JP2012181181A - Particle for immobilization of physiologically active substance, physiologically active substance immobilization particle and sugar affinity substance capturing particle - Google Patents
Particle for immobilization of physiologically active substance, physiologically active substance immobilization particle and sugar affinity substance capturing particle Download PDFInfo
- Publication number
- JP2012181181A JP2012181181A JP2011187699A JP2011187699A JP2012181181A JP 2012181181 A JP2012181181 A JP 2012181181A JP 2011187699 A JP2011187699 A JP 2011187699A JP 2011187699 A JP2011187699 A JP 2011187699A JP 2012181181 A JP2012181181 A JP 2012181181A
- Authority
- JP
- Japan
- Prior art keywords
- particle
- physiologically active
- active substance
- immobilizing
- particles
- 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.)
- Granted
Links
- 239000002245 particle Substances 0.000 title claims abstract description 170
- 239000013543 active substance Substances 0.000 title claims abstract description 98
- 235000000346 sugar Nutrition 0.000 title claims abstract description 50
- 239000000126 substance Substances 0.000 title claims description 44
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 65
- 239000000178 monomer Substances 0.000 claims abstract description 51
- 230000003100 immobilizing effect Effects 0.000 claims abstract description 44
- 229920000642 polymer Polymers 0.000 claims abstract description 21
- 229920001577 copolymer Polymers 0.000 claims abstract description 3
- 239000007771 core particle Substances 0.000 claims description 36
- 150000001720 carbohydrates Chemical class 0.000 claims description 24
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 11
- 229920003229 poly(methyl methacrylate) Polymers 0.000 claims description 10
- 239000004926 polymethyl methacrylate Substances 0.000 claims description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- 230000000975 bioactive effect Effects 0.000 claims description 8
- -1 antibody Substances 0.000 claims description 7
- 125000003827 glycol group Chemical group 0.000 claims description 7
- 239000003446 ligand Substances 0.000 claims description 6
- 239000000088 plastic resin Substances 0.000 claims description 6
- 150000003384 small molecules Chemical class 0.000 claims description 5
- NJNWCIAPVGRBHO-UHFFFAOYSA-N 2-hydroxyethyl-dimethyl-[(oxo-$l^{5}-phosphanylidyne)methyl]azanium Chemical group OCC[N+](C)(C)C#P=O NJNWCIAPVGRBHO-UHFFFAOYSA-N 0.000 claims description 4
- 108090001008 Avidin Proteins 0.000 claims description 4
- 239000000427 antigen Substances 0.000 claims description 4
- 108091007433 antigens Proteins 0.000 claims description 4
- 102000036639 antigens Human genes 0.000 claims description 4
- 125000004057 biotinyl group Chemical class [H]N1C(=O)N([H])[C@]2([H])[C@@]([H])(SC([H])([H])[C@]12[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(*)=O 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 4
- 229920001515 polyalkylene glycol Polymers 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- ZEWLHMQYEZXSBH-UHFFFAOYSA-M 4-[2-(2-methylprop-2-enoyloxy)ethoxy]-4-oxobutanoate Chemical compound CC(=C)C(=O)OCCOC(=O)CCC([O-])=O ZEWLHMQYEZXSBH-UHFFFAOYSA-M 0.000 claims description 3
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 3
- ZSZRUEAFVQITHH-UHFFFAOYSA-N 2-(2-methylprop-2-enoyloxy)ethyl 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CC(=C)C(=O)OCCOP([O-])(=O)OCC[N+](C)(C)C ZSZRUEAFVQITHH-UHFFFAOYSA-N 0.000 claims description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 claims description 2
- 125000000524 functional group Chemical group 0.000 abstract description 19
- 238000001179 sorption measurement Methods 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract 1
- 238000000034 method Methods 0.000 description 34
- 239000000463 material Substances 0.000 description 21
- 238000006116 polymerization reaction Methods 0.000 description 19
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 239000012488 sample solution Substances 0.000 description 13
- 239000007788 liquid Substances 0.000 description 12
- 239000000523 sample Substances 0.000 description 11
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 10
- 125000003277 amino group Chemical group 0.000 description 10
- 239000011324 bead Substances 0.000 description 10
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 238000003756 stirring Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- 108090001090 Lectins Proteins 0.000 description 6
- 102000004856 Lectins Human genes 0.000 description 6
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 239000006185 dispersion Substances 0.000 description 6
- 239000002523 lectin Substances 0.000 description 6
- 239000011325 microbead Substances 0.000 description 6
- 230000009257 reactivity Effects 0.000 description 6
- 125000006850 spacer group Chemical group 0.000 description 6
- 239000003513 alkali Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-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 4
- 239000002253 acid Substances 0.000 description 4
- 238000011049 filling Methods 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 239000006087 Silane Coupling Agent Substances 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 125000003172 aldehyde group Chemical group 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 150000002772 monosaccharides Chemical class 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 239000003505 polymerization initiator Substances 0.000 description 3
- 230000000379 polymerizing effect Effects 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 2
- 239000004925 Acrylic resin Substances 0.000 description 2
- 229920000178 Acrylic resin Polymers 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 2
- 229930186217 Glycolipid Natural products 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 2
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 229920006037 cross link polymer Polymers 0.000 description 2
- 239000002612 dispersion medium Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 238000005886 esterification reaction Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine hydrate Chemical compound O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 description 2
- 150000007857 hydrazones Chemical class 0.000 description 2
- 229910010272 inorganic material Inorganic materials 0.000 description 2
- 239000011147 inorganic material Substances 0.000 description 2
- 230000008611 intercellular interaction Effects 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 150000002739 metals Chemical class 0.000 description 2
- 125000005641 methacryl group Chemical group 0.000 description 2
- 150000002771 monosaccharide derivatives Chemical class 0.000 description 2
- 239000011368 organic material Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229910052709 silver Inorganic materials 0.000 description 2
- 239000004332 silver Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- UWFRVQVNYNPBEF-UHFFFAOYSA-N 1-(2,4-dimethylphenyl)propan-1-one Chemical compound CCC(=O)C1=CC=C(C)C=C1C UWFRVQVNYNPBEF-UHFFFAOYSA-N 0.000 description 1
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 1
- 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 1
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 1
- ROVPTGZDLJKYQV-UHFFFAOYSA-N 3-[diethoxy(ethyl)silyl]propyl 2-methylprop-2-enoate Chemical compound CCO[Si](CC)(OCC)CCCOC(=O)C(C)=C ROVPTGZDLJKYQV-UHFFFAOYSA-N 0.000 description 1
- DOYKFSOCSXVQAN-UHFFFAOYSA-N 3-[diethoxy(methyl)silyl]propyl 2-methylprop-2-enoate Chemical compound CCO[Si](C)(OCC)CCCOC(=O)C(C)=C DOYKFSOCSXVQAN-UHFFFAOYSA-N 0.000 description 1
- ISHXIPDXNPZCMQ-UHFFFAOYSA-N 3-[diethyl(methoxy)silyl]propyl 2-methylprop-2-enoate Chemical compound CC[Si](CC)(OC)CCCOC(=O)C(C)=C ISHXIPDXNPZCMQ-UHFFFAOYSA-N 0.000 description 1
- LZMNXXQIQIHFGC-UHFFFAOYSA-N 3-[dimethoxy(methyl)silyl]propyl 2-methylprop-2-enoate Chemical compound CO[Si](C)(OC)CCCOC(=O)C(C)=C LZMNXXQIQIHFGC-UHFFFAOYSA-N 0.000 description 1
- WUTSHINWYBIRDG-UHFFFAOYSA-N 3-[ethoxy(diethyl)silyl]propyl 2-methylprop-2-enoate Chemical compound CCO[Si](CC)(CC)CCCOC(=O)C(C)=C WUTSHINWYBIRDG-UHFFFAOYSA-N 0.000 description 1
- JSOZORWBKQSQCJ-UHFFFAOYSA-N 3-[ethoxy(dimethyl)silyl]propyl 2-methylprop-2-enoate Chemical compound CCO[Si](C)(C)CCCOC(=O)C(C)=C JSOZORWBKQSQCJ-UHFFFAOYSA-N 0.000 description 1
- PRWUMCUQZMKBHZ-UHFFFAOYSA-N 3-[ethyl(dimethoxy)silyl]propyl 2-methylprop-2-enoate Chemical compound CC[Si](OC)(OC)CCCOC(=O)C(C)=C PRWUMCUQZMKBHZ-UHFFFAOYSA-N 0.000 description 1
- JBDMKOVTOUIKFI-UHFFFAOYSA-N 3-[methoxy(dimethyl)silyl]propyl 2-methylprop-2-enoate Chemical compound CO[Si](C)(C)CCCOC(=O)C(C)=C JBDMKOVTOUIKFI-UHFFFAOYSA-N 0.000 description 1
- URDOJQUSEUXVRP-UHFFFAOYSA-N 3-triethoxysilylpropyl 2-methylprop-2-enoate Chemical compound CCO[Si](OCC)(OCC)CCCOC(=O)C(C)=C URDOJQUSEUXVRP-UHFFFAOYSA-N 0.000 description 1
- XDLMVUHYZWKMMD-UHFFFAOYSA-N 3-trimethoxysilylpropyl 2-methylprop-2-enoate Chemical compound CO[Si](OC)(OC)CCCOC(=O)C(C)=C XDLMVUHYZWKMMD-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 229920001342 Bakelite® Polymers 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-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
- 239000007995 HEPES buffer Substances 0.000 description 1
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 1
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 108091093037 Peptide nucleic acid Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 235000004443 Ricinus communis Nutrition 0.000 description 1
- 240000000528 Ricinus communis Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 235000016127 added sugars Nutrition 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000000956 alloy Substances 0.000 description 1
- 229910045601 alloy Inorganic materials 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 238000007630 basic procedure Methods 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000008619 cell matrix interaction Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 239000007810 chemical reaction solvent Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000007720 emulsion polymerization reaction Methods 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229920000578 graft copolymer Polymers 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 229910052809 inorganic oxide Inorganic materials 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229910052741 iridium Inorganic materials 0.000 description 1
- GKOZUEZYRPOHIO-UHFFFAOYSA-N iridium atom Chemical compound [Ir] GKOZUEZYRPOHIO-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 150000002597 lactoses Chemical class 0.000 description 1
- 238000011173 large scale experimental method Methods 0.000 description 1
- 125000005647 linker group Chemical group 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000000696 magnetic material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 229910052762 osmium Inorganic materials 0.000 description 1
- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium atom Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 description 1
- 125000005429 oxyalkyl group Chemical group 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229910052703 rhodium Inorganic materials 0.000 description 1
- 239000010948 rhodium Substances 0.000 description 1
- MHOVAHRLVXNVSD-UHFFFAOYSA-N rhodium atom Chemical compound [Rh] MHOVAHRLVXNVSD-UHFFFAOYSA-N 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 229910052814 silicon oxide Inorganic materials 0.000 description 1
- 238000011172 small scale experimental method Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
Description
本発明は、生理活性物質を固定するための粒子、及び該粒子上に生理活性物質を固定した生理活性物質固定粒子に関する。
また本発明は、該生理活性物質固定化用粒子に捕捉基としての糖類を固定化した糖親和性物質捕捉粒子に関する。
The present invention relates to a particle for immobilizing a physiologically active substance, and a physiologically active substance-fixed particle having a physiologically active substance immobilized on the particle.
The present invention also relates to a glycophilic substance-trapping particle in which a saccharide as a capturing group is immobilized on the physiologically active substance-immobilizing particle.
一般的に生理活性物質固定化のための粒子（マイクロビーズ）は、カラムや容器に充填して、生理活性物質の反応、分離、精製等にしばしば用いられるとともに、近年では、マイクロチップの流路の一部に充填して、反応のための表面積を確保して、診断ツールに用いるなどして使用されている。 In general, particles (microbeads) for immobilizing a physiologically active substance are often used for reaction, separation, purification, etc. of a physiologically active substance by filling a column or a container. It is used by, for example, filling a part of the surface of the substrate to secure a surface area for reaction and using it for a diagnostic tool.
このような目的の場合、粒子表面に確実に生理活性物質を固定化することが必須であり、そのため、以前は樹脂に対する物理化学吸着による生理活性物質の固定が主流であったが、現在では、粒子表面に官能基を導入し、生理活性物質を化学結合により固定化する方法がよく採用されている。
これにより、生理活性物質の剥離を防ぐとともに、分子量や構造によらず確実に生理活性物質を固定化することが可能となった。
For such a purpose, it is essential to immobilize the physiologically active substance on the particle surface. Therefore, the fixation of the physiologically active substance by physicochemical adsorption to the resin has been the mainstream before. A method in which a functional group is introduced on the particle surface and a physiologically active substance is immobilized by chemical bonding is often employed.
As a result, peeling of the physiologically active substance can be prevented, and the physiologically active substance can be reliably fixed regardless of the molecular weight or structure.
特許文献１には、磁性体を内包する樹脂製マイクロビーズを乳化重合により作製し、ビーズ表面の官能基にエチレングリコールジグリシジルエーテルを反応させ、モノクローナル抗体を結合させる方法が記載されている。しかしながら、この方法では、マイクロビーズを重合により作製する工程が必要で、それは煩雑であるとともに、必要な粒径、粒度分布を制御することが困難である。 Patent Document 1 describes a method in which a resin-made microbead enclosing a magnetic material is prepared by emulsion polymerization, and a functional group on the surface of the bead is reacted with ethylene glycol diglycidyl ether to bind a monoclonal antibody. However, this method requires a step of producing microbeads by polymerization, which is complicated and difficult to control the required particle size and particle size distribution.
一方、特許文献２には、入手容易な汎用樹脂製マイクロビーズを基材として、その表面を加水分解し、表面に精製したカルボン酸に親水性のスペーサー分子を結合させ、さらにスペーサー分子の官能基に生理活性物質を結合し、親水性により非特異吸着を抑制する方法が述べられている。
この方法により得られるマイクロビーズは、加水分解によりカルボン酸を生じる樹脂を基材として用いる必要がある。また、当該マイクロビーズを、そのスペーサー分子に固定した生理活性物質に親和性の高い物質を捕捉するために使用する場面において、生体由来の夾雑物を多く含む検体を接触させると非特異吸着を抑制しきれない可能性が高い。
On the other hand, in Patent Document 2, a general-purpose resin microbead, which is readily available, is used as a base, its surface is hydrolyzed, and a hydrophilic spacer molecule is bonded to the purified carboxylic acid on the surface. Describes a method of binding a physiologically active substance to the substance and suppressing non-specific adsorption due to hydrophilicity.
The microbeads obtained by this method need to use, as a base material, a resin that generates carboxylic acid by hydrolysis. In addition, when the microbead is used to capture a substance having a high affinity for a physiologically active substance immobilized on the spacer molecule, nonspecific adsorption is suppressed by contacting a specimen containing a large amount of contaminants derived from a living body. There is a high possibility that it will not fit.
ところで、糖鎖及び糖鎖親和性の生体物質は、生理活性物質の重要な一群として知られている。
糖鎖とは、グルコース、ガラクトース、マンノース、フコース、キシロース、Ｎ−アセチルグルコサミン、Ｎ−アセチルガラクトサミン、シアル酸などの単糖及びそれらの誘導体がグリコシド結合によって鎖状に結合した分子の総称である。
糖鎖は、生体内の重要な構成成分の一つであり、生体内でタンパク質や脂質などに結合した複合糖質として存在することが多く、非常に多様性に富んでいる。
糖鎖は、天然に存在する生物が有する様々な機能に関与する物質であり、生体内において細胞間情報伝達、タンパク質の機能や相互作用の調整などに深く関わっていることが明らかになりつつある。
By the way, sugar chains and sugar chain affinity biological substances are known as an important group of physiologically active substances.
The sugar chain is a general term for molecules in which monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and derivatives thereof are linked in a chain form by glycosidic bonds.
A sugar chain is one of the important components in a living body, and often exists as a complex carbohydrate bound to a protein, lipid, or the like in the living body, and is extremely diverse.
Sugar chains are substances that are involved in various functions of naturally occurring organisms, and it is becoming clear that they are deeply involved in cell-to-cell information transmission, protein functions and interaction adjustments in vivo. .
例えば、糖鎖を有する生体高分子としては、細胞の安定化に寄与する植物細胞の細胞壁のプロテオグリカン、細胞の分化、増殖、接着、移動等に影響を与える糖脂質、及び細胞間相互作用や細胞認識に関与している糖タンパク質等が挙げられるが、これらの高分子の糖鎖が、互いに機能を代行、補助、増幅、調節、あるいは阻害しあいながら高度で精密な生体反応を制御する機構が次第に明らかにされつつある。さらに、このような糖鎖と細胞の分化増殖、細胞接着、免疫、及び細胞の癌化との関係が明確にされれば、この糖鎖工学と、医学、細胞工学、あるいは臓器工学とを密接に関連させて新たな展開を図ることが期待できる。 For example, biopolymers having sugar chains include plant cell wall proteoglycans that contribute to cell stabilization, glycolipids that affect cell differentiation, proliferation, adhesion, migration, etc., and cell-cell interactions and cells. Glycoproteins involved in recognition can be mentioned, but the mechanism by which these high-molecular sugar chains control advanced and precise biological reactions while acting, assisting, amplifying, regulating, or inhibiting each other's functions gradually. It is being revealed. Furthermore, if the relationship between such sugar chains and cell differentiation / proliferation, cell adhesion, immunity, and cell carcinogenesis is clarified, this sugar chain engineering and medicine, cell engineering, or organ engineering are closely related. We can expect new developments related to
その一例として、細胞表面の糖鎖や、糖鎖−レセプター間の相互作用異常による疾病の発生、あるいはエイズなどのウイルス感染における糖鎖の役割等に関する研究が活発化してきている。また、細胞−細胞間相互作用、細胞−マトリックス間相互作用における糖鎖の関与に関する研究も、生体反応を理解する上で重要になってきている（たとえば、非特許文献１）。 As an example, research on the sugar chains on the cell surface, the occurrence of diseases due to abnormal interactions between sugar chains and receptors, or the role of sugar chains in viral infections such as AIDS, has become active. Studies on the involvement of sugar chains in cell-cell interactions and cell-matrix interactions have also become important in understanding biological reactions (for example, Non-Patent Document 1).
糖鎖が関与する生体反応を解明するために、糖鎖解析技術が開発されてきた。例えば、特許文献３には、糖鎖のアルデヒド基と特異的に反応する官能基を担持してなる支持体、及び、当該支持体から構成され、糖鎖を捕捉する担体に用いられる重合体粒子が記載されている。特許文献３に記載された支持体及び重合体粒子は、糖鎖解析用バイオチップとして好適に利用されるものであり、これによって糖鎖の捕捉、分離、精製を簡単な工程で行うことができる。 In order to elucidate biological reactions involving sugar chains, sugar chain analysis techniques have been developed. For example, Patent Document 3 discloses a support that carries a functional group that specifically reacts with an aldehyde group of a sugar chain, and polymer particles that are composed of the support and are used as a carrier that captures the sugar chain. Is described. The support and polymer particles described in Patent Document 3 are suitably used as a biochip for sugar chain analysis, whereby sugar chains can be captured, separated, and purified by simple steps. .
上記糖鎖解析用バイオチップは糖鎖の探索、構造解析といった糖鎖側からのアプローチを容易にしたが、糖鎖が関与する生体反応を解明するためには、糖親和性物質の探索、構造解析といったリガンド側からのアプローチも重要である。
糖親和性物質の探索、構造解析を行うため、或いは、糖親和性物質の生産、供給を行うためには、糖親和性物質の捕捉、分離、精製を簡易な方法で行う必要がある。特に、大規模な実験や工業的なプロセスへ適用することを考慮すると、大量の試料から糖親和性物質を効率よく捕捉、分離、精製できることが望まれる。
The above-mentioned biochip for sugar chain analysis has facilitated the approach from the sugar chain side, such as sugar chain search and structure analysis. However, in order to elucidate biological reactions involving sugar chains, the search and structure of sugar affinity substances An approach from the ligand side such as analysis is also important.
In order to search for a glycophilic substance and perform structural analysis, or to produce and supply a glycophilic substance, it is necessary to capture, separate, and purify the glycophilic substance by a simple method. In particular, considering application to large-scale experiments and industrial processes, it is desirable that glycophilic substances can be efficiently captured, separated and purified from a large amount of samples.
本発明は上記実情に鑑み成し遂げられたものであり、生理活性物質を固定化することができる粒子（生理活性物質固定化用粒子）、特に好ましくは、生理活性物質を固定化することができ且つ非特異吸着が抑制された粒子を簡便に作製し、提供することを目的とする。
また、本発明の他の目的は、上記した生理活性物質固定化用粒子の上に生理活性物質を固定した粒子を提供することにある。
さらに本発明の他の目的は、上記した生理活性物質固定化用粒子の上に捕捉基としての糖類を固定した糖親和性物質捕捉粒子を提供することにある。
The present invention has been accomplished in view of the above circumstances, and is capable of immobilizing a physiologically active substance (particularly for immobilizing a physiologically active substance), particularly preferably a physiologically active substance and It is an object to easily produce and provide particles in which non-specific adsorption is suppressed.
Another object of the present invention is to provide particles in which a physiologically active substance is immobilized on the aforementioned particles for immobilizing a physiologically active substance.
Still another object of the present invention is to provide a sugar affinity substance-capturing particle in which a saccharide as a capturing group is fixed on the above-mentioned particle for immobilizing a physiologically active substance.
本発明の生理活性物質固定化用粒子は、生理活性物質を固定化する粒子であって、コア粒子表面に、カルボキシル基を側鎖に有するポリマーを固定してなることを特徴とする。 The particle for immobilizing a physiologically active substance of the present invention is a particle for immobilizing a physiologically active substance, and is characterized in that a polymer having a carboxyl group in a side chain is immobilized on the core particle surface.
また、本発明の生理活性物質固定粒子は、上記本発明の生理活性物質固定化用粒子の上に、前記カルボキシル基から誘導される結合を介して生理活性物質が固定化されていることを特徴とする。
生理活性物質固定化用粒子の上に固定される生理活性物質としては、小分子リガンド、抗体、抗原、糖類、ＤＮＡ、ＲＮＡ、アビジン又はアミノ化ビオチンが好適に利用される。
生理活性物質固定化用粒子の上に生理活性物質として糖類を固定した場合には、糖親和性物質捕捉粒子として用いることができる。
The physiologically active substance-immobilized particle of the present invention is characterized in that the physiologically active substance is immobilized on the physiologically active substance-immobilized particle of the present invention via a bond derived from the carboxyl group. And
As the physiologically active substance immobilized on the particles for immobilizing the physiologically active substance, small molecule ligands, antibodies, antigens, saccharides, DNA, RNA, avidin or aminated biotin are preferably used.
When a saccharide is immobilized as a physiologically active substance on the particle for immobilizing a physiologically active substance, it can be used as a sugar affinity substance capturing particle.
本発明の生理活性物質固定化用粒子は、コア粒子表面にカボキシル基を側鎖に有するポリマーを固定したものであるから、簡便な方法で作製することができる。
また、本発明の生理活性物質固定粒子は、上記本発明の生理活性物質固定化用粒子のカルボキシル基に糖類等の生理活性物質を導入したものであり、カルボキシル基を元々は有しないコア粒子の表面に、カルボキシル基の反応性を利用して様々な生理活性物質を固定化したものを提供することができる。
また、本発明の生理活性物質固定化用粒子、粒子状の捕捉材であるから、例えば当該捕捉材粒子をカラム充填したり或いは試料溶液中に分散させる等の簡便な方法で、大量の試料を短時間で処理することができる。
Since the particles for immobilizing a physiologically active substance of the present invention are those in which a polymer having a carboxyl group in the side chain is immobilized on the surface of the core particle, it can be produced by a simple method.
Further, the physiologically active substance-immobilized particles of the present invention are obtained by introducing a physiologically active substance such as a saccharide into the carboxyl group of the particles for immobilizing the physiologically active substance of the present invention, and are core particles that originally have no carboxyl group. It is possible to provide a surface on which various physiologically active substances are immobilized utilizing the reactivity of carboxyl groups.
In addition, since the physiologically active substance-immobilizing particles and the particulate trapping material of the present invention are used, a large amount of sample can be prepared by a simple method such as filling the trapping material particles in a column or dispersing in the sample solution. It can be processed in a short time.
本発明において生理活性物質固定化用粒子とは、生理活性物質をその表面に固定化するための処理を施した粒子のことを意味する。
また、生理活性物質固定粒子とは、生理活性物質固定化用粒子に生理活性物質を固定化した粒子のことを意味する。生理活性物質固定粒子には、生理活性物質固定化用粒子のカルボキシル基によって生理活性物質を固定し、当該生理活性物質を捕捉サイトとして機能させ、当該生理活性物質に対して親和性を有する他の生理活性物質を捕捉（「固定」と言い換えることができる）するための捕捉材としての粒子と、生理活性物質固定化用粒子それ自体を捕捉材として用い、カルボキシル基を捕捉サイトとして機能させ、カルボキシル基に対して親和性を有する生理活性物質を捕捉した結果物としての粒子とが包含される。どちらの場合であっても、粒子上に捕捉された生理活性物質は、試料から粒子ごと回収した後、粒子から分離、精製し又は粒子上に固定したまま利用することができる。
また、糖親和性物質捕捉粒子とは、粒子上に糖類を含む捕捉基を有し、糖類を捕捉サイトとして利用し、糖親和性物質を捕捉するための粒子である。
In the present invention, the particle for immobilizing a physiologically active substance means a particle subjected to a treatment for immobilizing a physiologically active substance on the surface thereof.
Further, the physiologically active substance-immobilized particle means a particle in which a physiologically active substance is immobilized on a physiologically active substance-immobilized particle. In the physiologically active substance-immobilized particles, the physiologically active substance is immobilized by the carboxyl group of the particles for immobilizing the physiologically active substance, the physiologically active substance functions as a capture site, and other substances having affinity for the physiologically active substance Using a particle as a capturing material for capturing a physiologically active substance (which can be referred to as “fixation”) and a particle for immobilizing a physiologically active substance itself as a capturing material, a carboxyl group functions as a capturing site, and carboxyl And particles as a result of capturing a physiologically active substance having an affinity for a group. In either case, the physiologically active substance captured on the particles can be collected from the sample together with the particles and then separated from the particles, purified, or used while being fixed on the particles.
The sugar affinity substance capturing particles are particles for capturing a sugar affinity substance having a capturing group containing saccharide on the particle and using the saccharide as a capturing site.
なお、本発明において生理活性物質とは、免疫学的反応、内分泌的作用、薬理学的作用等の典型的な生理活性を有する物質に限定されず、広義の意味において生体に対し何らかの影響を与える物質のことを意味し、生体に由来する物質に限定されることも無く、人工的に合成された物質であってもよい。
また本発明において糖類とは、糖鎖、糖鎖誘導体、当該糖鎖又は糖鎖誘導体を含有する物質、単糖、単糖誘導体、当該単糖又は単糖誘導体を含有する物質のなかから選ばれる物質を意味する。
In the present invention, the physiologically active substance is not limited to a substance having typical physiological activity such as immunological reaction, endocrine action, pharmacological action, etc., and has some influence on the living body in a broad sense. It means a substance, and is not limited to a substance derived from a living body, and may be an artificially synthesized substance.
In the present invention, the saccharide is selected from among sugar chains, sugar chain derivatives, substances containing the sugar chains or sugar chain derivatives, monosaccharides, monosaccharide derivatives, and substances containing the monosaccharide or monosaccharide derivatives. Means a substance.
＜生理活性物質固定化用粒子＞
本発明の生理活性物質固定化用粒子は、コア粒子表面に生理活性物質を固定化するための官能基としてカルボキシル基を有することを特徴とした粒子である。
ここで官能基をカルボキシル基に限定している理由は、生理活性物質を固定化する種々の方法が確立しており、簡便に固定化ができること、さらに、カルボキシル基が反応性を持ちながら比較的安定で、保存時の失活に注意を払わなくてもよいことにある。
<Particles for immobilizing physiologically active substances>
The particle for immobilizing a physiologically active substance of the present invention is a particle characterized by having a carboxyl group as a functional group for immobilizing a physiologically active substance on the core particle surface.
The reason why the functional group is limited to the carboxyl group is that various methods for immobilizing physiologically active substances have been established, and can be easily immobilized, and the carboxyl group is relatively reactive while being reactive. It is stable and it is not necessary to pay attention to deactivation during storage.
コア粒子は、生理活性物質固定化用粒子の基材となる粒子であり、その材質は特に限定されるものではなく、有機材料、無機材料を問わず用いることができる。
有機材料としては、例えば、ポリメタクリル酸メチル（ＰＭＭＡ）等のアクリル系モノマーを主体とするアクリル系架橋重合体；ポリアクリルアミドゲル（商品名：Ｂｉｏ−Ｇｅｌ Ｐ、バイオラッド社）、ポリスチレン、エチレン−無水マレイン酸共重合物等のアクリル系樹脂以外のエチレン性二重結合含有モノマーを主体とする架橋重合体等のプラスチック樹脂が挙げられる。その他にもアフィニティクロマトグラフィーの担体として用いられる多孔性のアガロース粒子（商品名：Ｓｅｐｈａｒｏｓｅ）やデキストラン粒子（商品名：Ｓｅｐｈａｄｅｘ）等が挙げられる。
ここで、アクリル系モノマーを主体とするアクリル系樹脂とは、アクリル系モノマーの共重合割合が好ましくは５０モル％以上、さらに好ましくは７０モル％以上である重合体を意味する。
また、無機材料としては、ガラス；シリカ（酸化ケイ素）；金、銀、白金、パラジウム、イリジウム、ロジウム、オスミウム、鉄、銅、コバルト等の金属；これら金属の合金や無機酸化物等が挙げられる。
The core particle is a particle that becomes a base material for the particle for immobilizing a physiologically active substance, and the material thereof is not particularly limited, and any organic material or inorganic material can be used.
Examples of organic materials include acrylic cross-linked polymers mainly composed of acrylic monomers such as polymethyl methacrylate (PMMA); polyacrylamide gel (trade name: Bio-Gel P, Bio-Rad), polystyrene, ethylene- Examples thereof include a plastic resin such as a crosslinked polymer mainly composed of an ethylenic double bond-containing monomer other than an acrylic resin such as a maleic anhydride copolymer. Other examples include porous agarose particles (trade name: Sepharose) and dextran particles (trade name: Sephadex) used as carriers for affinity chromatography.
Here, the acrylic resin mainly composed of an acrylic monomer means a polymer having an acrylic monomer copolymerization ratio of preferably 50 mol% or more, more preferably 70 mol% or more.
Examples of the inorganic material include glass; silica (silicon oxide); metals such as gold, silver, platinum, palladium, iridium, rhodium, osmium, iron, copper, and cobalt; alloys of these metals, inorganic oxides, and the like. .
コア粒子としては、架橋プラスチック樹脂、ガラス、シリカが高い強度を有するので好ましい。これらのなかでも、捕捉した物質の分離精製プロセス段階において耐アルカリ性、耐酸性に強い点で、架橋プラスチック樹脂が好ましい。
架橋プラスチック樹脂のなかでも、架橋ポリメタクリル酸メチル（架橋ＰＭＭＡ）の粒子を使用することが特に好ましい。その理由としては、粒子の強度、耐アルカリ性に優れていることに加えて、（１）水に溶解せず、且つ、比重が１．２程度であり分散媒の比重調整により分散状態を維持させやすいため、水に分散させた状態から遠心分離で回収しやすいこと、（２）粒子の表面に捕捉基と親水基を付与するための重合反応を行う際にＰＭＭＡ粒子の場合には粒子の溶解を考慮せず任意の溶媒を選択できること、（３）粒子上に捕捉した生理活性物質を粒子から分離するために化学結合を切断する時に、耐酸性及び耐アルカリ性に優れているので粒子が溶解しない、（４）コストも安いこと等が理由として挙げられる。
As the core particles, a crosslinked plastic resin, glass, and silica are preferable because they have high strength. Among these, a cross-linked plastic resin is preferable in terms of resistance to alkali resistance and acid resistance in the process of separating and purifying the captured substance.
Among the crosslinked plastic resins, it is particularly preferable to use particles of crosslinked polymethyl methacrylate (crosslinked PMMA). The reason is that in addition to excellent particle strength and alkali resistance, (1) it does not dissolve in water and the specific gravity is about 1.2, and the dispersion state is maintained by adjusting the specific gravity of the dispersion medium. It is easy to collect by centrifugation from a state dispersed in water, and (2) dissolution of particles in the case of PMMA particles when carrying out a polymerization reaction to give capture groups and hydrophilic groups to the surface of the particles (3) When the chemical bond is cleaved to separate the physiologically active substance trapped on the particles from the particles, the particles do not dissolve because they are excellent in acid resistance and alkali resistance. (4) The cost is also low.
コア粒子の粒経は、医療用途や小規模実験のような少量の試料を取り扱うレベルから、工業プロセスのような大量の試料を取り扱うレベルまで、実施規模に適宜調節されるが、大量の試料を取り扱う場合には通常、平均粒子径１〜１００μｍのコア粒子が好適に使用され、さらに好適には１〜５０μｍであり、最も好適には１〜２０μｍの粒子を用いる。なお、当該コア粒子の平均粒子径とは、数平均粒子径である。 The particle size of the core particles is adjusted as appropriate from the level of handling a small amount of sample such as medical use or small-scale experiments to the level of handling a large amount of sample such as an industrial process. In the case of handling, core particles having an average particle diameter of 1 to 100 μm are usually preferably used, more preferably 1 to 50 μm, and most preferably 1 to 20 μm. The average particle size of the core particles is the number average particle size.
コア粒子の表面にカルボキシル基を付与する方法としては、コア粒子の表面に、カルボキシル基を側鎖に有するポリマーを固定する方法が好ましい。コア粒子表面にカルボキシル基を側鎖に有するポリマーを固定してなる粒子は、カルボキシル基を元々は有しないコア粒子を用いて簡便な方法で作製することができるから、コア粒子を幅広い材質から選択することができ、方法も簡便であるため、生産性及びコスト面で優れている。
カルボキシル基を側鎖に有するポリマーを固定するには、カルボキシル基を側鎖に有するポリマーを含有する溶液中に、コア粒子を分散させて表面に付着させる方法や、コア粒子表面において側鎖にカルボキシル基を有するモノマーを重合させる方法がある。
このうち、コア粒子表面において側鎖にカルボキシル基を有するモノマーを重合させる方法は、粒子表面でカルボキシル基を側鎖に有するポリマーをその場重合するから、特に簡便なプロセスであり、好ましい。
なお、本発明において「モノマー」とは、ポリマー鎖中において繰り返し単位となる比較的低分子の重合性化合物を意味し、厳密な意味でのモノマーだけに限られず、数分子のモノマーが結合したオリゴマー（それ自体が繰り返し単位を含む）であって重合反応性を有し、重合させたときにオリゴマー分子が繰り返し単位となるものも包含される。
As a method for imparting a carboxyl group to the surface of the core particle, a method of fixing a polymer having a carboxyl group in the side chain to the surface of the core particle is preferable. Particles made by fixing polymers with carboxyl groups in the side chain on the core particle surface can be made by a simple method using core particles that do not originally have carboxyl groups. Since the method can be simple, the productivity and cost are excellent.
In order to fix the polymer having a carboxyl group in the side chain, the core particle is dispersed in a solution containing the polymer having a carboxyl group in the side chain and adhered to the surface, or the side chain on the core particle surface is carboxylated. There is a method of polymerizing a monomer having a group.
Among these, the method of polymerizing a monomer having a carboxyl group in the side chain on the core particle surface is a particularly simple process because a polymer having a carboxyl group in the side chain on the particle surface is polymerized in situ, and is preferable.
In the present invention, “monomer” means a relatively low molecular weight polymerizable compound that becomes a repeating unit in a polymer chain, and is not limited to a monomer in a strict sense, but an oligomer in which several molecules of monomers are bonded. Also included are those (which themselves contain repeating units) that have polymerization reactivity and that when oligomerized, the oligomer molecules become repeating units.
また、上記重合反応の際に、側鎖にカルボキシル基を有するモノマーと側鎖に親水基を有するモノマーを粒子表面に共存させて共重合させることにより、粒子表面にカルボキシル基を有し生理活性物質を固定化することができ、且つそれ以外の物質、例えばタンパク質等の非特異吸着を抑制することができる粒子を得ることが可能となる。
なお、上記重合反応において、さらに他のモノマーを共重合させてもよい。
In the polymerization reaction, a monomer having a carboxyl group in the side chain and a monomer having a hydrophilic group in the side chain are allowed to coexist on the surface of the particle to copolymerize the physiologically active substance having a carboxyl group on the particle surface. It is possible to obtain particles capable of immobilizing and inhibiting nonspecific adsorption of other substances such as proteins.
In the above polymerization reaction, another monomer may be further copolymerized.
先ず、側鎖にカルボキシル基を有するモノマーについて述べる。このモノマーは、側鎖に官能基としてカルボキシル基を有するモノマーであれば特に限定するものではないが、通常は（メタ）アクリル系モノマー等のエチレン性不飽和結合を有するモノマーを用いる。
主鎖にカルボキシル基が直結している場合（例えばアクリル酸を用いた場合）は、重合後、カルボキシル基に反応させる生理活性物質が高分子量の物質である場合には、その立体障害によりカルボキシル基との反応が進まず、固定化量が理論量に達しないことが考えられる。この問題を解決するためには、カルボキシル基がスペーサーを介して、モノマーの重合性基から離れた位置に存在することが好ましい。
従って、側鎖の末端にカルボキシル基が位置することがモノマー形態として望ましい。スペーサーとなる側鎖の長さは、炭素原子１個以上に相当する長さであることが好ましい。
First, a monomer having a carboxyl group in the side chain will be described. Although this monomer will not be specifically limited if it is a monomer which has a carboxyl group as a functional group in a side chain, Usually, the monomer which has ethylenically unsaturated bonds, such as a (meth) acrylic-type monomer, is used.
When the carboxyl group is directly connected to the main chain (for example, when acrylic acid is used), if the physiologically active substance to be reacted with the carboxyl group after polymerization is a high molecular weight substance, the carboxyl group is caused by its steric hindrance. It is conceivable that the immobilized amount does not reach the theoretical amount. In order to solve this problem, the carboxyl group is preferably present at a position away from the polymerizable group of the monomer via a spacer.
Accordingly, it is desirable that the carboxyl group is located at the end of the side chain as a monomer form. The length of the side chain serving as the spacer is preferably a length corresponding to one or more carbon atoms.
スペーサーは親水性のものが好ましい。中でも、アルキレングリコール残基又はポリアルキレングリコール残基を含むものが好ましく、特に、エチレングリコール残基を含むものが好ましい。エチレングリコール残基の繰り返し数は１以上５０以下が好ましく、さらに好ましくは１以上３０以下であり、最も好ましくは１以上１０以下である。なお、繰り返し数が１の場合はポリアルキレングリコール残基とは言わず、アルキレングリコール残基またはオキシアルキル基と表記される。 The spacer is preferably hydrophilic. Among these, those containing an alkylene glycol residue or a polyalkylene glycol residue are preferred, and those containing an ethylene glycol residue are particularly preferred. The number of repeating ethylene glycol residues is preferably 1 or more and 50 or less, more preferably 1 or more and 30 or less, and most preferably 1 or more and 10 or less. When the number of repeats is 1, it is not called a polyalkylene glycol residue, but is expressed as an alkylene glycol residue or an oxyalkyl group.
具体的には、スペーサーを介して側鎖末端にカルボキシル基を有するモノマーとしては、以下の式１で表される、２−メタクリロイルオキシエチルサクシネートを用いることが、重合反応性、入手のし易さ、重合後の官能基の反応性の点で最も好ましい。 Specifically, as a monomer having a carboxyl group at the end of the side chain through a spacer, it is possible to use 2-methacryloyloxyethyl succinate represented by the following formula 1 for polymerization reactivity and easy availability. The most preferred is the reactivity of the functional group after polymerization.
次に、側鎖に親水基を有するモノマーについて述べる。これは、粒子表面に親水基を導入することを目的としており、当該親水基の存在により、粒子表面における目的物質以外の非特異吸着を抑制することが可能となる。
このモノマーは、側鎖に親水基を有するモノマーであれば特に限定するものではないが、通常は（メタ）アクリル系モノマー等のエチレン性不飽和結合を有するモノマーを用いる。
側鎖に用いる親水基は、特に限定するものでないが、ポリアルキレングリコール基や、ホスホリルコリン基を好適に用いることができ、特に、ポリエチレングリコール基、ホスホリルコリン基を用いることが好ましい。
Next, a monomer having a hydrophilic group in the side chain will be described. This is intended to introduce a hydrophilic group to the particle surface, and the presence of the hydrophilic group makes it possible to suppress nonspecific adsorption other than the target substance on the particle surface.
Although this monomer will not be specifically limited if it is a monomer which has a hydrophilic group in a side chain, Usually, the monomer which has ethylenically unsaturated bonds, such as a (meth) acrylic-type monomer, is used.
The hydrophilic group used for the side chain is not particularly limited, but a polyalkylene glycol group or a phosphorylcholine group can be suitably used, and a polyethylene glycol group or a phosphorylcholine group is particularly preferable.
側鎖にポリエチレングリコール基を有するモノマーとしては、以下の式２で表されるメトシキポリエチレングリコール（メタ）アクリレートを用いることが、重合反応性、入手のし易さ、重合後の非特異吸着特性の点で最も好ましい。式２中の、ポリエチレングリコール残基のｎ数は、１以上５０以下の整数であり、好ましくは１以上３０以下、最も好ましくは５以上１０以下である。 As a monomer having a polyethylene glycol group in the side chain, it is possible to use methoxypolyethylene glycol (meth) acrylate represented by the following formula 2, polymerization reactivity, availability, non-specific adsorption characteristics after polymerization Is most preferable. The n number of the polyethylene glycol residue in Formula 2 is an integer of 1 to 50, preferably 1 to 30 and most preferably 5 to 10.
（式２中、ｎは１以上、５０以下の整数） (In Formula 2, n is an integer of 1 to 50)
側鎖の親水基にホスホリルコリン基を有するモノマーとしては、以下の式３で表される２−メタクリロイルオキシエチルホスホリルコリンを用いることが好適である。 As a monomer having a phosphorylcholine group in the hydrophilic group of the side chain, 2-methacryloyloxyethyl phosphorylcholine represented by the following formula 3 is preferably used.
コア粒子表面で、側鎖にカルボキシル基を有するモノマーを重合、又は、側鎖にカルボキシル基を有するモノマーと側鎖に親水基を有するモノマーとを共重合する方法としては、特に限定されるものではないが、コア粒子を、側鎖にカルボキシル基を有するモノマー、重合開始剤、及び必要に応じて他のモノマーを含む溶液中に側鎖にカルボキシル基を有するモノマー、重合開始剤、及び必要に応じて他のモノマーを含む溶液中に分散させ、撹拌しながら重合を行う方法が簡便で、かつ生成したポリマーによってビーズ表面をムラなく被覆できるので好ましい。
特に、コア粒子表面に重合性官能基を有する場合には、上記重合反応により生成したポリマーが粒子表面に極めて強固に固定される。
The method of polymerizing a monomer having a carboxyl group in the side chain on the surface of the core particle, or copolymerizing a monomer having a carboxyl group in the side chain and a monomer having a hydrophilic group in the side chain is not particularly limited. Although there is no core particle, a monomer having a carboxyl group in the side chain, a polymerization initiator, and a monomer having a carboxyl group in the side chain in a solution containing another monomer if necessary, a polymerization initiator, and if necessary A method of dispersing in a solution containing other monomers and performing polymerization while stirring is preferable because the surface of the beads can be uniformly coated with the produced polymer.
In particular, when the surface of the core particle has a polymerizable functional group, the polymer generated by the polymerization reaction is extremely firmly fixed to the particle surface.
コア粒子表面に重合性官能基を付与する方法としては、コアとなるビーズに応じて適切に選択できる。
例えば、コア粒子が架橋ＰＭＭＡなど有機高分子化合物からなる場合、メタクリル基が導入されたビーズが市販されており、これを用いてもよい。架橋ＰＭＭＡを主体とするコア粒子を用いる場合には、当該粒子表面を酸・アルカリで加水分解することで、表面にカルボキシル基を生成させ、この生成させたカルボキシル基に対し、重合性官能基を併せ持つシランカップリング剤をさらに反応させる方法が、簡便で好ましい。
シランカップリング剤の例としては、３−メタクリロキシプロピルジメチルメトキシシラン、３−メタクリロキシプロピルジメチルエトキシシラン、３−メタクリロキシプロピルメチルジメトキシシラン、３−メタクリロキシプロピルメチルジエトキシシラン、３−メタクリロキシプロピルトリメトキシシラン、３−メタクリロキシプロピルトリエトキシシラン、３−メタクリロキシプロピルジエチルメトキシシラン、３−メタクリロキシプロピルジエチルエトキシシラン、３−メタクリロキシプロピルエチルジメトキシシラン、３−メタクリロキシプロピルエチルジエトキシシランなどが挙げられる。
一方、コア粒子がガラスやシリカからなる場合は、当該粒子表面に存在する水酸基等の官能基と、重合性官能基を持つカップリング剤、好ましくはシランカップリング剤を反応させることによって、重合性官能基を導入することができる。
A method for imparting a polymerizable functional group to the surface of the core particle can be appropriately selected depending on the core bead.
For example, when the core particle is made of an organic polymer compound such as cross-linked PMMA, beads introduced with a methacryl group are commercially available and may be used. When using core particles mainly composed of cross-linked PMMA, the surface of the particles is hydrolyzed with acid / alkali to generate a carboxyl group on the surface, and a polymerizable functional group is added to the generated carboxyl group. A method of further reacting the silane coupling agent having the combination is simple and preferable.
Examples of silane coupling agents include 3-methacryloxypropyldimethylmethoxysilane, 3-methacryloxypropyldimethylethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxy Propyltrimethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropyldiethylmethoxysilane, 3-methacryloxypropyldiethylethoxysilane, 3-methacryloxypropylethyldimethoxysilane, 3-methacryloxypropylethyldiethoxysilane Etc.
On the other hand, when the core particle is made of glass or silica, it is polymerizable by reacting a functional group such as a hydroxyl group present on the particle surface with a coupling agent having a polymerizable functional group, preferably a silane coupling agent. Functional groups can be introduced.
側鎖にカルボキシル基を有するモノマーと側鎖に親水基を有するモノマーとを共存させて共重合する場合、側鎖にカルボキシル基を有するモノマーと側鎖に親水基を有するモノマーの混合は、重量比（側鎖にカルボキシル基を有するモノマー：側鎖に親水基を有するモノマー）で１００：０〜１：９９まで可能であるが、カルボキシル基を用いた生理活性物質の固定化量の確保するためには、１００：０〜５：９５が好ましく、加えて非特異吸着の阻止効果を良好にするために、６０：４０〜１０：９０であることが最も好ましい。 When copolymerizing a monomer having a carboxyl group in the side chain and a monomer having a hydrophilic group in the side chain, the mixture of the monomer having a carboxyl group in the side chain and the monomer having a hydrophilic group in the side chain is a weight ratio. (Monomer having a carboxyl group in the side chain: monomer having a hydrophilic group in the side chain) is possible from 100: 0 to 1:99, but in order to ensure the amount of immobilization of the physiologically active substance using the carboxyl group Is preferably 100: 0 to 5:95, and most preferably 60:40 to 10:90 in order to improve the non-specific adsorption inhibiting effect.
重合反応に用いる溶媒は、モノマーおよび重合開始剤が溶解し、かつ粒子が溶解しないものであればよく、例えば、メタノール、エタノール、２−ブタノン（メチルエチルケトン）、ｔ−ブチルアルコール、ベンゼン、トルエン、テトラヒドロフラン、ジオキサン、ジクロロメタン、クロロホルム、ジメチルホルムアミド等を好適にも用いることができる。これらの溶媒は、単独または２種以上の組み合わせで用いられる。重合反応は、窒素またはアルゴンガス雰囲気下で行ってもよく、溶液の撹拌手段は、特に限定するものではないが、モーターに撹拌羽を連結した物を反応溶媒中に入れ、撹拌羽を回転させて撹拌する方法が、撹拌条件を一定にするのに好適である。また、小スケールで合成を行う場合は、撹拌子による撹拌も可能である。 The solvent used in the polymerization reaction may be any solvent that dissolves the monomer and the polymerization initiator and does not dissolve the particles. For example, methanol, ethanol, 2-butanone (methyl ethyl ketone), t-butyl alcohol, benzene, toluene, tetrahydrofuran , Dioxane, dichloromethane, chloroform, dimethylformamide and the like can be suitably used. These solvents are used alone or in combination of two or more. The polymerization reaction may be carried out in an atmosphere of nitrogen or argon gas, and the means for stirring the solution is not particularly limited. However, a product in which a stirring blade is connected to a motor is placed in a reaction solvent, and the stirring blade is rotated. The stirring method is suitable for making the stirring conditions constant. In addition, when the synthesis is performed on a small scale, stirring with a stirring bar is also possible.
コア粒子表面で合成されたポリマーは、コア粒子表面に強固且つ安定的に固定化されているため、洗浄によって除去されることはない。特に、コア粒子の表面に重合性官能基を有する場合には、モノマーの一部が粒子表面にグラフト状に付加重合するので、粒子表面で生成したポリマーが、このように粒子表面に化学結合したグラフト状ポリマーと絡み合って、より強固に固定されると考えられる。
したがって、たとえモノマーが残った状態で重合を終了させても、粒子表面に十分ポリマーが生成していれば、未反応モノマーを除去すればよく、重合開始後４時間以上経過していれば、任意の時間で重合を終了することができる。
重合終了後は、粒子をフィルターでろ過、もしくは遠心分離で粒子を回収した後、反応に用いた溶媒で、３〜６回洗浄することで、表面にカルボキシル基を有する粒子を得ることができる。
カルボキシル基は、比較的安定であるため、そのまま、乾燥させて保存することが可能だが、好ましくは、低温で保存することが望ましい。ただし、乾燥すると粒子の凝集が起こり易くなるので、再分散が難しい粒子の場合は、水などに分散させて低温で保存することも可能である。
Since the polymer synthesized on the surface of the core particle is firmly and stably immobilized on the surface of the core particle, it is not removed by washing. In particular, when the surface of the core particle has a polymerizable functional group, a part of the monomer is addition-polymerized in a graft form on the particle surface, so that the polymer formed on the particle surface is chemically bonded to the particle surface in this way. It is thought that it is entangled with the graft polymer and fixed more firmly.
Therefore, even if the polymerization is terminated with the monomer remaining, as long as the polymer is sufficiently formed on the particle surface, the unreacted monomer may be removed. The polymerization can be completed within
After completion of the polymerization, the particles are filtered through a filter or collected by centrifugation, and then washed 3 to 6 times with the solvent used in the reaction, whereby particles having a carboxyl group on the surface can be obtained.
Since the carboxyl group is relatively stable, it can be stored as it is, but it is preferable to store it at a low temperature. However, since the particles tend to aggregate when dried, the particles that are difficult to redisperse can be dispersed in water or the like and stored at a low temperature.
前記のように作製した生理活性物質固定化用粒子は、コア粒子表面に付与されたカルボキシル基の反応性を利用して様々な生理活性物質を固定化することができる。得られた生理活性物質固定化粒子は、コア粒子表面にカルボキシル基から誘導される結合を介して生理活性物質が固定されており、当該固定した生理活性物質に親和性を有する別の生理活性物質を捕捉するための捕捉材として用いることができる。捕捉サイトとするために固定される生理活性物質としては、例えば、小分子リガンド、抗体、抗原、糖類、ＤＮＡ、ＲＮＡ、アビジン、アミノ化ビオチン等が挙げられる。小分子リガンドとは、特定のタンパク質やレクチンと特異的に結合する比較的低分子の化合物である。
なお、コア粒子表面にカルボキシル基を有する生理活性物質固定化用粒子も、カルボキシル基に親和性を有する生理活性物質を捕捉するための捕捉材として、そのまま用いることができる。
The physiologically active substance immobilizing particles produced as described above can immobilize various physiologically active substances using the reactivity of the carboxyl group imparted to the core particle surface. In the obtained bioactive substance-immobilized particles, the bioactive substance is immobilized on the surface of the core particle through a bond derived from a carboxyl group, and another bioactive substance having affinity for the immobilized bioactive substance It can be used as a capturing material for capturing. Examples of the physiologically active substance that is immobilized to form a capture site include small molecule ligands, antibodies, antigens, saccharides, DNA, RNA, avidin, and aminated biotin. A small molecule ligand is a relatively small molecule compound that specifically binds to a specific protein or lectin.
The particle for immobilizing a physiologically active substance having a carboxyl group on the surface of the core particle can also be used as it is as a capturing material for capturing a physiologically active substance having affinity for the carboxyl group.
コア粒子表面のカルボキシル基を用いて生理活性物質を固定化するためには、カルボキシル基を活性化させた後、生理活性物質に含まれるアミノ基と反応させることが、最も簡便で、かつ確実な方法である。
カルボキシル基を活性化する方法は、特に限定するものではないが、カルボジイミド化合物を用いて活性エステル化する方法が好ましい。
In order to immobilize a physiologically active substance using the carboxyl group on the surface of the core particle, it is the simplest and most reliable method to react with an amino group contained in the physiologically active substance after activating the carboxyl group. Is the method.
The method for activating the carboxyl group is not particularly limited, but a method for active esterification using a carbodiimide compound is preferred.
カルボジイミド化合物としては、例えば１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド塩酸塩、ジシクロヘキシルカルボジイミド、ジイソプロピルカルボジイミドを使用することができる。特に、水溶性カルボジイミド（ＷＳＣ）と略される１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド塩酸塩は、水溶液中でカルボキシル基を活性化することが可能であり、基本的に水溶性である生理活性物質の固定化には最適である。 As the carbodiimide compound, for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, dicyclohexylcarbodiimide, and diisopropylcarbodiimide can be used. In particular, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, abbreviated as water-soluble carbodiimide (WSC), can activate a carboxyl group in an aqueous solution and is basically water-soluble. It is optimal for immobilizing a physiologically active substance.
具体的は、得られた生理活性物質固定化用粒子を５ｍｇ／ｍＬ〜飽和濃度、好ましくは１００〜２００ｍｇ／ｍＬのＷＳＣ水溶液（ｐＨ５〜６．５）に分散させ、３７℃で１〜３時間反応させる。３時間以上の反応は、生じた活性基が水により加水分解されるので、反応条件としては不適当である。
次に、前述の活性化した粒子を水溶液で１〜数回、水溶液を変えて洗浄し、生理活性物質を溶解、分散した溶液中に加え、０〜３７℃で、１〜２４時間反応させて、生理活性物質を固定化する。
Specifically, the obtained particles for immobilizing a physiologically active substance are dispersed in a WSC aqueous solution (pH 5 to 6.5) having a concentration of 5 mg / mL to a saturated concentration, preferably 100 to 200 mg / mL, and the temperature is 37 ° C. for 1 to 3 hours. React. A reaction of 3 hours or longer is not suitable as a reaction condition because the generated active group is hydrolyzed by water.
Next, the aforementioned activated particles are washed with an aqueous solution one to several times while changing the aqueous solution, added to a solution in which a physiologically active substance is dissolved and dispersed, and reacted at 0 to 37 ° C. for 1 to 24 hours. Immobilize physiologically active substances.
抗体やアミノ化糖のようにアミノ基を元々有する生理活性物質は、そのまま粒子上のカルボキシル基と反応させて固定できるが、アミノ基を元々は有しない生理活性物質を固定したい場合には、粒子上のカルボキシル基と反応できるアミノ基などの官能基、及び、生理活性物質が有する官能基と反応できる官能基を併せ持つ化合物を、リンカーとして用いる。
例えば、アミノ基を有しない糖を固定して糖親和性物質捕捉材を得たい場合には、ヒドラジンを用いることができる。ヒドラジンを用いる場合、先ずカルボキシル基を有する粒子をヒドラジド化した後で糖類を反応させても良いし、先ず糖類をヒドラジド化した後でカルボキシル基を有する粒子を反応させても良い。
A physiologically active substance originally having an amino group such as an antibody or an aminated sugar can be immobilized by reacting with the carboxyl group on the particle as it is, but if a physiologically active substance originally having no amino group is to be immobilized, the particle A compound having both a functional group such as an amino group capable of reacting with the above carboxyl group and a functional group capable of reacting with a functional group possessed by a physiologically active substance is used as a linker.
For example, hydrazine can be used when a sugar-affinity substance capturing material is to be obtained by immobilizing a sugar having no amino group. When hydrazine is used, the saccharide may be reacted first after hydrazide conversion of the carboxyl group-containing particles, or the carboxyl group-containing particles may be reacted first after saccharide formation.
前者の手順においては、先ず、ヒドラジンが有する２つのアミノ基のうち一方が粒子上のカルボキシル基と反応して、粒子表面にアミノ基が導入される。次に、粒子表面のアミノ基と糖類が有するアルデヒド基が反応して、糖類の残基が粒子上に導入される。 In the former procedure, first, one of the two amino groups of hydrazine reacts with a carboxyl group on the particle, and an amino group is introduced onto the particle surface. Next, the amino group on the particle surface reacts with the aldehyde group of the saccharide to introduce a saccharide residue onto the particle.
後者の手順においては、先ず、ヒドラジンが有する２つのアミノ基のうち一方が糖類のアルデヒド基と反応して、アミノ化体である糖ヒドラゾンとなる。次に、糖ヒドラゾンのアミノ基と粒子上のカルボキシル基が反応して、糖類の残基が粒子上に導入される。 In the latter procedure, first, one of the two amino groups of hydrazine reacts with the aldehyde group of the saccharide to form a sugar hydrazone that is an aminated product. Next, the amino group of the sugar hydrazone reacts with the carboxyl group on the particle to introduce a sugar residue onto the particle.
従って、上記ヒドラジンを用いる方法で得られた糖親和性物質捕捉粒子は、糖類が固定化されている部分の末端が下記結合１の構造を有している。
結合１：−Ｃ（＝Ｏ）−ＮＲ−Ｎ＝（糖類）
（上記構造において、Ｒは水素原子又はＮに結合可能な水素原子以外の基である。また、これらの構造に含まれるＮは４級アンモニウム化していても良い。）
Therefore, the sugar affinity substance-trapping particles obtained by the method using hydrazine have the structure of the following bond 1 at the end of the portion where the saccharide is immobilized.
Bond 1: —C (═O) —NR—N = (saccharide)
(In the above structure, R is a hydrogen atom or a group other than a hydrogen atom that can be bonded to N. Further, N contained in these structures may be quaternary ammoniumated.)
具体的な反応条件の一例を挙げれば、ヒドラジド化した粒子と糖類とを２％酢酸を含むアセトニトリル中で加熱する方法がある。２％酢酸を含むアセトニトリルの代わりに、脱水反応を促進する他の適切な溶媒を用いても良い。反応液中の糖の濃度は、通常０．０１ｍｍｏｌ／Ｌ〜１００ｍｍｏｌ／Ｌとし、好ましくは０．１ｍｍｏｌ／Ｌ〜５０ｍｍｏｌ／Ｌ、最も好ましくは０．１ｍｍｏｌ／Ｌ〜１０ｍｍｏｌ／Ｌとする。
このような反応混合液は、乾固するまで加熱しても良い。乾固するまで加熱することによって、粒子の単位表面積当りの糖固定量を大きくすることができる。
As an example of specific reaction conditions, there is a method of heating hydrazide particles and saccharides in acetonitrile containing 2% acetic acid. Instead of acetonitrile containing 2% acetic acid, other suitable solvents that promote the dehydration reaction may be used. The sugar concentration in the reaction solution is usually 0.01 mmol / L to 100 mmol / L, preferably 0.1 mmol / L to 50 mmol / L, and most preferably 0.1 mmol / L to 10 mmol / L.
Such a reaction mixture may be heated to dryness. By heating to dryness, the amount of sugar fixed per unit surface area of the particles can be increased.
前記のようにして得られた生理活性物質固定粒子は、そのまま、すぐに捕捉材として使用してもよいし、冷蔵、または冷凍保存して用いてどちらでもよい。冷凍保存の場合は、好ましくは凍結乾燥することで、生理活性物質を安定に保存することが可能である。ただし、乾燥により生理活性物質が劣化する場合や乾燥すると再分散が難しい粒子の場合は、ＰＢＳ、トリス塩酸バッファ、ＨＥＰＥＳバッファなど各種緩衝液に分散させて低温で保存することも可能である。 The physiologically active substance-immobilized particles obtained as described above may be used immediately as a capturing material, or may be used after refrigerated or frozen. In the case of frozen storage, the physiologically active substance can be stably stored, preferably by freeze-drying. However, when the physiologically active substance is deteriorated by drying or particles that are difficult to redisperse after drying, it can be dispersed in various buffer solutions such as PBS, Tris-HCl buffer, HEPES buffer, and stored at a low temperature.
本発明の生理活性物質固定化用粒子または生理活性物質固定粒子は、捕捉材粒子として用いられる。当該捕捉材粒子を、ターゲットとする生理活性物質を含む試料溶液と接触させた後、粒子上に捕捉されたターゲットの生理活性物質を、粒子ごと回収する。
試料溶液としては、血液，血清，組織の破砕物あるいは抽出物，細胞の破砕物あるいは抽出物，タンパク質，核酸，酵素，レクチン，ペプチド，ペプチド核酸，抗体，糖鎖，糖タンパク質，糖脂質，およびそれらの誘導体などを含む溶液が挙げられる。
本発明の生理活性物質固定化用粒子または生理活性物質固定粒子は、粒子状の捕捉材であるから、例えば当該捕捉材粒子をカラムに充填して試料溶液を連続通過させる或いは試料溶液中に分散させる等の簡便な方法で、大量の試料を短時間で処理することができる。
The particles for immobilizing a physiologically active substance or the physiologically active substance-immobilized particles of the present invention are used as capture material particles. After the capturing material particles are brought into contact with a sample solution containing a target physiologically active substance, the target physiologically active substance captured on the particles is collected together with the particles.
Sample solutions include blood, serum, tissue disruptions or extracts, cell disruptions or extracts, proteins, nucleic acids, enzymes, lectins, peptides, peptide nucleic acids, antibodies, sugar chains, glycoproteins, glycolipids, and Examples thereof include solutions containing derivatives thereof.
Since the particle for immobilizing a physiologically active substance or the particle for immobilizing a physiologically active substance of the present invention is a particulate trapping material, for example, the column is filled with the trapping material particles and the sample solution is continuously passed through or dispersed in the sample solution. It is possible to process a large amount of sample in a short time by a simple method such as making it occur.
粒子上に捕捉された生理活性物質は、捕捉サイトと捕捉された生理活性物質の間の結合又はその近傍を適宜の方法で切断することによって分離することができる。
例えば、捕捉材粒子上に糖ヒドラゾン結合によって固定された糖類に、糖親和性物質が固定されている場合には、当該捕捉材粒子の分散液中に、固定化した糖と同じ糖を過剰量添加することで、糖親和性物質が脱離する。添加する糖の濃度は１ｍＭ以上が望ましい。
また、当該捕捉材粒子の分散液を酸性条件又はアルカリ性条件にすることで、糖親和性物質を脱離させることが可能である。捕捉材粒子の分散液を酸性条件にする調節液としては、例えばグリシン−塩酸緩衝液（ｐＨ２〜４）があり、捕捉材粒子の分散液をアルカリ性条件にする調節液としては、例えばエタノールアミン‐塩酸緩衝液（ｐＨ７．５〜１１）がある。
なお、上記反応においては、捕捉材粒子を強い酸性又は強いアルカリ性の条件にすることで、粒子と生理活性物質の間の化学結合を切断するが、コア粒子として架橋プラスチック樹脂、特に架橋ＰＭＭＡを用いる場合には耐酸性及び耐アルカリ性に優れているので粒子が溶解することなく、生理活性物質を分離することができる。
The physiologically active substance captured on the particles can be separated by cutting the bond between the captured site and the captured physiologically active substance or the vicinity thereof by an appropriate method.
For example, when a sugar-affinity substance is immobilized on a saccharide immobilized on a capture material particle by a sugar hydrazone bond, an excess amount of the same sugar as the immobilized sugar is added to the dispersion liquid of the capture material particle. By adding, the glycophilic substance is eliminated. The concentration of added sugar is desirably 1 mM or more.
In addition, the sugar affinity substance can be desorbed by setting the dispersion liquid of the capturing material particles to an acidic condition or an alkaline condition. Examples of the adjusting liquid that makes the dispersion liquid of the capturing material particles acidic conditions include glycine-hydrochloric acid buffer (pH 2 to 4), and the adjusting liquid that makes the dispersion liquid of the capturing material particles alkaline conditions are, for example, ethanolamine There is a hydrochloric acid buffer (pH 7.5-11).
In the above reaction, the capture material particles are subjected to strong acidic or strong alkaline conditions to break the chemical bond between the particles and the physiologically active substance. However, a crosslinked plastic resin, particularly crosslinked PMMA is used as the core particle. In this case, since the acid resistance and alkali resistance are excellent, the physiologically active substance can be separated without dissolving the particles.
以下、本発明を実施例及び比較例に基づいて詳細に説明するが、本発明はこれに限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and a comparative example, this invention is not limited to this.
＜実施例１＞
（粒子の作製）
コア粒子として、東洋インキ製造株式会社製アクリル微粒子「リオスフィア」（品番ＲＳＰ−３００９、平均粒径２．６μｍ、メチルエチルケトン中に２０ｗｔ％の濃度で分散）を用いた。このリオスフィアは、架橋ＰＭＭＡ粒子であって、重合性官能基としてメタクリル基が導入された市販ビーズである。
このコア粒子１０ｇに、新中村化学工業株式会社製２−メタクリロイルオキシエチルサクシネート０．５５ｇ、新中村化学工業株式会社メトキシポリエチレングリコール＃４００メタクリレート（式（２）におけるエチレングリコール繰り返し単位の数（ｎ）＝９（カタログ値））４．５ｇ、和光純薬工業製アゾビスイソブチルニトリル０．０４ｇを加え、窒素雰囲気下で撹拌しながら６０℃２４時間重合を行った。得られた重合溶液を遠心分離して粒子を回収し、２−ブタノンに分散させ、再度遠心分離で粒子を回収した。この２−ブタノンへの分散と遠心分離による回収を計５回実施したのち、分散媒を水に置換することで、生理活性物質固定化用粒子を得た。
<Example 1>
(Production of particles)
As the core particles, acrylic fine particles “Riosphere” manufactured by Toyo Ink Manufacturing Co., Ltd. (product number RSP-3009, average particle size 2.6 μm, dispersed in methyl ethyl ketone at a concentration of 20 wt%) was used. This Riosphere is a cross-linked PMMA particle and is a commercially available bead in which a methacryl group is introduced as a polymerizable functional group.
To 10 g of the core particles, 0.55 g of 2-methacryloyloxyethyl succinate manufactured by Shin-Nakamura Chemical Co., Ltd., Shin-Nakamura Chemical Co., Ltd., methoxypolyethylene glycol # 400 methacrylate (the number of ethylene glycol repeating units in formula (2) (n ) = 9 (catalog value)) 4.5 g, 0.04 g of azobisisobutylnitrile manufactured by Wako Pure Chemical Industries, Ltd. was added, and polymerization was performed at 60 ° C. for 24 hours while stirring in a nitrogen atmosphere. The obtained polymerization solution was centrifuged to collect particles, dispersed in 2-butanone, and particles were collected again by centrifugation. The dispersion into 2-butanone and the recovery by centrifugation were carried out five times in total, and then the dispersion medium was replaced with water to obtain physiologically active substance immobilization particles.
（活性エステル化）
得られた生理活性物質固定化用粒子１００ｍｇを、２００ｍｇ/ｍＬのＷＳＣ（株式会社同仁化学研究所製）のＰＢＳ（ｐＨ５．８）溶液５ｍＬに分散し、３７℃にて２時間転倒混和した。その後、遠心分離で粒子を回収し、ＰＢＳ（ｐＨ５．８）で３回洗浄することで、活性エステル化した。
（１次抗体固定化）
得られた活性エステル化粒子２０ｍｇに対し、５０μｇ／ｍＬに調製したＣＲＰ抗体（Ａｂｎｏｖａ製）のりん酸水素二カリウム溶液１ｍＬを加え、室温にて１晩転倒混和した。０．０５％Ｔｗｅｅｎ２０含有ＰＢＳで３回洗浄した。
(Active esterification)
100 mg of the obtained particles for immobilizing a physiologically active substance was dispersed in 5 mL of a 200 mg / mL WSC (manufactured by Dojindo Laboratories) PBS (pH 5.8) and mixed by inverting at 37 ° C. for 2 hours. Thereafter, the particles were collected by centrifugation and washed three times with PBS (pH 5.8) to be active esterified.
(Primary antibody immobilization)
To 20 mg of the obtained active esterified particles, 1 mL of a dipotassium hydrogen phosphate solution of CRP antibody (manufactured by Abnova) adjusted to 50 μg / mL was added and mixed by inversion overnight at room temperature. Washed 3 times with PBS containing 0.05% Tween20.
（ＣＲＰとの反応）
ＣＲＰ抗体を固定化した粒子５ｍｇに３μｇ／ｍＬに調製したＣＲＰのＰＢＳ溶液１ｍＬを加え、室温にて１時間転倒混和した。遠心分離で粒子を回収後０．０５％Ｔｗｅｅｎ２０含有ＰＢＳで３回洗浄した。
(Reaction with CRP)
1 mL of CRP in PBS prepared at 3 μg / mL was added to 5 mg of particles having the CRP antibody immobilized thereon, and mixed by inverting at room temperature for 1 hour. The particles were collected by centrifugation and washed 3 times with PBS containing 0.05% Tween20.
（２次抗体との反応）
ＣＲＰを反応させた粒子に、１μｇ／ｍＬに調製したＨＲＰ標識化ＣＲＰ抗体（Ａｂｎｏｖａ製）溶液を１ｍＬ加え、室温にて１時間転倒混和した。遠心分離で粒子を回収後０．０５％Ｔｗｅｅｎ２０含有ＰＢＳで３回洗浄した。
(Reaction with secondary antibody)
1 mL of HRP-labeled CRP antibody (Abnova) solution prepared to 1 μg / mL was added to the particles reacted with CRP, and mixed by inverting at room temperature for 1 hour. The particles were collected by centrifugation and washed 3 times with PBS containing 0.05% Tween20.
（ＣＲＰ捕捉量の定量）
ＨＲＰ標識化ＣＲＰ抗体を反応させた粒子を、住友ベークライト株式会社製ペルオキシダーゼ発色キットを用いて発色させ、４５０ｎｍの吸光度を測定することによりＣＲＰの捕捉量を見積もった。
(Quantification of CRP capture amount)
The particles reacted with the HRP-labeled CRP antibody were colored using a peroxidase coloring kit manufactured by Sumitomo Bakelite Co., Ltd., and the amount of CRP captured was estimated by measuring the absorbance at 450 nm.
＜比較例１＞
ＷＳＣによる活性化を行ったのち、２−アミノエタノールで不活性化を行った粒子に対し、実施例と同様のＣＲＰ捕捉量測定を行った。
＜実施例１及び比較例１の結果＞
表１に示したとおり、実施例１におけるＣＲＰ捕捉量は、明らかに比較例１におけるＣＲＰ捕捉量よりも大きかった。
<Comparative Example 1>
After the activation by WSC, the CRP trapping amount measurement similar to that of the example was performed on the particles inactivated with 2-aminoethanol.
<Results of Example 1 and Comparative Example 1>
As shown in Table 1, the CRP trapping amount in Example 1 was clearly larger than the CRP trapping amount in Comparative Example 1.
＜実施例２＞
（ラクトース固定化粒子の作製）
実施例１と同様に生理活性物質固定化用粒子を作製し、粒子表面のカルボキシル基を活性化させたのち、ヒドラジン一水和物（和光純薬工業製）１ｍＬを加え、室温にて２時間転倒混和した。その後メタノールで３回洗浄し、さらに純水で３回洗浄した。次いで、１Ｍ ＨＣｌ（和光純薬工業製）を１ｍＬ加え、ボルテックスミキサーでよく撹拌し、純水で３回洗浄した。
得られたビーズ２ｍｇに対し、１０ｍＭラクトース水溶液２０μＬと２％酢酸を含むアセトニトリル１８０μＬを加え、８０℃にて１時間加熱し、乾固させた。これによりビーズ表面に糖（ラクトース）を固定した。
その後、４００μＬの純水で３回、４００μＬのメタノールで各３回ずつ洗浄した。次いで、１０％無水酢酸を含むメタノールを１００μＬ加え、室温にて３０分静置し、その後４００μＬのメタノールで３回洗浄した。
（レクチン捕捉）
得られたビーズ（糖親和性物質捕捉粒子）を、所定量のＶＥＣＴＯＲ ＬＡＢＯＲＡＴＯＲＩＥＳ製ＢＩＯＴＩＮＹＬＡＴＥＤ ＲＩＣＩＮＵＳ ＣＯＭＭＵＮＩＳ ＡＧＧＬＵＴＩＮＩＮＩ（ＲＣＡ１２０）溶液（溶媒：Ｔｒｉｓ−ＨＣｌバッファ）中に分散させ室温にて１時間静置し、ビーズにレクチンを捕捉させた。その後０．０５％Ｔｒｉｔｏｎ−Ｘ１００含有Ｔｒｉｓ−ＨＣｌバッファで３回洗浄した。
（ＳＤＳ−ＰＡＧＥ）
レクチンを捕捉させたビーズに飽和ラクトース溶液（溶媒：Ｔｒｉｓ−ＨＣｌバッファ）を加え、室温にて１時間静置し、ビーズと溶液を分離し、分離液をサンプル液として用いた。得られた各サンプル液の１／５量をＳＤＳ−ＰＡＧＥで電気泳動し、銀染色した。
（サンプル液のバリエーション）
上記一連の基本手順に従って少しずつ条件を変えて、実施例または比較例に該当するサンプル液（１）〜サンプル液（７）を得た。各サンプル液を得るための実験条件を表２に示した。
<Example 2>
(Production of lactose-immobilized particles)
After preparing particles for immobilizing a physiologically active substance in the same manner as in Example 1 and activating the carboxyl group on the particle surface, 1 mL of hydrazine monohydrate (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred for 2 hours at room temperature Mix by inversion. Thereafter, it was washed 3 times with methanol, and further washed 3 times with pure water. Next, 1 mL of 1M HCl (manufactured by Wako Pure Chemical Industries, Ltd.) was added, stirred well with a vortex mixer, and washed 3 times with pure water.
To 2 mg of the obtained beads, 20 μL of 10 mM lactose aqueous solution and 180 μL of acetonitrile containing 2% acetic acid were added, and heated at 80 ° C. for 1 hour to dry. As a result, sugar (lactose) was immobilized on the bead surface.
Then, it was washed 3 times with 400 μL of pure water and 3 times with 400 μL of methanol. Next, 100 μL of methanol containing 10% acetic anhydride was added, left at room temperature for 30 minutes, and then washed 3 times with 400 μL of methanol.
(Lectin capture)
The obtained beads (glycophilic substance capturing particles) were dispersed in a predetermined amount of BIOTINYLATED RICINUS COMMUNIS AGGLUTINI (RCA120) solution (solvent: Tris-HCl buffer) manufactured by VECTOR LABORATORIES, and allowed to stand at room temperature for 1 hour. Lectin was captured. Thereafter, the plate was washed 3 times with a Tris-HCl buffer containing 0.05% Triton-X100.
(SDS-PAGE)
A saturated lactose solution (solvent: Tris-HCl buffer) was added to the beads on which the lectin was captured, and the mixture was allowed to stand at room temperature for 1 hour to separate the beads and the solution, and the separated solution was used as a sample solution. 1/5 amount of each obtained sample liquid was electrophoresed by SDS-PAGE and silver-stained.
(Sample liquid variations)
The sample solution (1) to sample solution (7) corresponding to the examples or comparative examples were obtained by changing the conditions little by little according to the above-described series of basic procedures. Table 2 shows the experimental conditions for obtaining each sample solution.
＜実施例２の結果＞
実施例２における電気泳動の結果を図１に示す。
ＲＣＡ１２０溶液を直接電気泳動した結果（サンプル液（７）、図１における丸囲み番号７）と対比したところ、ラクトースを固定した粒子から分離したサンプル液（サンプル液（５）及び（６）、図１における丸囲み番号５及び６）でＲＣＡ１２０が検出されていることから、糖でレクチンを捕捉していることが示されている。
また、ラクトースを固定していない粒子から分離したサンプル液（サンプル液（２）及び（３）、図１における丸囲み番号２及び３）は、ＲＣＡ１２０がほとんど検出されなかったことから、本粒子への非特異吸着が極めて少ないことが示されている。
なお、ＲＣＡ１２０の処方時濃度が０（ゼロ）であったサンプル液（サンプル液（１）及び（４）、図１における丸囲み番号１及び４）は、当然ながらＲＣＡ１２０が全く検出されなかった。
<Results of Example 2>
The result of electrophoresis in Example 2 is shown in FIG.
As compared with the result of direct electrophoresis of the RCA120 solution (sample solution (7), circled number 7 in FIG. 1), the sample solution (sample solutions (5) and (6), FIG. RCA120 is detected in the circled numbers 5 and 6) in Fig. 1, indicating that the lectin is captured by sugar.
In addition, sample liquids (sample liquids (2) and (3), circled numbers 2 and 3 in FIG. 1) separated from the particles on which lactose is not fixed are not detected in RCA120. It has been shown that there is very little nonspecific adsorption.
Of course, RCA120 was not detected at all in the sample liquids (sample liquids (1) and (4), circled numbers 1 and 4 in FIG. 1) in which the concentration at the time of formulation of RCA120 was 0 (zero).
本発明の表面にカルボキシル基を有する粒子を、簡便に得ることができ、物質の分離精製、マイクロチップの充填剤をして使用することができる。 The particle | grains which have a carboxyl group on the surface of this invention can be obtained simply, can be used by separating and refining a substance and filling a microchip.
Claims (14)
コア粒子表面に、カルボキシル基を側鎖に有するポリマーを固定してなることを特徴とする生理活性物質固定化用粒子。 A particle for immobilizing a physiologically active substance,
A particle for immobilizing a physiologically active substance, wherein a polymer having a carboxyl group in a side chain is immobilized on the surface of a core particle.
結合１：−Ｃ（＝Ｏ）−ＮＲ−Ｎ＝（糖類）
（上記構造において、Ｒは水素原子又はＮに結合可能な水素原子以外の基である。また、これらの構造に含まれるＮは４級アンモニウム化していても良い。） 14. The physiologically active substance-immobilized particle according to claim 13, wherein a terminal of the part where the saccharide is immobilized has a structure of the following bond 1, and is used as a glycophilic substance-capturing particle.
Bond 1: —C (═O) —NR—N = (saccharide)
(In the above structure, R is a hydrogen atom or a group other than a hydrogen atom that can be bonded to N. Further, N contained in these structures may be quaternary ammoniumated.)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2011187699A JP5866880B2 (en) | 2011-02-10 | 2011-08-30 | Particles for immobilizing physiologically active substances, physiologically active substance-immobilized particles, and sugar affinity substance capturing particles |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2011026665 | 2011-02-10 | ||
| JP2011026665 | 2011-02-10 | ||
| JP2011187699A JP5866880B2 (en) | 2011-02-10 | 2011-08-30 | Particles for immobilizing physiologically active substances, physiologically active substance-immobilized particles, and sugar affinity substance capturing particles |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2012181181A true JP2012181181A (en) | 2012-09-20 |
| JP5866880B2 JP5866880B2 (en) | 2016-02-24 |
Family
ID=47012479
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2011187699A Expired - Fee Related JP5866880B2 (en) | 2011-02-10 | 2011-08-30 | Particles for immobilizing physiologically active substances, physiologically active substance-immobilized particles, and sugar affinity substance capturing particles |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5866880B2 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0839004A (en) * | 1994-08-03 | 1996-02-13 | Yamamoto Mfg Co Ltd | Discharge section for grain screening device |
| JP2014062798A (en) * | 2012-09-21 | 2014-04-10 | Sumitomo Bakelite Co Ltd | Analyzing carrier, and method for manufacturing and using the same |
| WO2014061371A1 (en) * | 2012-10-19 | 2014-04-24 | 住友ベークライト株式会社 | Analysis carrier and manufacturing method and use method therefor |
| JP2014160052A (en) * | 2013-01-25 | 2014-09-04 | Hitachi Chemical Co Ltd | Core shell type particle and manufacturing method for the same |
| JP2014210845A (en) * | 2013-04-17 | 2014-11-13 | 住友ベークライト株式会社 | Polymer particle to be used for producing sugar chain capturing particle, and sugar chain capturing particle |
| WO2016140216A1 (en) * | 2015-03-02 | 2016-09-09 | Jsr株式会社 | Solid-phase support, ligand-bonding solid-phase support, target-substance detecting or separating method, and method of manufacturing solid-phase support |
| WO2018186267A1 (en) * | 2017-04-06 | 2018-10-11 | 旭化成株式会社 | Hydrophilic coloring cellulose microparticles |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005524058A (en) * | 2002-04-23 | 2005-08-11 | ベックマン コールター インコーポレイテッド | Substrates coated with polymers for immobilizing biomolecules and cells |
| WO2005097844A1 (en) * | 2004-03-31 | 2005-10-20 | Sumitomo Bakelite Co., Ltd. | Polymer particle |
| JP2006201091A (en) * | 2005-01-21 | 2006-08-03 | Sumitomo Bakelite Co Ltd | Microparticles for capture beads and capture beads and biochips using the same |
| JP2009031130A (en) * | 2007-07-27 | 2009-02-12 | Toray Ind Inc | Manufacturing method of selective bondable substance fixed microbeads |
| JP2009286968A (en) * | 2008-05-30 | 2009-12-10 | Canon Inc | Graft polymer-containing substrate, its production method and magnetic biosensor |
| JP2010101550A (en) * | 2008-10-23 | 2010-05-06 | Nitto Electric Works Ltd | Ventilation system for double floor |
| JP2010530955A (en) * | 2006-12-29 | 2010-09-16 | ユニヴァーシティ オブ ワシントン | Dual functional non-polluting surfaces and materials |
-
2011
- 2011-08-30 JP JP2011187699A patent/JP5866880B2/en not_active Expired - Fee Related
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005524058A (en) * | 2002-04-23 | 2005-08-11 | ベックマン コールター インコーポレイテッド | Substrates coated with polymers for immobilizing biomolecules and cells |
| WO2005097844A1 (en) * | 2004-03-31 | 2005-10-20 | Sumitomo Bakelite Co., Ltd. | Polymer particle |
| JP2006201091A (en) * | 2005-01-21 | 2006-08-03 | Sumitomo Bakelite Co Ltd | Microparticles for capture beads and capture beads and biochips using the same |
| JP2010530955A (en) * | 2006-12-29 | 2010-09-16 | ユニヴァーシティ オブ ワシントン | Dual functional non-polluting surfaces and materials |
| JP2009031130A (en) * | 2007-07-27 | 2009-02-12 | Toray Ind Inc | Manufacturing method of selective bondable substance fixed microbeads |
| JP2009286968A (en) * | 2008-05-30 | 2009-12-10 | Canon Inc | Graft polymer-containing substrate, its production method and magnetic biosensor |
| JP2010101550A (en) * | 2008-10-23 | 2010-05-06 | Nitto Electric Works Ltd | Ventilation system for double floor |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0839004A (en) * | 1994-08-03 | 1996-02-13 | Yamamoto Mfg Co Ltd | Discharge section for grain screening device |
| JP2014062798A (en) * | 2012-09-21 | 2014-04-10 | Sumitomo Bakelite Co Ltd | Analyzing carrier, and method for manufacturing and using the same |
| WO2014061371A1 (en) * | 2012-10-19 | 2014-04-24 | 住友ベークライト株式会社 | Analysis carrier and manufacturing method and use method therefor |
| JPWO2014061371A1 (en) * | 2012-10-19 | 2016-09-05 | 住友ベークライト株式会社 | Analytical carrier, method for producing and using the same |
| JP2014160052A (en) * | 2013-01-25 | 2014-09-04 | Hitachi Chemical Co Ltd | Core shell type particle and manufacturing method for the same |
| JP2014210845A (en) * | 2013-04-17 | 2014-11-13 | 住友ベークライト株式会社 | Polymer particle to be used for producing sugar chain capturing particle, and sugar chain capturing particle |
| WO2016140216A1 (en) * | 2015-03-02 | 2016-09-09 | Jsr株式会社 | Solid-phase support, ligand-bonding solid-phase support, target-substance detecting or separating method, and method of manufacturing solid-phase support |
| JPWO2016140216A1 (en) * | 2015-03-02 | 2017-12-14 | Jsr株式会社 | Solid phase carrier, ligand-bound solid phase carrier, target substance detection or separation method, and method for producing the solid phase carrier |
| WO2018186267A1 (en) * | 2017-04-06 | 2018-10-11 | 旭化成株式会社 | Hydrophilic coloring cellulose microparticles |
| JPWO2018186267A1 (en) * | 2017-04-06 | 2019-11-21 | 旭化成株式会社 | Hydrophilized colored cellulose fine particles |
| US11353453B2 (en) | 2017-04-06 | 2022-06-07 | Asahi Kasei Kabushiki Kaisha | Hydrophilic colored cellulose microparticles |
Also Published As
| Publication number | Publication date |
|---|---|
| JP5866880B2 (en) | 2016-02-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5866880B2 (en) | Particles for immobilizing physiologically active substances, physiologically active substance-immobilized particles, and sugar affinity substance capturing particles | |
| Kalecki et al. | Oriented immobilization of protein templates: a new trend in surface imprinting | |
| Khumsap et al. | Epitope-imprinted polymers: applications in protein recognition and separation | |
| EP2230264B1 (en) | Particles containing multi-block polymers | |
| JP2012122073A (en) | Polymer particle | |
| JP6453590B2 (en) | Solid phase carrier, ligand-bound solid phase carrier, target substance detection or separation method, and method for producing the solid phase carrier | |
| JP6287847B2 (en) | Analytical carrier, method for producing and using the same | |
| JP6203295B2 (en) | Target substance capturing method, solid phase carrier for capturing target substance, and method for producing the solid phase carrier | |
| JP2007279028A (en) | Biological material structure having pores and method for producing the same, biological material carrier using the same, purification method of target substance, affinity chromatography container, separation chip, target substance analysis method, target substance analysis Separation device and sensor chip | |
| AU2011340263B2 (en) | Method | |
| JP6028488B2 (en) | Analytical carrier, method for producing and using the same | |
| Elaissari et al. | Biorelevant latexes and microgels for the interaction with nucleic acids | |
| KR102270242B1 (en) | Polymer microparticle for carrying physiologically active substance and method for preparing same | |
| JP2009031130A (en) | Manufacturing method of selective bondable substance fixed microbeads | |
| JP2003277455A (en) | Method for producing polymer particles, polymer particles and physiologically active substance carrier | |
| JP4873123B2 (en) | Method for producing carrier polymer particles | |
| JP2007101520A (en) | Biological substance complex, biological substance complex carrier, target substance purification method, affinity chromatography container, separation chip, target substance analysis method, target substance analysis separation apparatus and sensor chip | |
| JP6241564B1 (en) | Sugar chain-capturing polymer particles | |
| JP4866112B2 (en) | Biological material structure and manufacturing method of biological material structure, biological material carrier, purification method of target substance, affinity chromatography container, separation chip, target substance analysis method, target substance analysis separation device, And sensor chip | |
| JP2012093290A (en) | Particle for medical use and method of capturing physiologically active substance | |
| WO2006036003A1 (en) | Microparticle having, linked thereto, substance having biospecific affinity and use thereof | |
| JP2017149810A (en) | Copolymer, coating agent using the same, and coating method | |
| EP4083636A1 (en) | Biochip and detection method | |
| JP2016006405A (en) | Detection method and quantitative method of detection object, kit and preparation method of reagent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20140612 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20150115 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20150217 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20150417 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20150915 |
|
| 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: 20151208 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20151221 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 5866880 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| LAPS | Cancellation because of no payment of annual fees |