JP3932320B2 - Detection method of larvae adhering to oyster - Google Patents
Detection method of larvae adhering to oyster Download PDFInfo
- Publication number
- JP3932320B2 JP3932320B2 JP2005070009A JP2005070009A JP3932320B2 JP 3932320 B2 JP3932320 B2 JP 3932320B2 JP 2005070009 A JP2005070009 A JP 2005070009A JP 2005070009 A JP2005070009 A JP 2005070009A JP 3932320 B2 JP3932320 B2 JP 3932320B2
- Authority
- JP
- Japan
- Prior art keywords
- larvae
- react
- oyster
- mussel
- monoclonal antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000237502 Ostreidae Species 0.000 title claims description 100
- 235000020636 oyster Nutrition 0.000 title claims description 100
- 238000001514 detection method Methods 0.000 title claims description 30
- 241000237536 Mytilus edulis Species 0.000 claims description 100
- 235000020638 mussel Nutrition 0.000 claims description 98
- 210000004408 hybridoma Anatomy 0.000 claims description 57
- 235000020639 clam Nutrition 0.000 claims description 47
- 238000000034 method Methods 0.000 claims description 47
- 241000237519 Bivalvia Species 0.000 claims description 41
- 239000012634 fragment Substances 0.000 claims description 41
- 241000237858 Gastropoda Species 0.000 claims description 13
- 238000011084 recovery Methods 0.000 claims description 9
- 210000004027 cell Anatomy 0.000 description 28
- 239000000287 crude extract Substances 0.000 description 24
- 239000000126 substance Substances 0.000 description 22
- 239000002609 medium Substances 0.000 description 16
- 239000000427 antigen Substances 0.000 description 13
- 102000036639 antigens Human genes 0.000 description 13
- 108091007433 antigens Proteins 0.000 description 13
- 239000012091 fetal bovine serum Substances 0.000 description 12
- 238000002965 ELISA Methods 0.000 description 11
- 230000009257 reactivity Effects 0.000 description 11
- 206010035226 Plasma cell myeloma Diseases 0.000 description 8
- 238000002372 labelling Methods 0.000 description 8
- 201000000050 myeloid neoplasm Diseases 0.000 description 8
- 239000012228 culture supernatant Substances 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 6
- KPKZJLCSROULON-QKGLWVMZSA-N Phalloidin Chemical compound N1C(=O)[C@@H]([C@@H](O)C)NC(=O)[C@H](C)NC(=O)[C@H](C[C@@](C)(O)CO)NC(=O)[C@H](C2)NC(=O)[C@H](C)NC(=O)[C@@H]3C[C@H](O)CN3C(=O)[C@@H]1CSC1=C2C2=CC=CC=C2N1 KPKZJLCSROULON-QKGLWVMZSA-N 0.000 description 6
- 239000012980 RPMI-1640 medium Substances 0.000 description 6
- 230000007910 cell fusion Effects 0.000 description 6
- 239000006166 lysate Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 210000000628 antibody-producing cell Anatomy 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 230000001900 immune effect Effects 0.000 description 5
- 238000003127 radioimmunoassay Methods 0.000 description 5
- 239000013535 sea water Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 230000001464 adherent effect Effects 0.000 description 4
- 239000003593 chromogenic compound Substances 0.000 description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 210000004989 spleen cell Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 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 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- 241000238586 Cirripedia Species 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 108010009711 Phalloidine Proteins 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 229910052737 gold Inorganic materials 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 230000016784 immunoglobulin production Effects 0.000 description 3
- 239000004816 latex Substances 0.000 description 3
- 229920000126 latex Polymers 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 241000239250 Copepoda Species 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- 241000238424 Crustacea Species 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 241000237509 Patinopecten sp. Species 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108010004729 Phycoerythrin Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-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
- 239000002671 adjuvant Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- -1 etc.) Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000007667 floating Methods 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 229910001385 heavy metal Inorganic materials 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 235000020637 scallop Nutrition 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 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 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- 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 1
- QCQCHGYLTSGIGX-GHXANHINSA-N 4-[[(3ar,5ar,5br,7ar,9s,11ar,11br,13as)-5a,5b,8,8,11a-pentamethyl-3a-[(5-methylpyridine-3-carbonyl)amino]-2-oxo-1-propan-2-yl-4,5,6,7,7a,9,10,11,11b,12,13,13a-dodecahydro-3h-cyclopenta[a]chrysen-9-yl]oxy]-2,2-dimethyl-4-oxobutanoic acid Chemical compound N([C@@]12CC[C@@]3(C)[C@]4(C)CC[C@H]5C(C)(C)[C@@H](OC(=O)CC(C)(C)C(O)=O)CC[C@]5(C)[C@H]4CC[C@@H]3C1=C(C(C2)=O)C(C)C)C(=O)C1=CN=CC(C)=C1 QCQCHGYLTSGIGX-GHXANHINSA-N 0.000 description 1
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241001247197 Cephalocarida Species 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 108090000270 Ficain Proteins 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 241000711408 Murine respirovirus Species 0.000 description 1
- 241000133262 Nauplius Species 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 206010029719 Nonspecific reaction Diseases 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 108010053210 Phycocyanin Proteins 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 241000270666 Testudines Species 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000001467 acupuncture Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000002543 antimycotic Substances 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- XAHAOEIIQYSRHJ-UHFFFAOYSA-N benzene-1,2-diamine Chemical compound NC1=CC=CC=C1N.NC1=CC=CC=C1N XAHAOEIIQYSRHJ-UHFFFAOYSA-N 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012501 chromatography medium Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 235000019836 ficin Nutrition 0.000 description 1
- POTUGHMKJGOKRI-UHFFFAOYSA-N ficin Chemical compound FI=CI=N POTUGHMKJGOKRI-UHFFFAOYSA-N 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 239000012784 inorganic fiber Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002923 metal particle Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 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 1
- 125000000449 nitro group Chemical class [O-][N+](*)=O 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000004091 panning Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
Description
本発明は、マガキ付着期幼生の検出方法、検出キット、検出器、及び回収方法、並びにそれらに使用するモノクローナル抗体又はそのフラグメント、及び前記モノクローナル抗体を産生するハイブリドーマに関する。 The present invention relates to a detection method, a detection kit, a detector, and a recovery method for larvae adhering stage larvae, a monoclonal antibody or a fragment thereof used therein, and a hybridoma that produces the monoclonal antibody.
マガキの養殖においては、マガキの付着期幼生が発生する場所にホタテ貝の貝殻を吊して、前記貝殻にマガキ付着期幼生を効率よく付着させることが重要だとされている。 In culturing oysters, it is important to suspend scallop shells at places where larvae adherence-stage larvae occur and to attach oyster adhesion stage larvae efficiently to the shells.
マガキ付着期幼生が発生する場所の特定は、ある場所から採取した試料にマガキ付着期幼生が多く存在するか否かを調査することにより行われているが、マガキ付着期幼生は、マガキ以外の二枚貝類の付着期幼生（例えば、ムラサキイガイ、アサリ、バカガイ等の付着期幼生）とその形態が非常に似ているので、専門家であってもマガキ付着期幼生の特定は困難である。そのため、マガキ付着期幼生を特異的に検出する手法や、マガキ付着期幼生を特異的に回収する手法の開発が求められている。 The location of oyster-attached larvae is identified by examining whether there are many oyster-attached larvae in samples collected from a certain location. It is very similar to the bivalve attachment stage larvae (for example, mussels, clams, clams, etc.), and it is difficult for even an expert to identify the oyster attachment stage larvae. Therefore, the development of a method for specifically detecting larvae-attached larvae and a method for specifically recovering oyster-attached larvae is required.
しかしながら、二枚貝類に属するアサリの浮遊幼生を特異的に検出する方法は開発されているものの（特許文献１参照）、マガキ付着期幼生を特異的に検出する方法又は回収する方法は開発されていなかった。
本発明は、上記課題に鑑みてなされたものであり、マガキ付着期幼生を特異的に検出するための検出方法、検出キット、及び検出器、及びマガキ付着期幼生を特異的に回収するための回収方法、並びにそれらに使用するモノクローナル抗体又はそのフラグメント、及び前記モノクローナル抗体を産生するハイブリドーマを提供することを目的とする。 The present invention has been made in view of the above problems, and a detection method, a detection kit and a detector for specifically detecting oyster adhesion stage larvae, and for recovering oyster adhesion stage larvae specifically. It is an object of the present invention to provide a collection method, and a monoclonal antibody or a fragment thereof used therein, and a hybridoma that produces the monoclonal antibody.
本発明者らは、ムラサキイガイの付着期幼生（ペディベリジャー幼生）に特異的なモノクローナル抗体を産生するハイブリドーマを作製しようと試み、ムラサキイガイ付着期幼生を腹腔内に注射することにより免疫したマウスの脾臓細胞と、マウスミエローマ細胞とを融合し、得られたハイブリドーマの培養上清を用いて、ムラサキイガイ付着期幼生、アサリ付着期幼生、バカガイ付着期幼生、マガキ付着期幼生、甲殻類プランクトン（コペポーダ類、アルテミアノープリウス幼生等）、及びフジツボ類の付着期幼生（イワフジツボ、アカフジツボ等の付着期幼生（キプリス幼生））の粗抽出液（各幼生又はプランクトンを超音波装置、ホモジナイザー等で処理した溶解物）との反応性を調べていたところ、ムラサキイガイ付着期幼生には反応するが、マガキ付着期幼生には反応しないモノクローナル抗体を産生するハイブリドーマ以外に、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体を産生するハイブリドーマが存在することを見出した。このことから、ムラサキイガイ付着期幼生には反応するが、マガキ付着期幼生には反応しないモノクローナル抗体と、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体を利用することにより、マガキ付着期幼生を特異的に検出又は回収することができる方法を開発し、本発明を完成するに至った。 The inventors of the present invention attempted to produce a hybridoma that produces a monoclonal antibody specific for the mussel adhesion stage larvae (pediberger larvae), and spleen cells of mice immunized by intraperitoneally injecting the mussel adhesion stage larvae Using the hybridoma culture supernatant obtained by fusing the mouse myeloma cells, the mussel adherent stage larvae, clam adherence stage larvae, snail adherence stage larvae, oyster adherence stage larvae, crustacean plankton (Copepoda, Artemia) Nauplius larvae, etc.), and a crude extract of adhering larvae such as barnacles (Iwafuji acupuncture, red barnacles, etc.) (a lysate or a lysate obtained by treating each larva or plankton with an ultrasonic device, a homogenizer, etc.) When we examined the reactivity of mussel, However, in addition to hybridomas that produce monoclonal antibodies that do not react with oyster-adherent larvae, they produce monoclonal antibodies that react specifically with oyster and mussel-adhered larvae but not with clams and snails. It was found that a hybridoma exists. This indicates that it reacts specifically with the adhesion larvae of oysters and mussels, but reacts with the monoclonal antibody that does not react with oysters, but reacts with the adhesion larvae of oysters and mussels. By utilizing a monoclonal antibody that does not react, a method capable of specifically detecting or recovering larvae adhering stage larvae was developed, and the present invention was completed.
すなわち、本発明に係るマガキ付着期幼生の検出方法は、試料に対し、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体と、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントと、を作用させる工程を含む。 That is, the method for detecting oyster adhesion stage larvae according to the present invention specifically reacts with an antibody that reacts with mussel adhesion stage larvae but does not react with oyster adhesion stage larvae, and magaki and mussel adhesion stage larvae. It includes a step of reacting with a monoclonal antibody or a fragment thereof that reacts but does not react with the clams and clam larvae.
また、本発明に係るマガキ付着期幼生の検出キットは、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体と、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントと、を含む。 Further, the detection kit for oyster adhesion stage larvae according to the present invention reacts specifically with mussel adhesion stage larvae, but does not react with oyster adhesion stage larvae, and specifically reacts with oyster and mussel adhesion stage larvae, And a monoclonal antibody or a fragment thereof which does not react with clams and clams in the larvae.
さらに、本発明に係るマガキ付着期幼生の検出器は、前記検出器は、イムノクロマトグラフ媒体を含み、前記イムノクロマトグラフ媒体は、試料を滴下する又は浸す第一の部分と、ムラサキイガイ付着期幼生には反応するが、マガキ付着期幼生には反応しない抗体が固定化されている第二の部分と、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントが固定化されている第三の部分と、を含み、前記第二の部分は、前記第一の部分と前記第三の部分との間に備えられている。 Furthermore, the detector of a oyster adhering period larva according to the present invention, the detector includes an immunochromatographic medium, wherein the immunochromatographic medium includes a first portion in which a sample is dropped or immersed, and a mussel adhering period larva. Reacts, but reacts specifically with the second part of the immobilized antibody, which does not react with oyster-adhered larvae, and adhering-stage larvae of oysters and mussels A third portion to which a monoclonal antibody or fragment thereof is immobilized, wherein the second portion is provided between the first portion and the third portion.
本発明に係るマガキ付着期幼生の回収方法は、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体と、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントとを用いて、試料中のマガキ付着期幼生を回収する工程を含む。 The method for recovering oyster-adherent larvae according to the present invention reacts specifically with oyster-adherent larvae but reacts with antibodies that do not react with oyster-adherent larvae and adhering-stage larvae of oysters and mussels. A step of recovering oyster-adherent larvae in a sample using a monoclonal antibody or a fragment thereof that does not react with the larvae-adherent larvae.
また、本発明に係るハイブリドーマは、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体を産生することを特徴とする。 The hybridoma according to the present invention is characterized in that it produces a monoclonal antibody that specifically reacts with the larvae of the oysters and mussels but does not react with the larvae of the clams and the clams.
さらに、本発明に係るモノクローナル抗体又はそのフラグメントは、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないことを特徴とする。 Furthermore, the monoclonal antibody according to the present invention or a fragment thereof reacts specifically with the adhesion stage larvae of oysters and mussels, but does not react with the adhesion stage larvae of clams and snails.
ここで、前記マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体としては、例えば、独立行政法人産業技術総合研究所 特許生物寄託センターにおいて受託番号FERM P-20217で寄託されているハイブリドーマ細胞株から産生されるモノクローナル抗体を挙げることができる。また、前記ムラサキイガイ付着期幼生に反応するが、マガキの付着期幼生には反応しない抗体は、例えば、ポリクローナル抗体、モノクローナル抗体又はそのフラグメントなどである。前記ムラサキイガイ付着期幼生に反応するが、マガキの付着期幼生には反応しないモノクローナル抗体としては、例えば、独立行政法人産業技術総合研究所 特許生物寄託センターにおいて受託番号FERM P-20216、FERM P-20218、FERM P-20219、FERM P-20220等で寄託されているハイブリドーマ細胞株から産生されるモノクローナル抗体を挙げることができる。 Here, as a monoclonal antibody that reacts specifically with the adhesion larvae of the oysters and mussels, but does not react with the larvae of the clams and snails, for example, in the National Institute of Advanced Industrial Science and Technology patent biological deposit center Mention may be made of the monoclonal antibodies produced from the hybridoma cell line deposited under accession number FERM P-20217. The antibody that reacts with the mussel adhesion stage larvae but does not react with the oyster adhesion stage larvae is, for example, a polyclonal antibody, a monoclonal antibody, or a fragment thereof. Examples of the monoclonal antibody that reacts with the mussel adhesion stage larvae but does not react with the oyster adhesion stage larvae include, for example, Accession Nos. FERM P-20216, FERM P-20218 at the National Institute of Advanced Industrial Science and Technology (AIST). And monoclonal antibodies produced from hybridoma cell lines deposited with FERM P-20219, FERM P-20220, and the like.
本発明によれば、マガキ付着期幼生を特異的に検出するための検出方法、検出キット、及び検出器、及びマガキ付着期幼生を特異的に回収するための回収方法、並びにそれらに使用するモノクローナル抗体又はそのフラグメント、及び前記モノクローナル抗体を産生するハイブリドーマを提供することができる。 According to the present invention, a detection method, a detection kit, and a detector for specifically detecting a oyster adhering stage larva, a recovery method for specifically recovering a oyster adhering stage larva, and a monoclonal used for them Antibodies or fragments thereof, and hybridomas producing the monoclonal antibodies can be provided.
上記知見に基づき完成した本発明を実施するための形態を、実施例を挙げながら詳細に説明する。実施の形態及び実施例に特に説明がない場合には、J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J.G. Seidman, J. A. Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd.などの標準的なプロトコール集に記載の方法、あるいはそれを修飾したり、改変した方法を用いる。また、市販の試薬キットや測定装置を用いている場合には、特に説明が無い場合、それらに添付のプロトコールを用いる。 The form for implementing this invention completed based on the said knowledge is demonstrated in detail, giving an Example. Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.
＝＝マガキ付着期幼生の検出及び回収＝＝
ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体は、少なくともマガキ付着期幼生には結合しないことから、ムラサキイガイ付着期幼生を含む幼生の除去に有用であると考えられる。また、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体は、少なくともアサリやバカガイの付着期幼生には結合しないことから、マガキやムラサキイガイの付着期幼生のみを回収するのに有用であるといえる。従って、これらの抗体やモノクローナル抗体を順次又は同時に用いることにより、形態学的に特定が困難とされていたマガキ付着期幼生と、ムラサキイガイ、アサリ、バカガイ等のマガキ以外の二枚貝類の付着期幼生との分離が可能となり、マガキ付着期幼生の検出及び回収を特異的に行うことができる。
== Detection and recovery of oyster larvae ==
An antibody that reacts with mussel-adhered-stage larvae but does not react with oyster-adhered-stage larvae does not bind at least to oyster-adhered-stage larvae, and is considered useful for removing larvae including mussel-adhered-stage larvae. In addition, monoclonal antibodies that react specifically with oysters and mussels larvae, but do not react with clams and snails larvae do not bind at least to clams and mussels larvae. It can be said that it is useful for recovering only the attached larvae. Therefore, by using these antibodies and monoclonal antibodies sequentially or simultaneously, larvae adhering stage larvae that were difficult to identify morphologically, and adhering stage larvae of bivalves other than oysters such as mussels, clams, and snails Can be separated, and detection and recovery of larvae-attached larvae can be performed specifically.
＜マガキ付着期幼生の回収方法＞
本発明に係るマガキ付着期幼生の回収方法は、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体と、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントとを用いて、試料（例えば、海水、川水など）中のマガキ付着期幼生を回収する工程を含む。例えば、試料中のムラサキイガイ付着期幼生を、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体を用いて除去し、続いて試料中のマガキ付着期幼生を、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントを用いて回収することができる。また、試料中のマガキ及びムラサキイガイ付着期幼生を、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントを用いて回収し、続いてムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体を用いてムラサキイガイ付着期幼生を除去することによりマガキ付着期幼生を回収することができる。
<Recovery method of larvae adhering stage larva>
The method for recovering oyster-adherent larvae according to the present invention reacts specifically with oyster-adherent larvae but reacts with antibodies that do not react with oyster-adherent larvae and adhering-stage larvae of oysters and mussels. A step of recovering oyster-adherent larvae in a sample (for example, seawater, river water, etc.) using a monoclonal antibody or a fragment thereof that does not react with the larvae-adherent larvae. For example, the mussel-attached larvae in the sample are removed using an antibody that reacts with the mussel-attached larvae but does not react with the oyster-attached larvae, and then the oyster-attached larvae in the sample are removed from the oyster and mussel oysters. It can be recovered by using a monoclonal antibody or a fragment thereof that reacts specifically with the adhesion stage larvae but does not react with the clam or scallop adhesion stage larvae. In addition, the oyster and mussel adhesion stage larvae in the sample are recovered using a monoclonal antibody or fragment thereof that specifically reacts with the adhesion stage larvae of oyster and mussel, but does not react with clams and mussel adhesion stage larvae, Subsequently, the oyster-attached larvae can be recovered by removing the mussel-attached larvae using an antibody that reacts with the mussel-attached larvae but does not react with the oyster-attached larvae.
なお、前記幼生の除去や回収には、幼生と前記抗体又はモノクローナル抗体若しくはそのフラグメントとの親和性を利用して行うことができる。例えば、前記抗体又はモノクローナル抗体若しくはそのフラグメントが結合したゲルを用いたカラムクロマトグラフィー法、前記抗体又はモノクローナル抗体若しくはそのフラグメントが結合したゲルやラテックスを遠心分離等により除去・回収するバッチ法、前記抗体又はモノクローナル抗体若しくはそのフラグメントが結合した磁性体を磁石により除去・回収する方法などを利用することができる。また、前記幼生の除去や回収にpanning法を適用してもよい。その他、蛍光物質が結合した前記抗体と、前記蛍光物質と蛍光色素が異なる蛍光物質が結合した前記モノクローナル抗体又はそのフラグメントとを幼生に結合し、FACS（Fluorescence Activated Cell Sorter）を用いて、マガキ付着期幼生を特異的に回収することもできる。前記蛍光物質としては、FITC（フルオレセインイソチオシアネート）、ローダミン、ファロイジンなどの公知の物質を用いることができる。 The removal and recovery of the larvae can be performed by utilizing the affinity between the larvae and the antibody or monoclonal antibody or fragment thereof. For example, a column chromatography method using a gel to which the antibody or monoclonal antibody or fragment thereof is bound, a batch method for removing and collecting the gel or latex to which the antibody or monoclonal antibody or fragment thereof is bound by centrifugation, the antibody Alternatively, a method of removing and recovering a magnetic substance bound with a monoclonal antibody or a fragment thereof with a magnet can be used. A panning method may be applied to the removal and recovery of the larvae. In addition, the antibody to which the fluorescent substance is bound and the monoclonal antibody or fragment thereof to which the fluorescent substance and the fluorescent dye different from the fluorescent substance are bound are bound to larvae and attached to the oyster using FACS (Fluorescence Activated Cell Sorter) The stage larvae can also be specifically recovered. As the fluorescent substance, known substances such as FITC (fluorescein isothiocyanate), rhodamine, and phalloidin can be used.
以上のように、本発明に係るマガキ付着期幼生の回収方法により、マガキ付着期幼生の回収が可能となり、養殖産業等に大きく貢献することができるようになると考えられる。 As described above, it is considered that the collection method of larvae adhering stage larvae according to the present invention makes it possible to collect larvae adhering period larvae and greatly contribute to the aquaculture industry and the like.
＜マガキ付着期幼生の検出方法＞
本発明に係るマガキ付着期幼生の検出方法は、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体と、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントとを用いることにより行うことができる。例えば、上述のように、試料（例えば、海水、川水などのマガキ付着期幼生が存在すると思われる場所の水）中のムラサキイガイ付着期幼生を、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体を用いて除去し、その後、試料中のマガキ付着期幼生を、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントを用いて免疫学的手法により検出することができる。また、上述のように、試料中のマガキ及びムラサキイガイ付着期幼生を、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントを用いて回収し、その後、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体を用いてムラサキイガイ付着期幼生を免疫学的手法により検出することにより行うこともできる。前記免疫学的手法としては、EIA（Enzyme Immunoassay）、蛍光免疫測定法、ELISA（Enzyme-Linked Immunosorbent Assay）、RIA（Radioimmuno assay）、ラテックス凝集法、イムノクロマト法、サンドイッチ法等の公知の方法を用いることができる。その他、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメント、及びムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体をそれぞれ異なる蛍光でラベルし、上述のようにFACS（Fluorescence Activated Cell Sorter）を用いて検出することもできるし、蛍光顕微鏡で、蛍光色の違いによって検出することもできる。
<Detection method of larvae adhering stage>
The method for detecting the oyster adhesion stage larvae according to the present invention reacts specifically with the oyster adhesion stage larvae but does not react with the oyster adhesion stage larvae, and specifically reacts with the oyster and mussel adhesion stage larvae. It can be carried out by using a monoclonal antibody or a fragment thereof that does not react with the larvae of the snail. For example, as described above, the mussel adhesion stage larvae in a sample (for example, water in a place where magaki adhesion stage larvae such as seawater and river water are present) react with the mussel adhesion stage larvae. Monoclonal antibody that does not react with larvae, and then reacts specifically with oyster-adherent larvae in the sample to adherent larvae of oysters and mussels, but does not react with clam and mussel-adherent larvae It can be detected by immunological techniques using antibodies or fragments thereof. In addition, as described above, the oyster and mussel adhesion stage larvae in the sample specifically react with the oyster and mussel adhesion stage larvae, but do not react with the clam and mussel adhesion stage larvae. It is also possible to detect the mussel adhesion stage larvae by an immunological technique using an antibody that reacts with the mussel adhesion stage larvae but does not react with the oyster adhesion stage larvae. As the immunological technique, known methods such as EIA (Enzyme Immunoassay), fluorescent immunoassay, ELISA (Enzyme-Linked Immunosorbent Assay), RIA (Radioimmuno assay), latex agglutination method, immunochromatography method, sandwich method, etc. are used. be able to. In addition, it reacts specifically with adhesion larvae of oysters and mussels, but does not react with adhesion larvae of clams and mussel, and reacts with larvae of adhesions of oysters, Non-reacting antibodies can be labeled with different fluorescences and detected using FACS (Fluorescence Activated Cell Sorter) as described above, or can be detected with a fluorescent microscope based on the difference in fluorescent color.
なお、上述においては、本発明に係るマガキ付着期幼生の検出方法において、海水又は川水等の試料を用いることとしたが、マガキ付着期幼生が存在すると思われる場所の水を超音波装置、ホモジナイザー等で処理した溶解物を試料として用いることとしてもよい。このように溶解物を試料として用いる場合には、例えば、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体で吸収した後、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントを用いたウェスタンブロット法やELISA法により、試料中にマガキ付着期幼生が存在していたかどうかを調べることができる。 In the above description, in the method for detecting oyster adhesion stage larvae according to the present invention, a sample such as seawater or river water was used. A lysate treated with a homogenizer or the like may be used as a sample. When the lysate is used as a sample in this way, for example, it reacts with the larvae-adherent larvae but absorbs with an antibody that does not react with the oyster-adherent larvae, and then reacts specifically with the adhering larvae of oyster and mussel However, it is possible to examine whether or not oyster-adherent larvae were present in the sample by Western blotting or ELISA using a monoclonal antibody or fragment thereof that does not react with clams and oysters.
以上のように、本発明に係るマガキ付着期幼生の検出は、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体と、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントとを用いることにより行うことができる。従って、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体と、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントとを含むキット又は器具は、マガキ付着期幼生の検出キット及び検出器として有用であるといえる。 As described above, the detection of oyster adhesion stage larvae according to the present invention reacts specifically with mussel adhesion stage larvae, but reacts specifically with the adhesion stage larvae of oysters and mussels. However, it can be carried out by using a monoclonal antibody or a fragment thereof which does not react with clams and clams' larvae. Therefore, an antibody that reacts with larvae that adhere to the mussel, but does not react with larvae that adhere to the oyster, and a monoclonal antibody that reacts specifically with the larvae of oysters and mussels, but does not react with the larvae of clams and mussels Alternatively, a kit or a device containing the fragment thereof can be said to be useful as a detection kit and detector for larvae adhering stage larvae.
本発明に係るマガキ付着期幼生の検出器は、試料を滴下する又は浸す第一の部分と、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体が固定化されている第二の部分と、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントが固定化されている第三の部分とを含み、第二の部分は第一の部分と第三の部分との間に備えられているイムノクロマトグラフ媒体を含むものであればどのようなものでもよく、例えば、緩衝液（例えば、リン酸塩、炭酸塩、塩酸塩等の塩の溶液）、防腐剤（例えば、アジ化ナトリウムなど）、非特異的な反応を抑制するための物質（例えば、ブロックエース、ゲラチン、スキムミルクなど）、免疫学的手法により前記第三の部分に捕捉されたマガキ付着期幼生を検出するのに必要な物質（例えば、標識物質、発色基質、二次抗体、発色増強剤など）、安定剤（例えば、BSA、ヤギ血清など）等の、抗体又はそのフラグメント以外に抗原の検出器に含ませる一般的な物質が１又は２以上、さらに含ませることとしてもよい。前記イムノクロマトグラフ媒体としては、例えば、ガラスやシリカなどの無機繊維からなる濾紙、ニトロセルロースなどの変性セルロース等を用いることができる。 The detector for adhering larvae according to the present invention is a first portion where a sample is dropped or immersed, and an antibody that reacts with the larvae adhering larvae but does not react with the oyster adhering larvae. A second part and a third part immobilized with a monoclonal antibody or fragment thereof that reacts specifically with the larvae of the oyster and mussel, but does not react with the larvae of the clam or snail, The second part may be any one that includes an immunochromatographic medium provided between the first part and the third part, for example, a buffer (eg, phosphate, carbonate, Salt, hydrochloride salt solutions), preservatives (eg, sodium azide), non-specific substances (eg, block ace, gelatin, skim milk) ), A substance (for example, a labeling substance, a chromogenic substrate, a secondary antibody, a chromogenic enhancer, etc.) necessary to detect larvae adhering stage larvae captured by the third part by an immunological technique, a stabilizer ( For example, BSA, goat serum, etc.) may be further included in addition to one or more general substances to be included in the antigen detector in addition to the antibody or fragment thereof. Examples of the immunochromatographic medium include filter paper made of inorganic fibers such as glass and silica, and modified cellulose such as nitrocellulose.
本発明に係るマガキ付着期幼生の検出器の一例としては、上述のようなイムノクロマトグラフ媒体において、さらに、第三の部分のモノクローナル抗体が産生されたホストの動物種の免疫グロブリンに特異的に反応する抗免疫グロブリン抗体等の二次抗体が固定化された第四の部分を有し、第二の部分と第三の部分は第一の部分と第四の部分との間に備えられ、第一の部分には、金属コロイド粒子（例えば、金コロイド粒子など）、重金属（例えば、金、白金など）、蛍光物質（例えば、FITC、ローダミン、ファロイジンなど）、着色ラテックス粒子等の標識物質で標識された、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントを含むものを挙げることができる。このようなクロマトグラフ媒体を用いることにより、試料中に存在する全てのムラサキイガイ付着期幼生、又はその溶解物中のムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体に対する抗原が、第二の部分に補足されてその位置で標識物質により検出が可能となる。また、試料中に存在するマガキ付着期幼生、又はその溶解物中のマガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントに対する抗原は、第三の部分に補足されてその位置で標識物質により検出が可能となる。さらに、第四の部分を備えることにより、マガキやムラサキイガイの付着期幼生には反応しなかった、標識物質で標識されたモノクローナル抗体又はそのフラグメントが、第四の部分に固定化された二次抗体により補足されてその位置で標識物質により検出が可能となり、これをもって検出試験が終了したことの判断をすることが可能になる。なお、上記のイムノクロマト媒体において、第二の部分が濃く標識された場合には、試料中に存在するムラサキイガイ付着期幼生等の一部が、第三の部分に流れて捕捉されている可能性が考えられるので、再度、試料を希釈した後、検出試験を行うことが好ましい。これにより、試料中にマガキ付着期幼生が存在するかどうかを正確に調べることが可能となる。 As an example of the oyster adhesion stage larva detector according to the present invention, the immunochromatographic medium as described above further reacts specifically with the immunoglobulin of the host animal species in which the third part of the monoclonal antibody was produced. A secondary antibody, such as an anti-immunoglobulin antibody, has a fourth part immobilized thereon, the second part and the third part being provided between the first part and the fourth part, One part is labeled with a labeling substance such as colloidal metal particles (for example, colloidal gold particles), heavy metals (for example, gold, platinum), fluorescent materials (for example, FITC, rhodamine, phalloidin), colored latex particles, etc. Containing monoclonal antibodies or fragments thereof that react specifically with the larvae of oysters and mussels, but do not react with the larvae of clams and snails Can be mentioned. By using such a chromatographic medium, all mussel-adherent larvae present in the sample react with the mussel-adherent larvae in the lysate, but the antigen to the antibody that does not react with the oyster-adherent larvae Then, it is supplemented by the second part, and the labeling substance can be detected at that position. In addition, for monoclonal antibodies or fragments thereof that react specifically with the adhesion stage larvae of oysters and mussels in the oyster adhesion stage larvae present in the sample, but not in the adhesion stage larvae of clams and snails The antigen can be detected by the labeling substance at the position after being captured by the third part. Furthermore, a secondary antibody in which a monoclonal antibody labeled with a labeling substance or a fragment thereof, which has not reacted with the adhesion stage larvae of oysters and mussels by being provided with the fourth part, is immobilized on the fourth part. As a result, the labeling substance can be detected at the position, and it can be determined that the detection test has been completed. In the above-mentioned immunochromatographic medium, when the second part is darkly labeled, there is a possibility that some of the mussel-adhering larvae etc. present in the sample flow and are captured by the third part. Therefore, it is preferable to perform a detection test after diluting the sample again. As a result, it is possible to accurately check whether or not oyster adhesion stage larvae are present in the sample.
また、本発明に係るマガキ付着期幼生の検出キットは、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体と、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントとを含むものであればどのようなものでもよく、これら以外に、例えば、緩衝液（例えば、リン酸塩、炭酸塩、塩酸塩等の塩の溶液）、防腐剤（例えば、アジ化ナトリウムなど）、非特異的な反応を抑制するための物質（例えば、ブロックエース、ゲラチン、スキムミルクなど）、上述のムラサキイガイ付着期幼生を除去するための物質、上述のマガキ付着期幼生を回収するための物質、試料からムラサキイガイ付着期幼生を除去した後又は溶解物中の、ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体に対する抗原を除去した後に上述の免疫学的手法によりマガキ付着期幼生を検出するのに必要な物質（例えば、標識物質、発色基質、二次抗体、発色増強剤など）、安定剤（例えば、BSA、ヤギ血清など）等の抗原の検出キットに含ませる一般的な物質、若しくは上述のような検出器（ムラサキイガイ付着期幼生を別途除去する場合には、検出器に上述の第二の部分が含まれていなくてもよい。）、又はこれらのうち２以上を組み合わせたものがさらに含まれていてもよい。 Further, the detection kit for oyster adhesion stage larvae according to the present invention reacts specifically with mussel adhesion stage larvae, but does not react with oyster adhesion stage larvae, and specifically reacts with oyster and mussel adhesion stage larvae, Any monoclonal antibody or fragment thereof that does not react with the clams and snail larvae may be used. Besides these, for example, a buffer solution (for example, phosphate, carbonate, hydrochloride) Salt solution), preservatives (for example, sodium azide, etc.), substances for suppressing non-specific reactions (for example, block ace, gelatin, skim milk, etc.), and the above-mentioned mussel adhesion stage larvae are removed. Substance for recovering the above-mentioned oyster adhesion stage larvae, after removing mussel adhesion stage larvae from the sample or lysate Substances that are required to detect larvae-attached larvae by the immunological method described above after removing antigens against antibodies that react with mussel-attached larvae but do not react with oyster-attached larvae , Chromogenic substrates, secondary antibodies, chromogenic enhancers, etc.), stabilizers (eg, BSA, goat serum, etc.) and other common substances included in antigen detection kits, or detectors such as those described above When separately removing the larvae, the detector may not include the second portion described above), or a combination of two or more of these may be further included.
なお、前記標識物質としては、例えば、蛍光物質（例えば、FITC、ローダミン、ファロイジンなど）、金などのコロイド粒子、重金属（例えば、金、白金など）、色素タンパク質（例えば、フィコエリトリン（PE）、フィコシアニン（PC）など）、放射性同位元素（例えば、3H、32P、35S、125I、131Iなど）、酵素（例えば、ペルオキシダーゼ、アルカリフォスファターゼなど）、ビオチン、ストレプトアビジン等の物質を用いることができるがこれらに制限されるものではなく、公知の標識物質を用いてもよい。また、発色基質としては、上述の酵素に対する発色基質であれば特に制限されるものではなく、例えば、ジアミノベンジジン（DAB）、o-フェニレンジアミン(o-Phenylenediamine)、過酸化水素水、BCIP/Nitro-TB（5-Bromo-4-chloro-3-indolylphosphate/Nitrotetrazolium blue）、pNPP（para-nitorophenylphosphate）などを用いることができる。前記発色増強剤としては、上述の基質の発色を増強させることができるものであればどのようなものでもよく、例えば、硫酸などを用いることができ、前記二次抗体としては、例えば、マガキ及びムラサキイガイの付着期幼生に特異的に反応し、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体などが産生されたホストの動物種の免疫グロブリンに特異的に反応する抗免疫グロブリン抗体、抗Ig(H+L)、抗Ig(Fc)などを用いることができる。 Examples of the labeling substance include fluorescent substances (eg, FITC, rhodamine, phalloidin, etc.), colloidal particles such as gold, heavy metals (eg, gold, platinum, etc.), chromoproteins (eg, phycoerythrin (PE), phycocyanin). (PC) etc.), radioisotopes (eg 3 H, 32 P, 35 S, 125 I, 131 I etc.), enzymes (eg peroxidase, alkaline phosphatase etc.), biotin, streptavidin etc. However, it is not limited to these, and a known labeling substance may be used. Further, the chromogenic substrate is not particularly limited as long as it is a chromogenic substrate for the above-mentioned enzyme. For example, diaminobenzidine (DAB), o-phenylenediamine (o-Phenylenediamine), hydrogen peroxide solution, BCIP / Nitro -TB (5-Bromo-4-chloro-3-indolylphosphate / Nitrotetrazolium blue), pNPP (para-nitorophenylphosphate), etc. can be used. The color development enhancer may be any one that can enhance the color development of the above-mentioned substrate. For example, sulfuric acid or the like can be used. Examples of the secondary antibody include oyster and Anti-immunoglobulin antibodies that react specifically with the immunoglobulins of the host animal species that produced monoclonal antibodies that react specifically with the larvae of the mussel, but do not react with the larvae of the clams and mussel (H + L), anti-Ig (Fc), and the like can be used.
以上のように、本発明に係るマガキ付着期幼生の検出方法、検出器、又は検出キットを用いることにより、マガキ付着期幼生の存在の有無の確認、マガキ付着期幼生の同定、マガキ付着期幼生の量の測定が可能となる。 As described above, by using the detection method, detector, or detection kit of a oyster adhering stage larva according to the present invention, confirmation of the presence or absence of a oyster adhering stage larva, identification of a oyster adhering stage larva, magaki attaching stage larva Can be measured.
なお、本実施の形態において用いられる上述のムラサキイガイ付着期幼生に反応し、マガキ付着期幼生には反応しない抗体は、モノクローナル抗体であってもかまわないが、ポリクローナル抗体であってもよい。なお、このポリクローナル抗体は、ムラサキイガイ付着期幼生を抗原として用い、常法により製造することができる。 The antibody that reacts with the above-mentioned mussel-adhered larvae and does not react with the oyster-adherent larvae used in the present embodiment may be a monoclonal antibody or a polyclonal antibody. This polyclonal antibody can be produced by a conventional method using mussel adhesion stage larva as an antigen.
＝＝モノクローナル抗体又はそのフラグメントの製造方法＝＝
上述のムラサキイガイ付着期幼生又はその粗抽出液に反応し、マガキ付着期幼生又はその粗抽出液には反応しないモノクローナル抗体や、上述のマガキ及びムラサキイガイの付着期幼生又はそれらの粗抽出液に特異的に反応し、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体は、それぞれのモノクローナル抗体を産生するハイブリドーマを用いることにより得ることができる。
== Method for producing monoclonal antibody or fragment thereof ==
Monoclonal antibody that reacts with the above-mentioned mussel-adhered larvae or crude extract thereof and does not react with oyster-adherent larvae or crude extract thereof, or specific to the aforementioned oyster and mussel adherent-stage larvae or crude extract thereof Monoclonal antibodies that react with the above and do not react with clams and turtle larvae can be obtained by using hybridomas that produce the respective monoclonal antibodies.
上述のハイブリドーマを用いたモノクローナル抗体の製造は、該ハイブリドーマを適当な培養培地で培養し、培養上清を回収することにより行ってもよいが、上述のハイブリドーマを哺乳類動物（例えば、マウス、ラット、ハムスター、ウサギ、ブタ、ウシ、ウマ、イヌ、サルなど）の腹腔内に投与することにより行ってもよい。なお、モノクローナル抗体の精製は、培養上清又は培養したハイブリドーマを超音波装置、ホモジナイザー等で処理した溶解物、又は哺乳類動物から採取した血液若しくは腹水を、常法、例えば、硫安塩析、クロマトグラフィー（例えば、イオン交換クロマトグラフィーやアフィニティークロマトグラフィーなど）、ゲル濾過等の方法、又はこれらの方法を適宜組み合わせた方法により行うことができる。 Production of the monoclonal antibody using the above-mentioned hybridoma may be carried out by culturing the hybridoma in an appropriate culture medium and collecting the culture supernatant. However, the above-mentioned hybridoma can be produced in a mammal (eg, mouse, rat, (Hamster, rabbit, pig, cow, horse, dog, monkey, etc.) may be administered intraperitoneally. The monoclonal antibody can be purified by lysing a culture supernatant or a cultivated hybridoma treated with an ultrasonic device, a homogenizer, etc., or blood or ascites collected from a mammal, using a conventional method such as ammonium sulfate salting-out, chromatography. (For example, ion exchange chromatography, affinity chromatography, etc.), a method such as gel filtration, or a method in which these methods are appropriately combined.
上述のムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しないモノクローナル抗体を産生するハイブリドーマとしては、例えば、独立行政法人産業技術総合研究所 特許生物寄託センターにおいて受託番号FERM P-20216、FERM P-20218、FERM P-20219、又はFERM P-20220で寄託されている細胞株を用いることができる。また、上述のマガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体を産生するハイブリドーマとしては、例えば、独立行政法人産業技術総合研究所 特許生物寄託センターにおいて受託番号FERM AP-20217で寄託されている細胞株を用いることができる。 As a hybridoma that produces a monoclonal antibody that reacts with the mussel adhesion stage larvae described above but does not react with the oyster adhesion stage larvae, for example, accession number FERM P-20216 at the National Institute of Advanced Industrial Science and Technology patent biological depository center, Cell lines deposited with FERM P-20218, FERM P-20219, or FERM P-20220 can be used. In addition, as a hybridoma that produces a monoclonal antibody that reacts specifically with the adhesion larvae of the above-mentioned oysters and mussels but does not react with the adhesion larvae of clams and snails, for example, National Institute of Advanced Industrial Science and Technology Patent A cell line deposited under the accession number FERM AP-20217 at the biological deposit center can be used.
上述のモノクローナル抗体のフラグメントとしては、該モノクローナル抗体の一部からなり、可変領域を含む抗原結合部位であれば特に制限されるものではないが、例えば、Fabフラグメント、F(ab’)2フラグメントなどを用いることができる。これらのフラグメントは、例えば、上述のモノクローナル抗体をタンパク質分解酵素によって部分消化することにより得ることができる。なお、タンパク質分解酵素としては、FabフラグメントやF(ab’)2フラグメントを得ることができるものであればどのようなものであってもよいが、例えば、ペプシン、フィシン等の分解酵素を用いることができる。 The above-described monoclonal antibody fragment is not particularly limited as long as it is an antigen-binding site comprising a part of the monoclonal antibody and including a variable region. For example, Fab fragment, F (ab ′) 2 fragment, etc. Can be used. These fragments can be obtained, for example, by partially digesting the above-described monoclonal antibody with a proteolytic enzyme. The proteolytic enzyme may be any as long as it can obtain Fab fragments and F (ab ′) 2 fragments. For example, a protease such as pepsin or ficin should be used. Can do.
＝＝＝ハイブリドーマの作製＝＝＝
上述のハイブリドーマは、例えば、以下の方法により作製することができる。まず、ムラサキイガイ付着期幼生又はその粗抽出液を抗原として用い、適当な量の抗原（アジュバントと併用してもよい。）を哺乳類動物（例えば、マウス、ラット、ハムスター、ウサギ、ブタ、ウシ、ウマ、イヌ、サルなど）の静脈内、皮下、腹腔内等に（１〜複数回）投与して免疫する。その後、免疫した動物から抗体産生細胞を採取し、骨髄腫細胞（ミエローマ細胞）と融合させてハイブリドーマを作製する。得られたハイブリドーマの培養上清等を用いて、ムラサキイガイ、マガキ、アサリ、及びバカガイ等の二枚貝類の付着期幼生又はそれらの粗抽出液との反応性を確認することにより、得ることができる。
=== Production of Hybridoma ===
The above-mentioned hybridoma can be produced, for example, by the following method. First, the mussel adherent stage larva or a crude extract thereof is used as an antigen, and an appropriate amount of antigen (which may be used in combination with an adjuvant) is used as a mammal (eg, mouse, rat, hamster, rabbit, pig, cow, horse). Intravenous, subcutaneous, intraperitoneal (1 to several times) administration (1 to several times) in a dog, monkey, etc. Thereafter, antibody-producing cells are collected from the immunized animal and fused with myeloma cells (myeloma cells) to produce hybridomas. Using the obtained hybridoma culture supernatant, etc., it can be obtained by confirming the reactivity of bivalves such as mussels, oysters, clams, and clams and their reactivity with the crude extract.
前記抗体産生細胞とミエローマ細胞との細胞融合は、例えば、ポリエチレングリコール（ＰＥＧ）、センダイウイルス（ＨＶＪ）などの融合促進剤を含む培養培地で抗体産生細胞とミエローマ細胞とを一緒に培養することにより行うことができるが、エレクトロポレーション等の電気刺激を利用して行うこともできる。なお、細胞融合の効率を高めるために、ジメチルスルホキシドやレシチンなどの補助剤を培養培地に含ませることとしてもよい。 Cell fusion between the antibody-producing cells and myeloma cells is performed by, for example, culturing the antibody-producing cells and myeloma cells together in a culture medium containing a fusion promoter such as polyethylene glycol (PEG) or Sendai virus (HVJ). Although it can be performed, it can also be performed using electrical stimulation such as electroporation. In order to increase the efficiency of cell fusion, adjuvants such as dimethyl sulfoxide and lecithin may be included in the culture medium.
前記抗体産生細胞としては、例えば、脾臓細胞、リンパ節細胞、胸腺細胞、末梢血細胞などを用いることができる。これらの細胞は、動物から脾臓、リンパ節、胸腺、又は末梢血を摘出し、摘出した組織を破砕、濾過、遠心分離等することにより得ることができる。また、前記ミエローマ細胞としては、各種動物由来の細胞株を用いてもよいが、それ自身薬剤に対して抵抗性を示さないが、融合すると薬剤に対して抵抗性を示す細胞株を用いることが好ましい。これにより、細胞融合した後、薬剤を添加した培養培地（例えば、ＨＡＴ培地など）で培養することにより、細胞融合によって得られたハイブリドーマの選択が容易となる。なお、融合させる抗体産生細胞とミエローマ細胞は、同種の動物由来の細胞を用いることが望ましいが、異なる種の動物由来の細胞を用いることとしてもよい。 Examples of the antibody-producing cells that can be used include spleen cells, lymph node cells, thymocytes, and peripheral blood cells. These cells can be obtained by removing spleen, lymph nodes, thymus, or peripheral blood from an animal, and crushing, filtering, centrifuging, etc. the removed tissue. In addition, as the myeloma cells, cell lines derived from various animals may be used. However, cell lines that do not themselves show resistance to drugs but that are resistant to drugs when fused may be used. preferable. Thereby, after cell fusion, selection of a hybridoma obtained by cell fusion is facilitated by culturing in a culture medium (for example, HAT medium) to which a drug is added. The antibody-producing cells and myeloma cells to be fused are preferably cells of the same species, but cells derived from animals of different species may be used.
上述のハイブリドーマは、常法のスクリーニングやクローニングを繰り返し行って、より特異性の高いモノクローナル抗体を産生するハイブリドーマを選択したものであることが望ましい。ハイブリドーマのスクリーニングには、例えば、酵素免疫測定法（EIA：Enzyme ImmunoAssay、ELISA：Enzyme-Linked Immunosorbent Assays）、放射線免疫測定法（RIA：Radio Immuno Assay）、ウェスタンブロット法等を用いることができ、ハイブリドーマのクローニングには、例えば、限界希釈法、軟寒天法、フィブリンゲル法、蛍光励起セルソーター法等を用いることができる。 It is desirable that the above-mentioned hybridoma is selected by repeating conventional screening and cloning and selecting a hybridoma that produces a monoclonal antibody with higher specificity. For the screening of hybridomas, for example, enzyme immunoassay (EIA: Enzyme ImmunoAssay, ELISA: Enzyme-Linked Immunosorbent Assays), radioimmunoassay (RIA: Radio Immuno Assay), Western blotting, etc. can be used. For cloning, for example, limiting dilution method, soft agar method, fibrin gel method, fluorescence excitation cell sorter method and the like can be used.
なお、上述のハイブリドーマのスクリーニングでは、最低限ムラサキイガイ、マガキ、アサリ、及びバカガイ等の二枚貝類の付着期幼生又はそれらの粗抽出液との反応性を調べればよいが、さらに、二枚貝類の付着期幼生以外に、フジツボの付着期幼生（キプリス幼生）、甲殻類プランクトン等の他の幼生若しくはプランクトン又はそれらの粗抽出液との反応性を調べる方が好ましい。これにより、より特異性の高いモノクローナル抗体を産生するハイブリドーマを得ることが可能となる。 In the above hybridoma screening, at least the adhesion period of bivalves such as mussels, oysters, clams, and clams or their reactivity with the crude extract may be examined. In addition to larvae, it is preferable to examine reactivity with other larvae such as barnacle attachment stage larvae (Cyprus larvae), crustacean plankton, or plankton, or their crude extracts. This makes it possible to obtain a hybridoma that produces a monoclonal antibody with higher specificity.
以上のようにして得られたハイブリドーマから産生される、ムラサキイガイ付着期幼生又はその粗抽出液に反応し、マガキ付着期幼生又はその粗抽出液には反応しないモノクローナル抗体や、マガキ及びムラサキイガイの付着期幼生又はそれらの粗抽出液に特異的に反応し、アサリやバカガイの付着期幼生又はそれらの粗抽出液には反応しないモノクローナル抗体を用いることにより、様々な幼生又はプランクトンを含む試料（例えば、海水など）中からマガキ付着期幼生を特異的に回収することが可能となり、また、様々な幼生又はプランクトンを含む試料（例えば、海水など）中からマガキ付着期幼生を特異的に検出、すなわち、マガキ付着期幼生の存在の有無の確認、マガキ付着期幼生の同定、マガキ付着期幼生の量の測定等を行うことが可能になる。 Monoclonal antibodies produced from hybridomas obtained as described above, which react with the mussel adhesion stage larvae or the crude extract thereof, and do not react with the oyster adhesion stage larvae or the crude extract thereof, or the adhesion stage of oysters and mussels Samples containing various larvae or plankton (for example, seawater) can be obtained by using monoclonal antibodies that react specifically with larvae or their crude extracts and do not react with the clams or clams' attached stage larvae or their crude extracts. It is possible to specifically collect larvae adhering stage larvae from inside, and also to detect oyster adhering stage larvae specifically from samples containing various larvae or plankton (for example, seawater). Confirm the presence or absence of attached larvae, identify larvae attached larvae, measure the amount of larvae attached larvae, etc. It becomes possible.
以下に本発明を実施例によって具体的に説明する。なお、これらの実施例は本発明を説明するためのものであって、本発明の範囲を限定するものではない。 Hereinafter, the present invention will be specifically described by way of examples. These examples are for explaining the present invention, and do not limit the scope of the present invention.
［実施例１］
室内飼育により得られたムラサキイガイ付着期幼生（ペディベリジャー幼生）を抗原として用いた。
[Example 1]
The mussel adherent stage larvae (pediberger larvae) obtained by indoor breeding were used as antigens.
7週齢のBALB/cマウスの腹腔内に抗原を6回注射し（1〜5回目の注射：幼生300個/100〜150μl PBS、6回目の注射（最終免疫）：幼生1000個/200μl PBS；1回目の投与から22日目に2回目の投与を、2回目の投与から24日目に3回目の投与を、3回目の投与からおよそ半年後に4回目の投与を、4回目の投与から半月後に5回目の投与を、5回目の投与からおよそ80日目に6回目の投与を行った。）、マウスを免疫した。 Inject the antigen into the abdominal cavity of 7-week-old BALB / c mice 6 times (1-5th injection: 300 larvae / 100-150 μl PBS, 6th injection (final immunization): 1000 larvae / 200 μl PBS The second dose on day 22 from the first dose, the third dose on day 24 from the second dose, the fourth dose approximately half a year after the third dose, from the fourth dose Half a month later, the fifth dose was given and the sixth dose was given approximately 80 days after the fifth dose.) The mice were immunized.
最終免疫から3日後にマウスを解剖して脾臓を摘出し、10％のFBS（Fetal Bovine Serum；GIBCO）及び1％の抗生物質（Antibiotic-Antimycotic；GIBCO）を含むRPMI1640（GIBCO）培地中で滅菌ステンレスメッシュにより裏漉した後、分散・懸濁することにより浮遊細胞を得た。この浮遊細胞を10％のFBS を含むRPMI1640培地（FBS+培地）で洗浄・遠心した後、RPMI1640培地（FBS-培地）で3回遠心・洗浄し、脾臓細胞（108個程度）を回収した。 Three days after the final immunization, the mice were dissected and the spleen was removed and sterilized in RPMI1640 (GIBCO) medium containing 10% FBS (Fetal Bovine Serum; GIBCO) and 1% antibiotics (Antibiotic-Antimycotic; GIBCO). The cells were lined with a stainless mesh and then suspended and dispersed to obtain floating cells. The suspension cells were washed and centrifuged with RPMI1640 medium (FBS + medium) containing 10% FBS, and then centrifuged and washed three times with RPMI1640 medium (FBS - medium) to collect spleen cells (about 10 8 cells). .
次に、ミエローマ細胞株P3U1（5×107個）と脾臓細胞（108個程度）を混合し、その後遠心して上清を除去し、細胞ペレット（沈殿）を作製した。これに1gのPEG4000（ポリエチレングリコール；MERCK社 Article No.9727）とFBS-培地1mlとを混合した溶液をゆっくりと添加しながら撹拌し、さらに１分間緩やかに撹拌した。その後、FBS-培地2mlをゆっくり加えながら１分間撹拌して、細胞融合を行った。 Next, the myeloma cell line P3U1 (5 × 10 7 cells) and spleen cells (about 10 8 cells) were mixed, and then centrifuged to remove the supernatant to prepare a cell pellet (precipitate). A solution obtained by mixing 1 g of PEG4000 (polyethylene glycol; Article No. 9727 of MERCK) and 1 ml of FBS - medium was stirred while slowly adding, and further stirred for 1 minute. Thereafter, 2 ml of FBS - medium was slowly added and stirred for 1 minute to perform cell fusion.
細胞融合処理後、さらにFBS-培地8mlを１分間かけてゆっくりと添加し、続いて遠心分離（1000rpm×5分間）して上澄みを吸引除去した。これにFBS-培地10mlを添加して懸濁し、FBS+HAT培地（10％のFBS、1％の抗生物質、及びHAT supplement medium（SIGMA；粉末1バイアルを50倍に希釈したものを使用）×0.5を含むRPMI1640培地（GIBCO））が入った分室シャーレ5枚にそれぞれ2ml播種し、37 ℃，5% CO2の条件下で5日間培養した。 After cell fusion treatment, 8 ml of FBS - medium was slowly added over 1 minute, followed by centrifugation (1000 rpm × 5 minutes), and the supernatant was removed by suction. This FBS - were suspended by addition of media 10 ml, FBS + HAT medium (10% FBS, 1% antibiotics, and HAT supplement medium (SIGMA; used after diluting powder 1 vial 50 times) × 2 ml of each of the five laboratory petri dishes containing RPMI1640 medium (GIBCO) containing 0.5 was inoculated and cultured for 5 days under conditions of 37 ° C. and 5% CO 2 .
各セル内で明瞭な増殖が確認されたハイブリドーマ細胞のコロニーをピックアップし、HT培地（10％の細胞増殖因子（conditioned medium from J774A.1 cells，HYBRIMAX；SIGMA）、100μMヒポキサンチン、及び16μMチミジンを含むRPMI1640培地）の入った96穴ウェルプレートに移し、ハイブリドーマの培養を行った。その後、明瞭なハイブリドーマの増殖が確認されたウェルについて、その培養上清を採取し、ELISA法により抗体の産生を確認した。 Pick colonies of hybridoma cells that have been clearly seen growing in each cell, and add HT medium (10% conditioned medium from J774A.1 cells, HYBRIMAX; SIGMA), 100 μM hypoxanthine, and 16 μM thymidine. It was transferred to a 96-well plate containing RPMI1640 medium), and the hybridoma was cultured. Thereafter, the culture supernatant was collected from wells in which the growth of clear hybridoma was confirmed, and antibody production was confirmed by ELISA.
（１）ムラサキイガイのペディベリジャー幼生30000個体に600μlの50mM Tris-HCl（pH 7.5）を加え、氷中でホモジナイズ・超音波（数秒×5回）処理を行い、5000rpm×20分間遠心することにより、ムラサキイガイ幼生粗抽出液（タンパク質濃度：39.5mg/ml）を調製した。
（２）（１）で得られたムラサキイガイ幼生粗抽出液（100μg/ml）50μlを、96穴イワキELISAプレートの各ウェルに加え、４℃で抗原をウェル底面に吸着させた。
（３）（２）で吸着処理した各ウェルをTBS（0.5 M NaCl及び20 mM Tris-HCl（pH7.5））200μlで洗浄した。
（４）各ウェルを1％ BSAを含むTBS 100μlで1時間ブロッキングした。
（５）各ウェルをTTBS（0.05％ Tween20を含むTBS）200μlで3回洗浄した。
（６）ハイブリドーマを培養することにより得られた培養上清（1次抗体）50μlを各ウェルに加え、1時間反応させた。
（７）各ウェルをTTBS 200μlで4回洗浄した。
（８）各ウェルに2次抗体（アルカリフォスファターゼ標識抗マウスIgG（H+L）ヤギ抗体；ZYMED laboratory）溶液（TBSで1000倍に希釈した溶液）50μlを加えて1時間反応させた。
（９）各ウェルをTTBS 200μlで5回洗浄した。
（10）各ウェルに基質（アルカリフォスファターゼ基質キット；BIO-RAD）溶液 100μlを加え、室温で1時間振盪することにより発色させた。
（11）マイクロプレートリーダー（BIO-RAD Model550；405nm）を用いて、各ウェルの溶液の吸光度を測定した。
(1) By adding 600 μl of 50 mM Tris-HCl (pH 7.5) to 30,000 mussel pediberger larvae, homogenizing and sonicating (several seconds x 5 times) in ice, and centrifuging at 5000 rpm for 20 minutes, A blue mussel larvae crude extract (protein concentration: 39.5 mg / ml) was prepared.
(2) 50 μl of the blue mussel larvae crude extract (100 μg / ml) obtained in (1) was added to each well of a 96-well Iwaki ELISA plate, and the antigen was adsorbed to the bottom of the well at 4 ° C.
(3) Each well subjected to the adsorption treatment in (2) was washed with 200 μl of TBS (0.5 M NaCl and 20 mM Tris-HCl (pH 7.5)).
(4) Each well was blocked with 100 μl of TBS containing 1% BSA for 1 hour.
(5) Each well was washed 3 times with 200 μl of TTBS (TBS containing 0.05% Tween20).
(6) 50 μl of the culture supernatant (primary antibody) obtained by culturing the hybridoma was added to each well and allowed to react for 1 hour.
(7) Each well was washed 4 times with 200 μl of TTBS.
(8) To each well was added 50 μl of a secondary antibody (alkaline phosphatase-labeled anti-mouse IgG (H + L) goat antibody; ZYMED laboratory) solution (1000-fold diluted with TBS) and allowed to react for 1 hour.
(9) Each well was washed 5 times with 200 μl of TTBS.
(10) 100 μl of substrate (alkaline phosphatase substrate kit; BIO-RAD) solution was added to each well, and color was developed by shaking for 1 hour at room temperature.
(11) The absorbance of the solution in each well was measured using a microplate reader (BIO-RAD Model 550; 405 nm).
上述のELISA法により高い抗体産生能が確認されたハイブリドーマについて、段階的に希釈した培養液をさらにELISA法により抗体価を測定し、抗体産生能が高いハイブリドーマをピックアップするという操作を3回繰り返して行い（2回目の培養は、HTを含まない、10％ FBS及び1％ 抗生物質を含むRPMI1640を用いた。）、ムラサキイガイ幼生粗抽出液に対してより高い抗体産生能を示すハイブリドーマ（A12-1-2b、B2-3-4-4a、C4-3-4c、C4-3-4b、及びF10-4-1c）を得た。 For the hybridoma whose high antibody production ability was confirmed by the ELISA method described above, the antibody titer of the culture solution diluted in stages was further measured by ELISA method, and the operation of picking up the hybridoma having high antibody production ability was repeated three times. (The second culture used RPMI1640 containing 10% FBS and 1% antibiotics without HT.) A hybridoma (A12-1) showing higher antibody-producing ability against the crude extract of mussel larvae -2b, B2-3-4-4a, C4-3-4c, C4-3-4b, and F10-4-1c).
［実施例２］
次に、実施例１により得られた５つのハイブリドーマの培養上清に含まれるモノクローナル抗体が、ムラサキイガイペディベリジャー幼生に対して特異的に反応するかいなかを調べるため、ムラサキイガイペディベリジャー幼生、ムラサキイガイ以外の二枚貝類（アサリ、バカガイ、マガキなど）のペディベリジャー幼生、甲殻類プランクトン（コペポーダ類、アルテミア（Artemia salina）ノープリウス幼生）、イワフジツボ（Chthamalus challengeri Hoek）キプリス幼生、アカフジツボキプリス幼生の粗抽出液（タンパク質の濃度；100μg/ml）との反応性を、実施例１に記載のELISA法と同様に確認した。なお、各粗抽出液は、各幼生又はプランクトンを50mM Tris-HCl（PH7.5）に加えてホモジナイズ・超音波（数秒×5回）処理し、5000rpm×20分間の遠心を行うことにより調製した。ELISAの結果を表１に示す。
[Example 2]
Next, in order to investigate whether or not the monoclonal antibodies contained in the culture supernatants of the five hybridomas obtained in Example 1 react specifically with the mussel pediberger larvae, other than the mussel pediberger larvae and mussels Crude extract (protein) (The concentration of 100 μg / ml) was confirmed in the same manner as in the ELISA method described in Example 1. Each crude extract was prepared by adding each larvae or plankton to 50 mM Tris-HCl (PH7.5), homogenizing and sonicating (several seconds × 5 times), and centrifuging at 5000 rpm × 20 minutes. . The results of ELISA are shown in Table 1.
［表１］
表１に示すように、ハイブリドーマ細胞株A12-1-2b、C4-3-4c、及びC4-3-4bの上清は、ムラサキイガイペディベリジャー幼生の粗抽出液に対して非常に強い反応性を示し、形態学的に非常に似ているマガキ、アサリ、バカガイ等のムラサキイガイ以外の二枚貝類のペディベリジャー幼生の粗抽出液や、二枚貝類以外の幼生やプランクトンの粗抽出液に対して反応性を示さなかった。また、ハイブリドーマ細胞株F10-4-1cの上清は、ムラサキイガイペディベリジャー幼生の粗抽出液に対して非常に強い反応性を示すとともに、形態学的にムラサキイガイペディベリジャー幼生と全く異なるコペポーダ類の粗抽出液に対して非常に弱い反応性を示し、その他の幼生の粗抽出液には反応性を示さないことが明らかになった。 As shown in Table 1, the supernatants of the hybridoma cell lines A12-1-2b, C4-3-4c, and C4-3-4b have a very strong reactivity to the crude extract of the mussel pediberger larvae. Reactive to crude extracts of pediberger larvae of bivalves other than mussels such as oysters, clams, and mussels, and larvae other than bivalves and crude extracts of plankton. Not shown. In addition, the supernatant of the hybridoma cell line F10-4-1c shows a very strong reactivity to the crude extract of the mussel pediberger larvae, and the morphologically different crude copepoda larvae from the mussel pediberger larvae. It became clear that it showed very weak reactivity to the extract and no reactivity to other larvae crude extracts.
一方、ハイブリドーマ細胞株B2-3-4-4aの上清は、ムラサキイガイペディベリジャー幼生の粗抽出液以外にマガキペディベリジャー幼生の粗抽出液に非常に強く反応性を示し、ムラサキイガイ及びマガキのペディベリジャー幼生以外の幼生及びプランクトンの粗抽出液全てに対して反応性を示さないことがわかった。 On the other hand, the supernatant of the hybridoma cell line B2-3-4-4a showed very strong reactivity to the crude extract of oyster pediberger larvae in addition to the crude extract of mussel pediberger larvae. It was found that there was no reactivity to all larvae other than larvae and the crude plankton extract.
以上のことから、ハイブリドーマ細胞株A12-1-2b、C4-3-4c、C4-3-4b、及びF10-4-1cは、ムラサキイガイペディベリジャー幼生に特異的に反応するモノクローナル抗体を産生し、ハイブリドーマ細胞株B2-3-4-4aは、マガキ及びムラサキイガイペディベリジャー幼生に特異的に反応するモノクローナル抗体を産生することが明らかになった。また、ハイブリドーマ細胞株B2-3-4-4aが産生するモノクローナル抗体は、ハイブリドーマ細胞株A12-1-2b、C4-3-4c、C4-3-4b、F10-4-1c等が産生するモノクローナル抗体とその特異性が異なることから、それぞれの抗原認識部位が異っていることが示唆された。さらに、ハイブリドーマ細胞株B2-3-4-4aが産生するモノクローナル抗体と、ハイブリドーマ細胞株A12-1-2b、C4-3-4c、C4-3-4b、F10-4-1c等が産生するモノクローナル抗体とを用いることにより、マガキペディベリジャー幼生を特異的に検出したり、回収したりすることができるのではないかと考えられた。そこで、これらのハイブリドーマ細胞株を独立行政法人産業技術総合研究所 特許生物寄託センターに寄託した（A12-1-2b：受託番号FERM P-20216、B2-3-4-4a：FERM P-20217、C4-3-4c：受託番号FERM P-20218、C4-3-4b：FERM P-20219、F10-4-1c：FERM P-20220）。 From the above, the hybridoma cell lines A12-1-2b, C4-3-4c, C4-3-4b, and F10-4-1c produce monoclonal antibodies that react specifically with mussel pediberger larvae, The hybridoma cell line B2-3-4-4a was found to produce monoclonal antibodies that react specifically with oyster and mussel pediberger larvae. The monoclonal antibodies produced by the hybridoma cell line B2-3-4-4a are monoclonal antibodies produced by the hybridoma cell lines A12-1-2b, C4-3-4c, C4-3-4b, F10-4-1c, etc. The antibody and its specificity are different, suggesting that each antigen recognition site is different. Furthermore, monoclonal antibodies produced by the hybridoma cell line B2-3-4-4a and monoclonal antibodies produced by the hybridoma cell lines A12-1-2b, C4-3-4c, C4-3-4b, F10-4-1c, etc. It was thought that oyster pediberger larvae could be specifically detected or recovered by using antibodies. Therefore, these hybridoma cell lines were deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (A12-1-2b: accession number FERM P-20216, B2-3-4-4a: FERM P-20217, C4-3-4c: accession number FERM P-20218, C4-3-4b: FERM P-20219, F10-4-1c: FERM P-20220).
Claims (20)
ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体と、
マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントと、
を作用させることを特徴とする検出方法。 A method for detecting larvae adhering stage larvae,
An antibody that reacts with mussel lagoons but does not react with oyster larvae,
A monoclonal antibody or fragment thereof that specifically reacts to the larvae of the oysters and mussels but does not react to the larvae of the clams and mussel;
The detection method characterized by making act.
ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体と、
マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントと、
を含むことを特徴とする検出キット。 A detection kit for larvae adhering stage larvae,
An antibody that reacts with mussel lagoons but does not react with oyster larvae,
A monoclonal antibody or fragment thereof that specifically reacts to the larvae of the oysters and mussels but does not react to the larvae of the clams and mussel;
A detection kit comprising:
前記検出器は、イムノクロマトグラフ媒体を含み、
前記イムノクロマトグラフ媒体は、
試料を滴下する又は浸す第一の部分と、
ムラサキイガイ付着期幼生に反応し、マガキ付着期幼生には反応しない抗体が固定化されている第二の部分と、
マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントが固定化されている第三の部分と、を含み、
前記第二の部分は、前記第一の部分と前記第三の部分との間に備えられていることを特徴とする検出器。 A detector of larvae adhering stage larvae,
The detector comprises an immunochromatographic medium;
The immunochromatographic medium is
A first part in which a sample is dropped or immersed;
A second part in which an antibody that reacts with the mussel adhesion stage larvae and does not react with the oyster adhesion stage larvae is immobilized;
A third part immobilized with a monoclonal antibody or fragment thereof that reacts specifically with the adhesion stage larvae of oysters and mussels, but does not react with the adhesion stage larvae of clams and snails,
The detector is characterized in that the second part is provided between the first part and the third part.
ムラサキイガイ付着期幼生に反応するが、マガキ付着期幼生には反応しない抗体と、マガキ及びムラサキイガイの付着期幼生に特異的に反応するが、アサリやバカガイの付着期幼生には反応しないモノクローナル抗体又はそのフラグメントとを用いて、試料中のマガキ付着期幼生を回収する工程を含むことを特徴とする回収方法。 A method for collecting larvae adhering stage larvae,
An antibody that reacts with larvae that adhere to the mussel, but does not react with larvae that adhere to the oyster, and a monoclonal antibody that reacts specifically with the larvae that adhere to the oyster and mussel, but does not react with the larvae that adhere to clams and mussel A recovery method comprising a step of recovering oyster adhesion stage larvae in a sample using a fragment.
The monoclonal antibody according to claim 19, wherein the monoclonal antibody is produced from a hybridoma deposited under accession number FERM P-20217.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2005070009A JP3932320B2 (en) | 2005-03-11 | 2005-03-11 | Detection method of larvae adhering to oyster |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2005070009A JP3932320B2 (en) | 2005-03-11 | 2005-03-11 | Detection method of larvae adhering to oyster |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2006250825A JP2006250825A (en) | 2006-09-21 |
| JP3932320B2 true JP3932320B2 (en) | 2007-06-20 |
Family
ID=37091487
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2005070009A Active JP3932320B2 (en) | 2005-03-11 | 2005-03-11 | Detection method of larvae adhering to oyster |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3932320B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009149533A (en) * | 2007-12-18 | 2009-07-09 | Chugoku Electric Power Co Inc:The | Monoclonal antibody specific to larva of blue sea mussel belonging to genus mytilus |
| JP5425832B2 (en) * | 2011-03-30 | 2014-02-26 | 中国電力株式会社 | How to identify Tamami Hydra Planula larvae |
| JP5474860B2 (en) * | 2011-03-30 | 2014-04-16 | 中国電力株式会社 | Monoclonal antibodies specific to Benicida hydra actinula larvae, Tamami hydraplanula larvae, and moon jellyfish planula larvae |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2913026B1 (en) * | 1998-01-21 | 1999-06-28 | 水産庁瀬戸内海区水産研究所長 | Clam floating larva specific monoclonal antibody |
| JP3743619B2 (en) * | 2000-12-08 | 2006-02-08 | 松下電器産業株式会社 | Microbe count / microbe concentration measuring device and microbe count / microbe concentration measuring method |
-
2005
- 2005-03-11 JP JP2005070009A patent/JP3932320B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2006250825A (en) | 2006-09-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4139818B2 (en) | Monoclonal antibody specific for mussel larvae | |
| AU2007291077B2 (en) | Antibodies to an epitope of AGR2. assays and hybridomas | |
| JP4084808B2 (en) | Monoclonal antibodies specific for red larvae adhering larvae | |
| WO2008035527A1 (en) | ANTI-C1q MONOCLONAL ANTIBODY | |
| JP3932320B2 (en) | Detection method of larvae adhering to oyster | |
| JP5269136B2 (en) | How to identify jellyfish planula larvae | |
| JP4194570B2 (en) | Monoclonal antibodies specific for barnacles' adherent larvae | |
| JP5425832B2 (en) | How to identify Tamami Hydra Planula larvae | |
| US7410771B2 (en) | Reagent and method for detecting a Cryptosporidium parvum sporozoite antigen | |
| JP5007321B2 (en) | Monoclonal antibody specific for mussel larva belonging to the genus Perna | |
| JP5577018B2 (en) | Monoclonal antibodies specific for mussel larvae | |
| JP2009149533A (en) | Monoclonal antibody specific to larva of blue sea mussel belonging to genus mytilus | |
| KR20120132227A (en) | Monoclonal antibody for detecting multiple type Foot and Mouth Disease Virus and method for detecting Foot and Mouth Disease Virus using the same | |
| JP6033894B2 (en) | Monoclonal antibody specific for mussel larva belonging to the genus Mytilus | |
| JP5474860B2 (en) | Monoclonal antibodies specific to Benicida hydra actinula larvae, Tamami hydraplanula larvae, and moon jellyfish planula larvae | |
| JP6026718B2 (en) | Monoclonal antibody specific for venicida hydra actinula larvae | |
| JPH04502363A (en) | Detection of basement membrane components for diagnosis of cancer and other diseases | |
| US9395370B2 (en) | Purification of monoclonal antibodies | |
| JP5781804B2 (en) | Monoclonal antibodies specific for Benicum hydra actinula larvae and Tamami hydraplanula larvae. | |
| JPH01153960A (en) | Red corpuscle agglutination assay | |
| WO2002066631A1 (en) | Antibodies recognizing helicobacter pylori and method of detecting helicobacter pylori | |
| EP4342989A1 (en) | Anti-norovirus antibody | |
| EP4342909A1 (en) | Anti-norovirus antibody | |
| CN115850498A (en) | Monoclonal anti-fluorescein thiocyanate antibody and application thereof | |
| KR20130135590A (en) | Lawsonia intracellularis-specific monoclonal antibody and hybridoma cell producing the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20061220 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20070109 |
|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20070129 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20070205 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20070228 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 3932320 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100330 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20110330 Year of fee payment: 4 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20120330 Year of fee payment: 5 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20130330 Year of fee payment: 6 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20140330 Year of fee payment: 7 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |