンパク質抗体-インフルエンザウィルス核タンパク質の複合体、及び、ヒト抗インフルエンザウィルス核タンパク質抗体-標識化抗原類似物の複合体を形成させる工程;
(2)工程(1)後の担体を洗浄し、担体上に結合していない物質を除去する工程;
(3)工程(2)後の担体上の複合体中の標識を測定する工程
を含有することを特徴とする、インフルエンザウィルスの検出又は定量方法。 - (1)検体と、担体上に固定化されているインフルエンザウィルス核タンパク質抗原類似物とを、ヒト抗インフルエンザウィルス核タンパク質抗体に標識が結合した標識化抗体の共存下に反応させて、担体上に、インフルエンザウィルス核タンパク質抗原類似物-標識化抗体の複合体を形成させる工程;
(2)工程(1)後の担体を洗浄し、担体上に結合していない物質を除去する工程;
(3)工程(2)後の担体上の複合体中の標識を測定する工程
を含有することを特徴とする、インフルエンザウィルスの検出又は定量方法。 - ヒト抗インフルエンザウィルス核タンパク質抗体が、ヒト抗A型インフルエンザウィルス核タンパク質抗体である請求項22~28のいずれかに記載の方法。
- ヒト抗A型インフルエンザウィルス核タンパク質抗体が、A型インフルエンザウィルス核タンパク質と特異的に反応し、B型インフルエンザウィルスの核タンパク質とは反応しないヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体である請求項29記載の方法。
- ヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体の重鎖可変領域のアミノ酸配列が、配列番号:8~13のいずれかで表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:22~27のいずれかで表されるアミノ酸配列を含む請求項30記載の方法。
- ヒト抗インフルエンザウィルス核タンパク質抗体が、ヒト抗B型インフルエンザウィルス核タンパク質抗体である請求項22~28のいずれかに記載の方法。
- ヒト抗B型インフルエンザウィルス核タンパク質抗体が、B型インフルエンザウィルス核タンパク質と特異的に反応し、A型インフルエンザウィルスの核タンパク質とは反応しないヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体である請求項32記載の方法。
- ヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体の重鎖可変領域のアミノ酸配列が、配列番号:14で表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:28で表されるアミノ酸配列を含む請求項33記載の方法。
- 重鎖可変領域のアミノ酸配列が、配列番号:8~13のいずれかで表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:22~27のいずれかで表されるアミノ酸配列を含む、A型インフルエンザウィルス核タンパク質と特異的に反応し、B型インフルエンザウィルスの核タンパク質と反応しないヒト抗A型インフルエンザウィルス核タンパク質モノクローナル抗体。
- B型インフルエンザウィルス核タンパク質と特異的に反応し、A型インフルエンザウィルスの核タンパク質と反応しないヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体。
- 重鎖可変領域のアミノ酸配列が、配列番号:14で表されるアミノ酸配列を含み、軽鎖可変領域のアミノ酸配列が、配列番号:28で表されるアミノ酸配列を含む、B型インフルエンザウィルス核タンパク質と特異的に反応し、A型インフルエンザウィルスの核タンパク質と反応しないヒト抗B型インフルエンザウィルス核タンパク質モノクローナル抗体。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP09758417A EP2309264A4 (en) | 2008-06-06 | 2009-06-05 | FLU VIRUS DETECTION DEVICE |
US12/996,340 US20110129816A1 (en) | 2008-06-06 | 2009-06-05 | Device for detection of influenza virus |
JP2010515934A JPWO2009148150A1 (ja) | 2008-06-06 | 2009-06-05 | インフルエンザウィルス検出用デバイス |
CN2009801304891A CN102112878A (zh) | 2008-06-06 | 2009-06-05 | 用于检测流感病毒的装置 |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2008-149300 | 2008-06-06 | ||
JP2008149300 | 2008-06-06 | ||
JP2009050066 | 2009-03-04 | ||
JP2009-050066 | 2009-03-04 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2009148150A1 true WO2009148150A1 (ja) | 2009-12-10 |
Family
ID=41398223
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2009/060328 WO2009148150A1 (ja) | 2008-06-06 | 2009-06-05 | インフルエンザウィルス検出用デバイス |
Country Status (5)
Country | Link |
---|---|
US (1) | US20110129816A1 (ja) |
EP (1) | EP2309264A4 (ja) |
JP (1) | JPWO2009148150A1 (ja) |
CN (1) | CN102112878A (ja) |
WO (1) | WO2009148150A1 (ja) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012018057A1 (ja) * | 2010-08-03 | 2012-02-09 | 田中貴金属工業株式会社 | イムノクロマトグラフィー用試薬組成物およびそれを用いた測定方法 |
WO2013145767A1 (ja) | 2012-03-30 | 2013-10-03 | 田中貴金属工業株式会社 | インフルエンザa型ウイルスの検出キット |
WO2014010184A1 (ja) * | 2012-07-09 | 2014-01-16 | 富士フイルム株式会社 | 呈色測定装置および方法 |
JP5469783B1 (ja) * | 2013-09-26 | 2014-04-16 | 株式会社 富山研究所 | 薬剤耐性インフルエンザウイルス検出キット |
JP2018127443A (ja) * | 2017-02-09 | 2018-08-16 | 国立大学法人山形大学 | ヒト抗fxiii(f13、凝固第xiii因子)−aモノクローナル抗体 |
CN112334481A (zh) * | 2018-06-11 | 2021-02-05 | 葛兰素史克消费保健(美国)控股有限责任公司 | 用于快速乙型流感诊断测试的抗体对 |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DK2872891T3 (da) * | 2012-07-10 | 2019-05-13 | Nepean Blue Mountains Local Health Distr | Risikostratificering ved influenza |
CN104297215A (zh) * | 2014-11-14 | 2015-01-21 | 山东出入境检验检疫局检验检疫技术中心 | 表面离子共振技术检测禽法氏囊病毒的非诊断性方法 |
CN104316691A (zh) * | 2014-11-14 | 2015-01-28 | 山东出入境检验检疫局检验检疫技术中心 | 表面离子共振技术检测新城疫病毒的非诊断性方法 |
KR101821956B1 (ko) | 2016-07-28 | 2018-01-26 | 원광대학교산학협력단 | 인플루엔자 a 바이러스의 뉴클레오단백질에 특이적인 단클론항체 및 이를 이용한 신속 형광면역 진단키트 |
CN106814189B (zh) * | 2016-12-20 | 2018-10-26 | 成都金思唯生物技术有限公司 | 一种检测艰难梭菌的试剂盒及其用途 |
CN107192823B (zh) * | 2017-06-08 | 2024-03-19 | 杭州遂真生物技术有限公司 | 一种b族链球菌酶联免疫检测试剂盒 |
US11525830B2 (en) * | 2017-10-13 | 2022-12-13 | Ji Hoon Lee | Biosensor for detecting influenza A virus using Au—FE3O4 composite |
US20210341475A1 (en) * | 2018-06-11 | 2021-11-04 | Glaxosmithkline Consumer Healthcare Holdings (Us) Llc | Antibody pairs for use in a rapid influenza a diagnostic test |
JPWO2020196298A1 (ja) * | 2019-03-25 | 2020-10-01 | ||
EP3951393A4 (en) * | 2019-03-25 | 2022-12-28 | Sekisui Medical Co., Ltd. | IMMUNOLOGICAL TESTING METHOD FOR RESPIRATORY TRACT INFECTION VIRUS AND DETECTION KIT |
CN114252621A (zh) * | 2020-09-23 | 2022-03-29 | 中国科学院大连化学物理研究所 | 一种基于rbd与ace2相互作用实时检测新型冠状病毒刺突蛋白的方法 |
WO2023224618A1 (en) * | 2022-05-18 | 2023-11-23 | Academia Sinica | Recombinant antibodies, kits comprising the same, and uses thereof in diagnosing influenza virus |
Citations (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH02257891A (ja) | 1989-03-31 | 1990-10-18 | Kyowa Hakko Kogyo Co Ltd | 組換え動物細胞による蛋白質の製造 |
JPH0388698A (ja) | 1989-08-30 | 1991-04-15 | Sakagami Masao | 長尺可撓体の巻上装置 |
JP2000055918A (ja) | 1998-08-03 | 2000-02-25 | Otsuka Pharmaceut Co Ltd | 抗体アッセイ装置 |
WO2001062907A1 (en) | 2000-02-22 | 2001-08-30 | Medical & Biological Laboratories Co., Ltd. | Antibody library |
JP2003530543A (ja) * | 1999-12-06 | 2003-10-14 | バイオサイト インコーポレイテッド | 検出試薬としてのヒト抗体 |
JP2004173681A (ja) | 2002-11-14 | 2004-06-24 | Atsushi Muraguchi | 抗原特異的リンパ球検出用マイクロウェルアレイチップ、抗原特異的リンパ球の検出法及び製造方法 |
JP2004187676A (ja) | 2002-11-29 | 2004-07-08 | Atsushi Muraguchi | 抗原特異的リンパ球抗原受容体遺伝子のクローニング方法 |
JP2004279158A (ja) | 2003-03-14 | 2004-10-07 | Denka Seiken Co Ltd | フロースルー型リガンド検出装置及びリガンド検出方法 |
JP2004279208A (ja) | 2003-03-14 | 2004-10-07 | Denka Seiken Co Ltd | フロースルー型リガンド検出装置及びその製造方法 |
WO2005007697A1 (ja) | 2003-07-23 | 2005-01-27 | Fujirebio Inc. | 抗インフルエンザa型ウイルスモノクローナル抗体及び該抗体を用いる免疫測定器具 |
WO2005007698A1 (ja) | 2003-07-23 | 2005-01-27 | Fujirebio Inc. | 抗インフルエンザb型ウイルスモノクローナル抗体及び該抗体を用いる免疫測定器具 |
WO2005015217A1 (ja) | 2003-08-11 | 2005-02-17 | Kyowa Medex Co., Ltd. | 測定対象物測定器具、測定装置および測定方法 |
JP2005261339A (ja) | 2004-03-19 | 2005-09-29 | Atsushi Muraguchi | 生物試料の取得方法 |
JP2006067979A (ja) | 2004-09-06 | 2006-03-16 | Bl:Kk | インフルエンザa型ウイルスの免疫検出法 |
JP2006153523A (ja) | 2004-11-25 | 2006-06-15 | Mitsubishi Kagaku Iatron Inc | 非浸襲性検体を用いたイムノクロマトグラフ法 |
JP2006189317A (ja) | 2005-01-06 | 2006-07-20 | Sysmex Corp | イムノクロマトグラフ法用試験具 |
JP2006194688A (ja) | 2005-01-12 | 2006-07-27 | Sysmex Corp | イムノクロマトグラフィー用キット |
JP2006194687A (ja) | 2005-01-12 | 2006-07-27 | Sysmex Corp | イムノクロマトグラフィー用試験具 |
JP2007014267A (ja) | 2005-07-07 | 2007-01-25 | Toyama Univ | B型肝炎ウイルスのHBs抗原に対するモノクローナル抗体、それに関連する遺伝子およびペプチド、並びにB型肝炎ウイルスの検定方法、B型肝炎の診断方法、治療方法 |
JP2007033293A (ja) | 2005-07-28 | 2007-02-08 | Mizuho Medy Co Ltd | 検出装置 |
JP2007093292A (ja) | 2005-09-27 | 2007-04-12 | Sysmex Corp | イムノクロマトグラフィー用キット |
WO2007055226A1 (ja) * | 2005-11-10 | 2007-05-18 | National University Corporation University Of Toyama | 抗原特異的リンパ球の検出方法および調製方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CH652145A5 (de) * | 1982-01-22 | 1985-10-31 | Sandoz Ag | Verfahren zur in vitro-herstellung von hybridomen welche humane monoklonale antikoerper erzeugen. |
AU2006329276B2 (en) * | 2005-12-26 | 2012-09-27 | Bl Co., Ltd. | Method for detection of virulent strain of influenza type-A virus |
-
2009
- 2009-06-05 WO PCT/JP2009/060328 patent/WO2009148150A1/ja active Application Filing
- 2009-06-05 JP JP2010515934A patent/JPWO2009148150A1/ja active Pending
- 2009-06-05 EP EP09758417A patent/EP2309264A4/en not_active Withdrawn
- 2009-06-05 CN CN2009801304891A patent/CN102112878A/zh active Pending
- 2009-06-05 US US12/996,340 patent/US20110129816A1/en not_active Abandoned
Patent Citations (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH02257891A (ja) | 1989-03-31 | 1990-10-18 | Kyowa Hakko Kogyo Co Ltd | 組換え動物細胞による蛋白質の製造 |
JPH0388698A (ja) | 1989-08-30 | 1991-04-15 | Sakagami Masao | 長尺可撓体の巻上装置 |
JP2000055918A (ja) | 1998-08-03 | 2000-02-25 | Otsuka Pharmaceut Co Ltd | 抗体アッセイ装置 |
JP2003530543A (ja) * | 1999-12-06 | 2003-10-14 | バイオサイト インコーポレイテッド | 検出試薬としてのヒト抗体 |
WO2001062907A1 (en) | 2000-02-22 | 2001-08-30 | Medical & Biological Laboratories Co., Ltd. | Antibody library |
JP2004173681A (ja) | 2002-11-14 | 2004-06-24 | Atsushi Muraguchi | 抗原特異的リンパ球検出用マイクロウェルアレイチップ、抗原特異的リンパ球の検出法及び製造方法 |
JP2004187676A (ja) | 2002-11-29 | 2004-07-08 | Atsushi Muraguchi | 抗原特異的リンパ球抗原受容体遺伝子のクローニング方法 |
JP2004279158A (ja) | 2003-03-14 | 2004-10-07 | Denka Seiken Co Ltd | フロースルー型リガンド検出装置及びリガンド検出方法 |
JP2004279208A (ja) | 2003-03-14 | 2004-10-07 | Denka Seiken Co Ltd | フロースルー型リガンド検出装置及びその製造方法 |
WO2005007698A1 (ja) | 2003-07-23 | 2005-01-27 | Fujirebio Inc. | 抗インフルエンザb型ウイルスモノクローナル抗体及び該抗体を用いる免疫測定器具 |
WO2005007697A1 (ja) | 2003-07-23 | 2005-01-27 | Fujirebio Inc. | 抗インフルエンザa型ウイルスモノクローナル抗体及び該抗体を用いる免疫測定器具 |
WO2005015217A1 (ja) | 2003-08-11 | 2005-02-17 | Kyowa Medex Co., Ltd. | 測定対象物測定器具、測定装置および測定方法 |
JP2005261339A (ja) | 2004-03-19 | 2005-09-29 | Atsushi Muraguchi | 生物試料の取得方法 |
JP2006067979A (ja) | 2004-09-06 | 2006-03-16 | Bl:Kk | インフルエンザa型ウイルスの免疫検出法 |
JP2006153523A (ja) | 2004-11-25 | 2006-06-15 | Mitsubishi Kagaku Iatron Inc | 非浸襲性検体を用いたイムノクロマトグラフ法 |
JP2006189317A (ja) | 2005-01-06 | 2006-07-20 | Sysmex Corp | イムノクロマトグラフ法用試験具 |
JP2006194688A (ja) | 2005-01-12 | 2006-07-27 | Sysmex Corp | イムノクロマトグラフィー用キット |
JP2006194687A (ja) | 2005-01-12 | 2006-07-27 | Sysmex Corp | イムノクロマトグラフィー用試験具 |
JP2007014267A (ja) | 2005-07-07 | 2007-01-25 | Toyama Univ | B型肝炎ウイルスのHBs抗原に対するモノクローナル抗体、それに関連する遺伝子およびペプチド、並びにB型肝炎ウイルスの検定方法、B型肝炎の診断方法、治療方法 |
JP2007033293A (ja) | 2005-07-28 | 2007-02-08 | Mizuho Medy Co Ltd | 検出装置 |
JP2007093292A (ja) | 2005-09-27 | 2007-04-12 | Sysmex Corp | イムノクロマトグラフィー用キット |
WO2007055226A1 (ja) * | 2005-11-10 | 2007-05-18 | National University Corporation University Of Toyama | 抗原特異的リンパ球の検出方法および調製方法 |
Non-Patent Citations (20)
Title |
---|
ANAL. CHEM., vol. 77, no. 24, 2005, pages 8050 - 8056 |
BIOCHEM. BIOPHYS. RES. COMUN., vol. 149, 1987, pages 960 |
CELL, vol. 33, 1983, pages 717 |
CELL, vol. 41, 1985, pages 479 |
CYTOMETRY A, vol. 71, no. 11, 2007, pages 961 - 967 |
CYTOMETRY A, vol. 71, no. 12, 2007, pages 1003 - 1010 |
CYTOTECHNOLOGY, vol. 3, 1990, pages 133 |
CYTOTECHNOLOGY, vol. 4, 1990, pages 173 |
GENE, vol. 27, 1984, pages 223 |
IN VITRO CELL DEV BIOL., vol. 21, no. 10, 1985, pages 593 - 596 |
J. BIOCHEM, vol. 101, 1987, pages 1307 |
J. GEN. VIROL., vol. 64, 1983, pages 697 - 700 |
JOURNAL OF GENERAL VIROLOGY, vol. 64, 1983, pages 697 - 700 |
NATURE, vol. 227, 1970, pages 680 |
NATURE, vol. 453, 2008, pages 667 - 672 |
PROC. NATL. ACAD. SCI., vol. 77, 1980, pages 4216 |
PROC. NATL., ACAD. SCI., vol. 78, 1981, pages 1527 |
SAMBROOK J: "Russell DW in Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS |
See also references of EP2309264A4 |
TANPAKUSHITSU KAKUSAN KOSO, PROTEIN, NUCLEIC ACID AND ENZYME, vol. 31, 1985, pages 37 - 45 |
Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012018057A1 (ja) * | 2010-08-03 | 2012-02-09 | 田中貴金属工業株式会社 | イムノクロマトグラフィー用試薬組成物およびそれを用いた測定方法 |
JP2012037253A (ja) * | 2010-08-03 | 2012-02-23 | Tanaka Kikinzoku Kogyo Kk | イムノクロマトグラフィー用試薬組成物およびそれを用いた測定方法 |
CN103052884A (zh) * | 2010-08-03 | 2013-04-17 | 田中贵金属工业株式会社 | 免疫层析用试剂组合物以及使用该组合物的测定方法 |
EP2602619A1 (en) * | 2010-08-03 | 2013-06-12 | Tanaka Kikinzoku Kogyo K.K. | Immunochromatography reagent composition, and measurement method using same |
US9952207B2 (en) | 2010-08-03 | 2018-04-24 | Tanaka Kikinzoku Kogyo K.K. | Immunochromatography reagent comprising a nonionic surfactant, bicine, and casein, and measurement method using the same |
EP2602619A4 (en) * | 2010-08-03 | 2013-12-25 | Tanaka Precious Metal Ind | IMMUNOCHROMATOGRAPHIC REAGENT COMPOSITION AND MEASURING METHOD USING THE SAME |
CN103052884B (zh) * | 2010-08-03 | 2015-07-15 | 田中贵金属工业株式会社 | 免疫层析用试剂组合物以及使用该组合物的测定方法 |
KR20140145142A (ko) | 2012-03-30 | 2014-12-22 | 다나카 기킨조쿠 고교 가부시키가이샤 | 인플루엔자 a형 바이러스의 검출 키트 |
JPWO2013145767A1 (ja) * | 2012-03-30 | 2015-12-10 | 田中貴金属工業株式会社 | インフルエンザa型ウイルスの検出キット |
WO2013145767A1 (ja) | 2012-03-30 | 2013-10-03 | 田中貴金属工業株式会社 | インフルエンザa型ウイルスの検出キット |
WO2014010184A1 (ja) * | 2012-07-09 | 2014-01-16 | 富士フイルム株式会社 | 呈色測定装置および方法 |
JP5917693B2 (ja) * | 2012-07-09 | 2016-05-18 | 富士フイルム株式会社 | 呈色測定装置および方法 |
JPWO2014010184A1 (ja) * | 2012-07-09 | 2016-06-20 | 富士フイルム株式会社 | 呈色測定装置および方法 |
US9404865B2 (en) | 2012-07-09 | 2016-08-02 | Fujifilm Corporation | Developed-color measurement apparatus and method |
JP5469783B1 (ja) * | 2013-09-26 | 2014-04-16 | 株式会社 富山研究所 | 薬剤耐性インフルエンザウイルス検出キット |
WO2015045053A1 (ja) * | 2013-09-26 | 2015-04-02 | 株式会社 富山研究所 | 薬剤耐性インフルエンザウイルス検出キット |
JP2018127443A (ja) * | 2017-02-09 | 2018-08-16 | 国立大学法人山形大学 | ヒト抗fxiii(f13、凝固第xiii因子)−aモノクローナル抗体 |
CN112334481A (zh) * | 2018-06-11 | 2021-02-05 | 葛兰素史克消费保健(美国)控股有限责任公司 | 用于快速乙型流感诊断测试的抗体对 |
Also Published As
Publication number | Publication date |
---|---|
EP2309264A4 (en) | 2011-11-23 |
US20110129816A1 (en) | 2011-06-02 |
CN102112878A (zh) | 2011-06-29 |
JPWO2009148150A1 (ja) | 2011-11-04 |
EP2309264A1 (en) | 2011-04-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2009148150A1 (ja) | インフルエンザウィルス検出用デバイス | |
JP4595810B2 (ja) | 抗インフルエンザa型ウイルスモノクローナル抗体及び該抗体を用いる免疫測定器具 | |
JP5661572B2 (ja) | インスリン測定方法 | |
KR102099370B1 (ko) | 인플루엔자 a형 바이러스의 검출 키트 | |
EP2063270A1 (en) | Rapid diagnosis method specific to avian influenza virus | |
JP5340575B2 (ja) | イムノクロマトグラフィー用試験具 | |
JP5575377B2 (ja) | 抗rsウイルスモノクローナル抗体を用いたrsウイルス検出用キット及びイムノクロマトグラフィー用試験具、並びに新規な抗rsウイルスモノクローナル抗体 | |
JP6979941B2 (ja) | Peg化分析物特異的結合剤を用いた粒子に基づくイムノアッセイ | |
JP2010122205A (ja) | 麻疹ウイルス検出方法、メンブレンアッセイ用試験具およびメンブレンアッセイ用試験キット | |
JPWO2007074811A1 (ja) | A型インフルエンザウイルス強毒株の検出法 | |
RU2636822C2 (ru) | Способ обнаружения мультиспецифического связывающего агента | |
WO2015170844A1 (ko) | 마그네틱(magnetic)을 이용한 형광다중면역검사 | |
KR102528090B1 (ko) | 트롬빈 안티트롬빈 복합체의 측정 시약 및 측정 방법 | |
JP2024012473A (ja) | Rsウイルスを検出するための検出用試薬又はキット及びrsウイルスを検出する方法 | |
WO2023017817A1 (ja) | 免疫測定方法、検体希釈液、及びイムノクロマトグラフィーキット | |
JPWO2009084369A1 (ja) | Hiv−1抗原の検出用試薬及び検出方法 | |
US20220034885A1 (en) | Compositions and methods for determining coronavirus neutralization titers | |
WO2011093217A1 (ja) | 抗Pandemic (H1N1) 2009抗体及びそれを用いた免疫測定方法 | |
JP2021505906A (ja) | HBsAgの定量検出のためのキット及び方法 | |
JP5630153B2 (ja) | A型インフルエンザウイルス核タンパク質の第100番アミノ酸がアルギニンであるか否かを判定する方法 | |
CN117715931A (zh) | 针对SARS-CoV-2的核衣壳蛋白的抗体及其用途 | |
JP7273195B2 (ja) | 干渉抑制薬物動態イムノアッセイ | |
JP2023536737A (ja) | Sars-cov-2を検出するためのアッセイ | |
JP2012046443A (ja) | 抗np−h289抗体及びそれを用いた免疫測定方法 | |
JP2024003083A (ja) | Rsウイルスを検出するための検出用試薬又はキット及びrsウイルスを検出する方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 200980130489.1 Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 09758417 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2010515934 Country of ref document: JP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2009758417 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 12996340 Country of ref document: US |