JPS6315553B2 - - Google Patents
Info
- Publication number
- JPS6315553B2 JPS6315553B2 JP17256380A JP17256380A JPS6315553B2 JP S6315553 B2 JPS6315553 B2 JP S6315553B2 JP 17256380 A JP17256380 A JP 17256380A JP 17256380 A JP17256380 A JP 17256380A JP S6315553 B2 JPS6315553 B2 JP S6315553B2
- Authority
- JP
- Japan
- Prior art keywords
- particles
- polymer
- immobilized
- antibody
- antigen
- 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.)
- Expired
Links
- 239000000126 substance Substances 0.000 claims description 25
- 239000010419 fine particle Substances 0.000 claims description 15
- 239000011859 microparticle Substances 0.000 claims description 13
- 239000000178 monomer Substances 0.000 claims description 11
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 claims description 9
- RPQRDASANLAFCM-UHFFFAOYSA-N oxiran-2-ylmethyl prop-2-enoate Chemical compound C=CC(=O)OCC1CO1 RPQRDASANLAFCM-UHFFFAOYSA-N 0.000 claims description 7
- 125000003277 amino group Chemical group 0.000 claims description 5
- 229920001577 copolymer Polymers 0.000 claims description 5
- 239000011203 carbon fibre reinforced carbon Substances 0.000 claims description 3
- CREMABGTGYGIQB-UHFFFAOYSA-N carbon carbon Chemical compound C.C CREMABGTGYGIQB-UHFFFAOYSA-N 0.000 claims description 2
- 239000002245 particle Substances 0.000 description 33
- 229920000642 polymer Polymers 0.000 description 30
- 239000000427 antigen Substances 0.000 description 24
- 102000036639 antigens Human genes 0.000 description 24
- 108091007433 antigens Proteins 0.000 description 24
- 238000006243 chemical reaction Methods 0.000 description 17
- 238000006116 polymerization reaction Methods 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 125000003700 epoxy group Chemical group 0.000 description 11
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- 210000004027 cell Anatomy 0.000 description 10
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- 230000004520 agglutination Effects 0.000 description 7
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- 208000006379 syphilis Diseases 0.000 description 6
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 5
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 5
- 239000006185 dispersion Substances 0.000 description 5
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 5
- 229920000126 latex Polymers 0.000 description 5
- 239000004816 latex Substances 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 4
- 238000004220 aggregation Methods 0.000 description 4
- 230000002776 aggregation Effects 0.000 description 4
- 210000001124 body fluid Anatomy 0.000 description 4
- 239000010839 body fluid Substances 0.000 description 4
- 150000001718 carbodiimides Chemical class 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
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- 238000001514 detection method Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- 238000012673 precipitation polymerization Methods 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- 108091006905 Human Serum Albumin Proteins 0.000 description 3
- 102000008100 Human Serum Albumin Human genes 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- VVQNEPGJFQJSBK-UHFFFAOYSA-N Methyl methacrylate Chemical compound COC(=O)C(C)=C VVQNEPGJFQJSBK-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000012491 analyte Substances 0.000 description 3
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- -1 styrene Chemical class 0.000 description 3
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 2
- PFHOSZAOXCYAGJ-UHFFFAOYSA-N 2-[(2-cyano-4-methoxy-4-methylpentan-2-yl)diazenyl]-4-methoxy-2,4-dimethylpentanenitrile Chemical compound COC(C)(C)CC(C)(C#N)N=NC(C)(C#N)CC(C)(C)OC PFHOSZAOXCYAGJ-UHFFFAOYSA-N 0.000 description 2
- HWSSEYVMGDIFMH-UHFFFAOYSA-N 2-[2-[2-(2-methylprop-2-enoyloxy)ethoxy]ethoxy]ethyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCCOCCOCCOC(=O)C(C)=C HWSSEYVMGDIFMH-UHFFFAOYSA-N 0.000 description 2
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical group C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 2
- 108010074051 C-Reactive Protein Proteins 0.000 description 2
- 102100032752 C-reactive protein Human genes 0.000 description 2
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 description 2
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- 208000030452 Transient pseudohypoaldosteronism Diseases 0.000 description 2
- 241000589884 Treponema pallidum Species 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000012736 aqueous medium Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000012888 bovine serum Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000006482 condensation reaction Methods 0.000 description 2
- 238000007720 emulsion polymerization reaction Methods 0.000 description 2
- FKRCODPIKNYEAC-UHFFFAOYSA-N ethyl propionate Chemical compound CCOC(=O)CC FKRCODPIKNYEAC-UHFFFAOYSA-N 0.000 description 2
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000003100 immobilizing effect Effects 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
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- 230000009257 reactivity Effects 0.000 description 2
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- 238000007086 side reaction Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 238000010557 suspension polymerization reaction Methods 0.000 description 2
- JHPBZFOKBAGZBL-UHFFFAOYSA-N (3-hydroxy-2,2,4-trimethylpentyl) 2-methylprop-2-enoate Chemical compound CC(C)C(O)C(C)(C)COC(=O)C(C)=C JHPBZFOKBAGZBL-UHFFFAOYSA-N 0.000 description 1
- KOMNUTZXSVSERR-UHFFFAOYSA-N 1,3,5-tris(prop-2-enyl)-1,3,5-triazinane-2,4,6-trione Chemical compound C=CCN1C(=O)N(CC=C)C(=O)N(CC=C)C1=O KOMNUTZXSVSERR-UHFFFAOYSA-N 0.000 description 1
- QRIMLDXJAPZHJE-UHFFFAOYSA-N 2,3-dihydroxypropyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCC(O)CO QRIMLDXJAPZHJE-UHFFFAOYSA-N 0.000 description 1
- BJELTSYBAHKXRW-UHFFFAOYSA-N 2,4,6-triallyloxy-1,3,5-triazine Chemical compound C=CCOC1=NC(OCC=C)=NC(OCC=C)=N1 BJELTSYBAHKXRW-UHFFFAOYSA-N 0.000 description 1
- JQXYBDVZAUEPDL-UHFFFAOYSA-N 2-methylidene-5-phenylpent-4-enoic acid Chemical group OC(=O)C(=C)CC=CC1=CC=CC=C1 JQXYBDVZAUEPDL-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- 102000009109 Fc receptors Human genes 0.000 description 1
- 108010087819 Fc receptors Proteins 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- 241000223996 Toxoplasma Species 0.000 description 1
- WRVRNZNDLRUXSW-UHFFFAOYSA-N acetic acid;prop-2-enoic acid Chemical compound CC(O)=O.OC(=O)C=C WRVRNZNDLRUXSW-UHFFFAOYSA-N 0.000 description 1
- XECAHXYUAAWDEL-UHFFFAOYSA-N acrylonitrile butadiene styrene Chemical compound C=CC=C.C=CC#N.C=CC1=CC=CC=C1 XECAHXYUAAWDEL-UHFFFAOYSA-N 0.000 description 1
- 229920000122 acrylonitrile butadiene styrene Polymers 0.000 description 1
- 239000004676 acrylonitrile butadiene styrene Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 150000001346 alkyl aryl ethers Chemical class 0.000 description 1
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 1
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 230000002494 anti-cea effect Effects 0.000 description 1
- 230000003172 anti-dna Effects 0.000 description 1
- 229940046836 anti-estrogen Drugs 0.000 description 1
- 230000001833 anti-estrogenic effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000012300 argon atmosphere Substances 0.000 description 1
- 238000010420 art technique Methods 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- HABAXTXIECRCKH-UHFFFAOYSA-N bis(prop-2-enyl) butanedioate Chemical compound C=CCOC(=O)CCC(=O)OCC=C HABAXTXIECRCKH-UHFFFAOYSA-N 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 108010018927 conglutinin Proteins 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- QCUFYOBGGZSFHY-UHFFFAOYSA-N depsidomycin Chemical compound O=C1C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(NC=O)C(C)CC)C(C)OC(=O)C2CCCNN2C(=O)C(CC(C)C)NC(=O)C2CCCNN21 QCUFYOBGGZSFHY-UHFFFAOYSA-N 0.000 description 1
- 150000004985 diamines Chemical class 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000328 estrogen antagonist Substances 0.000 description 1
- FFYWKOUKJFCBAM-UHFFFAOYSA-N ethenyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OC=C FFYWKOUKJFCBAM-UHFFFAOYSA-N 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- GSEWLHCVMFWKQO-UHFFFAOYSA-N hydrazinylidenemethanimine Chemical compound NN=C=N GSEWLHCVMFWKQO-UHFFFAOYSA-N 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000010954 inorganic particle Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 description 1
- 229920003145 methacrylic acid copolymer Polymers 0.000 description 1
- 229940117841 methacrylic acid copolymer Drugs 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- 229920006173 natural rubber latex Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920002239 polyacrylonitrile Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920002959 polymer blend Polymers 0.000 description 1
- 229920002689 polyvinyl acetate Polymers 0.000 description 1
- 239000011118 polyvinyl acetate Substances 0.000 description 1
- 229920002717 polyvinylpyridine Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- FBCQUCJYYPMKRO-UHFFFAOYSA-N prop-2-enyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCC=C FBCQUCJYYPMKRO-UHFFFAOYSA-N 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 201000005404 rubella Diseases 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 108010075210 streptolysin O Proteins 0.000 description 1
- 229920003048 styrene butadiene rubber Polymers 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
Description
本発明は免疫学的検査用試薬として有効な免疫
活性微粒子に関し、特に粒子状担体に免疫活性物
質を固定化してなる免疫活性粒子を用いてヒト又
は動物の体液中の成分を検出若しくは測定又は細
胞を識別する免疫学的検査用試薬として有効な免
疫活性微粒子の改良に関する。
抗原と抗体との反応を利用してその一方を免疫
学的に検出又は定量する場合に、測定したい物質
に結合する側の物質を粒子状担体に固定化させて
おき、その粒子が被測定物質の存在下で凝集を起
こす現象を利用して高感度の測定を行なう方法は
免疫学的臨床検査の重要な手段となつている。ま
た逆に測定したい物質を粒子状担体に固定化させ
ておき、その被測定物質と特異的に反応する抗原
又は抗体の存在による被測定物質固定化粒子の凝
集が、被検液中の被測定物質の存在により阻止さ
れることにより被測定物質を検出又は定量する方
法も免疫学的臨床検査において広く用いられてい
る。また特定の細胞と選択的に結合する物質を粒
子状担体に固定化させておき、その粒子が細胞に
結合するか否かによつて細胞の識別を行なう方法
も免疫学的検査の手段としてしばしば採用されて
いる。
このような免疫活性粒子を用いた免疫学的検査
用試薬における粒子状担体としては、従来、ヒト
を含む哺乳動物や鳥類の赤血球、カオリンや炭素
など無機物の粒子、天然ゴムラテツクスやポリス
チレンなどの有機高分子化合物のラテツクスが凝
集反応用として広く用いられている。これらのう
ち赤血球は多種類の抗原・抗体を固定化すること
が可能で応用範囲が最も広い。しかし採取する動
物個体によつて品質等に差があること、安定性に
難があり保存が難しいこと、またヒト血清により
非特異的に凝集する場合があることなどの問題点
がある。非生物由来の粒子として最も広く用いら
れているのはポリスチレン粒子であり、これは合
成高分子化合物であるところから品質を一定にす
ることが可能でまたそれ自体では安定である。ポ
リスチレンは疎水性で種々の蛋白質を吸着する性
質があるため、通常ポリスチレンへの抗原又は抗
体の固定は物理吸着によつて行なわれる。しかし
物理吸着によつて抗原又は抗体を固定化した場合
には固定化した抗原（又は抗体）と遊離の抗原
（又は抗体）との間に平衡が存在し、そのため測
定の目的物質である対応する抗体（又は抗原）に
対し粒子に固定化した抗原（又は抗体）と遊離の
抗原（又は抗体）との間に競争反応が起こり、そ
の競争反応は凝集に対して抑制的に作用する。そ
の結果、多くの例において感度と安定性の不足が
指摘されている。また当然のことながらポリスチ
レンに対して物理的に吸着されにくい物質は固定
化することができない。これらの問題点のために
ポリスチレン粒子は赤血球を担体とする場合に比
較して限られた範囲でしか実用に供されていな
い。これらの問題点の解決を図る目的で最近、ス
チレン−メタクリル酸コポリマーラテツクスにヒ
ト絨毛性ゴナドトロピンをカルボジイミドを使用
して結合させた試薬（DT2649218）、カルボキシ
ル化スチレン−ブタジエンコポリマー、カルボキ
シル化ポリスチレン、アミノ基をもつカルボキシ
ル化ポリスチレン、アクリル酸ポリマー、アクリ
ロニトリルポリマー、メタクリル酸ポリマー、ア
クリロニトリル−ブタジエン−スチレンコポリマ
ー、ポリ酢酸ビニルアクリレート、ポリビニルピ
リジン、塩化ビニル−アクリレートコポリマーな
ど種々のラテツクスポリマーにカルボジイミドを
縮合剤としてヒト絨毛性コーナドトロピン、ヒト
血清アルブミン又は変性ガンマグロブリンをアミ
ド結合を介して縮合させた粒径0.01〜0.9ミクロ
ンの粒子からなる試薬（特公昭53−12966）、メタ
クリル酸、２−ヒドロキシエチルメタクリレート
及びメチルメタクリレートを共重合して製造した
ヒドロキシル基とカルボキシル基を含有するメチ
ルメタクリレート系ラテツクスにトレポネーマ抗
原を臭化シアノゲン又はカルボジイミド法で結合
させた試薬（臨床病理27、補冊、522頁（1978））、
ポリスチレン粒子を芯として、それをスチレン−
グリシジルメタクリレートコポリマーの外皮で被
覆したラテツクスの遊離エポキシ基とヒト絨毛ゴ
ナドトロピン又はインシユリンを反応させ、それ
らをラテツクスに結合させた試薬（特公開昭55−
110118）など、共有結合により抗原又は抗体を担
体に結合させた試薬が提案されている。これら先
行技術においてはカルボジイミドにより免疫活性
物質を粒子に結合させる方法が多用されている
が、カルボジイミドを使用すると免疫活性物質分
子間および分子内の縮合反応を惹起する。これは
のぞましくない副反応であつて免疫活性物質の活
性を損なうものである。臭化シアノゲンを用いれ
ば免疫活性物質分子間および分子内の縮合反応を
回避することはできるが、この場合にはヒドロキ
シル基を有するポリマーと臭化シアノゲンとの反
応の再現性を得ることが難かしく、その結果免疫
活性物質を固定化した粒子の免疫活性が変動しす
い。これらの免疫活性物質固定化法に比較して重
合体に導入された遊離エポキシ基と蛋白質又はポ
リペプチドを反応させる方法は免疫活性の失活も
少なく再現性も良好である。しかしエポキシ基を
利用する上記先行技術においては重合体粒子表面
がスチレンのような疎水性化合物の共重合体であ
るため蛋白質を非特異的に吸着する傾向を有して
いる。一般にヒト又は動物の体液中には多種類の
蛋白質が含まれ、とくに血漿又は血清中にはこれ
が高濃度で含有されている。検体体液から蛋白質
が担持粒子に吸着されると、それが目的とする抗
原−抗体反応などの免疫学的反応に干渉し、凝集
反応の選択性や感度の低下をもたらすおそれがあ
る。本発明者らはこのような問題点を解消するこ
とを目的に検討を行なつた結果、本発明に到達し
た。
本発明は、(1)グリシジルアクリレートおよび
（または）グリシジルメタクリレートと、(2)炭素
炭素不飽和二重結合を有する水溶性単量体との共
重合体からなる平均直径が0.03〜10μｍの微粒子
に、アミノ基を有する免疫活性物質が共有結合に
よつて固定化されていることを特徴とする免疫活
性微粒子を提供するものであり、該微粒子は免疫
学的検査用試薬として著効を示す。本発明によれ
ば免疫活性物質はそのアミノ基と微粒子表面の遊
離エポキシ基との反応によつて生成する共有結合
によつて担体微粒子上に固定化される。免疫活性
物質との反応によつて遊離エポキシ基が全部消費
されずに変性を有するまま残存する可能性がある
場合には血清アルブミン、ゼラチンなど目的とす
る免疫学的検査に干渉しない親水性蛋白質を遊離
エポキシ基と反応させることによつて、エポキシ
基の反応性を失わせることができる。その際エポ
キシ基消去用の新水性蛋白質は固定化の目的であ
る免疫活性物質と混合して同時に反応させてもよ
く、また免疫活性物質を先に単独で反応させその
後で反応させてもよい。また上記アルブミンやゼ
ラチンなどの親水性蛋白質の代りにグリシン、ア
ラニンなどのアミノ酸を用いることも可能であ
る。このようにして免疫活性物を固定化した後に
重合体微粒子表面で何らの結合物によつて覆われ
ることなく露出しているのは、水溶性単量体に由
来する親水性部分である。蛋白質は水性媒体中で
は親水性重合体には吸着しにくいので、本発明に
よる免疫活性物質固定化微粒子は、検体体液に対
して安定で非特異的凝集を起こしにくく、また細
胞に対する非特異的付着がない。
本発明において共重合に用いる水溶性単量体と
しては、例えば２−オキシエチルアクリレート、
２−オキシエチルメタクリレート、２−オキシプ
ロピルアクリレート、２−オキシプロピルメタク
リレート、重合度２ないし25のポリエチレングリ
コールモノアルキルエーテルのアクリル酸エステ
ル又はメタクリル酸エステル、アクリルアミド、
メタクリルアミド、Ｎ−ビニルピロリドン、グリ
セロールメタクリレートなどが好ましく用いられ
る。これらの水溶性単量体は２種以上併用しても
よい。グリシジルアクリレートとグリシジルメタ
クリレートとの和に対する水溶性単量体の和の比
率はモル比で95：５ないし５：95の範囲で変える
ことができる。またグリシジルアクリレートとグ
リシジルメタクリレートの使用比は100：０ない
し０：100（モル比）すななわち任意でよい。遊離
エポキシ基はアミノ基のみでなく、カルボキシル
基、アルコール性ヒドロキシル基、フエノール性
ヒドロキシル基及びメルカプト基などとも反応し
得るが、重合体粒子の製造条件を適当に選べばグ
リシジルアクリレート又はグリシジルメタクリレ
ートのエポキシ基を副反応によつて失なうことな
く免疫活性物質の固定化に利用することができ
る。
本発明の重合体微粒子は例えば次の方法によつ
て製造することができる。
重合反応は通常乳化重合、沈澱重合又は懸濁重
合によつて好ましく行なわれる。これらいずれの
方法も重合と同時に重合体が粒子状になつて析出
するので本発明の目的に適している。とくに好ま
しいのは沈澱重合である。沈澱重合は、単量体は
溶解するが重合によつて生成する重合体は溶解し
ない媒体中で重合を行なう方法であつて、単量体
と重合媒体との組合せを選択することによつて生
成する重合体粒子の平均直径を0.03ないし10μｍ
の範囲に入るよう調節することが比較的容易であ
り、粒径の分布も比較的狭い。また沈澱重合は、
乳化重合や懸濁重合の場合と異なつて、乳化剤や
懸濁安定剤を使用しないので、重合反応後これら
の添加剤を除去する必要がないのも利点の一つで
ある。
架橋剤を重合系に添加することは必須ではない
が、通常重合に当つて重合性炭素炭素二重結合を
分子内に２基以上含む多官能性単量体を添加して
積極的に重合体を架橋させることがのぞましい。
そのような目的で重合系に添加するに適した多官
能性単量体は多数存在するが、若干例をあげれ
ば、ジビニルベンゼン、エチレングリコールジメ
タクリレート、Ｎ，N′−メチレンビスアクリル
アミド、コハク酸ジビニル、コハク酸ジアリル、
メタクリル酸ビニル、メタクリル酸アリル、トリ
アリルシアヌレート、トリアリルイソシアヌレー
トなどである。また架橋結合は重合反応後生成重
合体の反応性を利用してこれを多官能化合物と反
応させることによつて導入することもできる。例
えば生成重合体に含まれるエポキシ基とエチレン
ジアミンなどのジアミンとを反応させることによ
り重合体を架橋させることができる。
粒子の形状は多くの場合球形であるが球形であ
ることは必要条件ではなく不規則な形状であつて
も差し支えない。不規則な形状の粒子の直径は最
大径と最小径の和の1/2とする。平均直径は式(1)
によつて定義されるによつて表わされる。ただ
しdiはｉ番目の粒子の直径、Ｎは粒子の総数であ
る。
＝N
〓i=1
di／Ｎ ……(1)
凝集反応が判定しやすいのは経験的に平均直径
が0.1μｍ以上10μｍ以下の場合である。また細胞
標識の目的には平均直径は0.03μｍ以上5μｍ以下
の範囲が好ましい。また染料ないし顔料により適
当に着色した粒子は凝集反応、細胞標識いずれの
目的に対しても好都合である。また細胞標識に対
しては螢光を付与した粒子も好ましい。
免疫活性物質の微粒子への固定化反応は水性媒
体中で行ない、PHは7.0〜9.0、温度は０℃〜40℃
の範囲が適当である。反応液中の免疫活性物質の
濃度は個々の免疫活性物質の性質によつて増減す
る必要があり一律には決められない。その際すで
に述べたように血清アルブミン、ゼラチンなどの
親水性蛋白質を反応液に添加することはしばしば
免疫活性物質固定化微粒子の分散安定性を改良す
る上で有効である。また固定化反応後にグリシ
ン、アラニンなどのアミノ酸で処理することもし
ばしば同様に好ましい効果をもたらす。
本発明において使用する免疫活性物質はアミノ
基を有することが必要であるが、免疫活性物質の
大部分は蛋白質であるかまたはポリペプチド部分
を含んでいるのでその条件に適合する。ここで免
疫活性物質とは抗原および抗体のみでなく、補
体、Fcレセプター、C3レセプターなど液性免疫
反応ないし細胞性免疫反応に関与してある物質に
特異的に結合する物質を意味するものとする。具
体例を若干あげれば、梅毒トレポネーマ抗原、Ｂ
型肝炎表面抗原（HBs抗原）、Ｂ型肝炎表面抗原
に対する抗体（抗HBs抗体）、風疹抗原、トキソ
プラズマ抗原、ストレプトリジンＯ、抗ストレプ
トリジンＯ抗体、マイコプラズマ抗原、ヒト絨毛
性ゴナドトロピン（HCG）、抗HCG抗体、熱凝
集ヒトIgG、リウマチ因子、核蛋白、DNA、抗
DNA抗体、Ｃ反応性蛋白質（CRP）、抗CRP抗
体、抗エストロゲン抗体、α−フエトプロテイン
（α−FP）、抗α−FP抗体、癌胎児性抗原
（CEA）、抗CEA抗体、C1q、抗C1q抗体、C3、
抗C3抗体、抗C3b抗体、抗C3bi抗体、C4、抗C4
抗体、プロテイン−Ａ、コングルチニン、イムノ
コングルチニンなどである。
実施例 １
グリシジルメタクリレート、２−オキシエチル
メタクリレートおよびエチレングリコールジメタ
クリレートの３者を85.7：9.5：4.8のモル比で混
合し、その単量体混合物24部（重量、以下同じ）、
プロピオン酸エチル76部および２，2′−アゾビス
（２，４−ジメチル−４−メトキシバレロニトリ
ル）0.13部（4.7ｍmol／）の混合物を窒素ガス
雰囲気下40℃で３時間重合させた。白濁した重合
混合物をアセトンに注ぎ、1500×ｇで10分間遠心
分離し、沈降した粒子をエタノールで再分散して
洗浄、次いで再び遠心分離した。減圧下に乾燥し
て微粒子状重合体11.3部を得た。この微粒子状重
合体は球形でその直径は1.8μｍと4.2μｍの間にあ
り平均3.3μｍ、標準偏差は0.45μｍであつた。
上記のようにして調製した重合体微粒子にTP
抗原を下記のようにして固定化した。先ず梅毒病
原体Treponema pallidum（以下TPと略記）
Nichols株菌体成分をリン酸塩緩衡生理食塩水
（リン酸水素２ナトリウム＋リン酸水素１カリウ
ム0.01mol／、塩化ナトリウム0.14mol／、
PH7.2、以下PBSと略記）の中に109cell／mlの割
合で分散させた分散液を氷水で冷却しながら10K
Hzの超音波で20分間処理して菌体を破壊し、これ
をTP抗原液とした。このTP抗原液１容とウシ血
清グリコプロテインを１mg／mlの濃度でPBSに
溶解した溶液３容とを混合し、その混合液１mlと
前記重合体50mgをPBS1mlに分散した分散液とを
混合して30℃で２時間撹拌した。次に遠沈により
微粒子を分離してPBSで遠沈により３回洗浄し
た後、ウシ血清アルブミン（以下BSAと略記）
を１％添加したPBS20mlに分散した。このTP抗
原固定化微粒子分散液を３日間４℃の冷蔵庫中で
放置した後、下記のようにして活性を検定した。
Ｕ字型管底を有するポリスチレン製のマイクロ
タイタープレートに、TPHA力価640の梅毒陽性
血清希釈液を希釈倍率10倍を起点とする2n希釈系
列で各50μずつ入れた。ただし希釈用液として
はPBSにBSAを１％、梅毒補体結合反応用ライ
ター抗原溶液KW（日本凍結乾燥研究所）を５％、
塩化アンモニウムを0.73mol／の濃度で添加し
た溶液を使用した。またコントロールとして梅毒
陰性血清についても同様の希釈液をマイクロタイ
タープレートに入れた。次に血清希釈液の入つて
いるマイクロタイタープレートの各穴にTP抗原
固定化微粒子分散液を各50μずつ加え、３分間
振盪して両液を混合した後窒温で２時間静置し、
沈降像によつて凝集の程度を判定した。その結果
は表１の通りでTPHA以上の感度で血清中の梅
毒抗体を検出できることがわかる。
The present invention relates to immunoactive microparticles that are effective as reagents for immunological testing, and in particular to immunoactive particles formed by immobilizing an immunoactive substance on a particulate carrier to detect or measure components in human or animal body fluids or to The present invention relates to improvements in immunoactive microparticles that are effective as immunological test reagents for identifying. When using the reaction between an antigen and an antibody to immunologically detect or quantify one of them, the substance that binds to the substance to be measured is immobilized on a particulate carrier, and the particles become the substance to be measured. A highly sensitive measurement method that takes advantage of the phenomenon of agglutination in the presence of ions has become an important means for immunological clinical testing. Conversely, when the substance to be measured is immobilized on a particulate carrier, aggregation of the particles immobilized with the analyte due to the presence of antigens or antibodies that specifically react with the analyte can cause the analyte to be measured in the sample solution. Methods of detecting or quantifying a substance to be measured by inhibiting the presence of the substance are also widely used in immunological clinical tests. In addition, a method in which a substance that selectively binds to specific cells is immobilized on a particulate carrier and cells are identified based on whether or not the particles bind to the cells is often used as a means of immunological testing. It has been adopted. Particulate carriers in immunological test reagents using such immunoactive particles have conventionally been red blood cells of mammals including humans and birds, inorganic particles such as kaolin and carbon, and organic polymers such as natural rubber latex and polystyrene. Latexes of molecular compounds are widely used for agglutination reactions. Among these, red blood cells can immobilize many types of antigens and antibodies and have the widest range of applications. However, there are problems such as differences in quality depending on the individual animal collected, poor stability and difficulty in storage, and non-specific agglutination by human serum. The most widely used particles of non-biological origin are polystyrene particles, which can be made of a synthetic polymer compound, have a constant quality, and are stable in themselves. Since polystyrene is hydrophobic and has the property of adsorbing various proteins, antigens or antibodies are usually immobilized on polystyrene by physical adsorption. However, when an antigen or antibody is immobilized by physical adsorption, an equilibrium exists between the immobilized antigen (or antibody) and the free antigen (or antibody), so that the corresponding target substance of measurement A competitive reaction occurs between the antigen (or antibody) immobilized on particles and the free antigen (or antibody), and the competitive reaction acts to suppress agglutination. As a result, a lack of sensitivity and stability has been noted in many cases. Also, as a matter of course, substances that are difficult to physically adsorb to polystyrene cannot be immobilized. Due to these problems, polystyrene particles have been put to practical use only to a limited extent compared to when red blood cells are used as a carrier. In order to solve these problems, we have recently developed a reagent (DT2649218) in which human chorionic gonadotropin is bonded to a styrene-methacrylic acid copolymer latex using carbodiimide, carboxylated styrene-butadiene copolymer, carboxylated polystyrene, amino carbodiimide as a condensing agent in various latex polymers such as carboxylated polystyrene, acrylic acid polymer, acrylonitrile polymer, methacrylic acid polymer, acrylonitrile-butadiene-styrene copolymer, polyvinyl acetate acrylate, polyvinylpyridine, vinyl chloride-acrylate copolymer, etc. A reagent consisting of particles with a particle size of 0.01 to 0.9 microns condensed with human chorionic cornadotropin, human serum albumin or modified gamma globulin via an amide bond (Japanese Patent Publication No. 12966/1989), methacrylic acid, 2-hydroxyethyl methacrylate A reagent in which a treponemal antigen is bound by the cyanogen bromide or carbodiimide method to a methyl methacrylate latex containing hydroxyl and carboxyl groups produced by copolymerizing methyl methacrylate and methyl methacrylate (Clinical Pathology 27, Supplement, p. 522 (1978) ),
With polystyrene particles as the core, it is made of styrene.
A reagent in which the free epoxy groups of a latex coated with a glycidyl methacrylate copolymer coat are reacted with human chorionic gonadotropin or insulin, and these are bonded to the latex (Japanese Patent Publication No. 1986-1999).
110118), reagents in which an antigen or antibody is bound to a carrier through a covalent bond have been proposed. In these prior art methods, a method of binding an immunoactive substance to particles using carbodiimide is often used, but when carbodiimide is used, condensation reactions occur between and within molecules of the immunoactive substance. This is an undesirable side reaction that impairs the activity of the immunologically active substance. If cyanogen bromide is used, it is possible to avoid condensation reactions between and within molecules of the immunoactive substance, but in this case, it is difficult to obtain reproducibility of the reaction between a polymer having a hydroxyl group and cyanogen bromide. As a result, the immunoactivity of particles immobilized with immunoactive substances tends to fluctuate. Compared to these methods of immobilizing immunoactive substances, the method of reacting a free epoxy group introduced into a polymer with a protein or polypeptide causes less deactivation of immune activity and has good reproducibility. However, in the above-mentioned prior art techniques that utilize epoxy groups, since the polymer particle surface is a copolymer of a hydrophobic compound such as styrene, it tends to non-specifically adsorb proteins. In general, human or animal body fluids contain many types of proteins, and plasma or serum in particular contains these proteins at high concentrations. When proteins from a sample body fluid are adsorbed onto the supported particles, they may interfere with the target immunological reaction such as an antigen-antibody reaction, leading to a decrease in the selectivity and sensitivity of the agglutination reaction. The present inventors conducted studies aimed at solving these problems, and as a result, they arrived at the present invention. The present invention provides fine particles with an average diameter of 0.03 to 10 μm, which are made of a copolymer of (1) glycidyl acrylate and/or glycidyl methacrylate and (2) a water-soluble monomer having a carbon-carbon unsaturated double bond. The present invention provides immunoactive fine particles characterized in that an immunoactive substance having an amino group is immobilized by a covalent bond, and the fine particles exhibit remarkable efficacy as a reagent for immunological testing. According to the present invention, an immunoactive substance is immobilized on a carrier microparticle by a covalent bond generated by a reaction between its amino group and a free epoxy group on the surface of the microparticle. If there is a possibility that free epoxy groups may not be completely consumed and remain denatured due to reaction with immunologically active substances, use hydrophilic proteins such as serum albumin and gelatin that do not interfere with the target immunological test. By reacting with free epoxy groups, the reactivity of the epoxy groups can be lost. In this case, the new aqueous protein for eliminating epoxy groups may be mixed with the immunoactive substance to be immobilized and reacted at the same time, or the immunoactive substance may be reacted alone first and then reacted. It is also possible to use amino acids such as glycine and alanine in place of the hydrophilic proteins such as albumin and gelatin. After the immunoactive substance is immobilized in this manner, what is exposed on the surface of the polymer fine particles without being covered with any bond is the hydrophilic moiety derived from the water-soluble monomer. Since proteins are difficult to adsorb to hydrophilic polymers in aqueous media, the immunoactive substance-immobilized microparticles according to the present invention are stable to sample body fluids, do not easily cause non-specific aggregation, and do not adhere non-specifically to cells. There is no. In the present invention, water-soluble monomers used for copolymerization include, for example, 2-oxyethyl acrylate,
2-oxyethyl methacrylate, 2-oxypropyl acrylate, 2-oxypropyl methacrylate, acrylic ester or methacrylic ester of polyethylene glycol monoalkyl ether with a degree of polymerization of 2 to 25, acrylamide,
Methacrylamide, N-vinylpyrrolidone, glycerol methacrylate and the like are preferably used. Two or more of these water-soluble monomers may be used in combination. The ratio of the sum of water-soluble monomers to the sum of glycidyl acrylate and glycidyl methacrylate can be varied in the molar ratio from 95:5 to 5:95. The ratio of glycidyl acrylate to glycidyl methacrylate used may be 100:0 to 0:100 (molar ratio), ie, any ratio. Free epoxy groups can react not only with amino groups but also with carboxyl groups, alcoholic hydroxyl groups, phenolic hydroxyl groups, mercapto groups, etc., but if the manufacturing conditions of the polymer particles are appropriately selected, the epoxy group of glycidyl acrylate or glycidyl methacrylate can be reacted with. It can be used to immobilize immunoactive substances without losing the group due to side reactions. The polymer fine particles of the present invention can be produced, for example, by the following method. The polymerization reaction is usually preferably carried out by emulsion polymerization, precipitation polymerization or suspension polymerization. All of these methods are suitable for the purpose of the present invention because the polymer is precipitated in the form of particles at the same time as the polymerization. Particularly preferred is precipitation polymerization. Precipitation polymerization is a method in which polymerization is carried out in a medium that dissolves the monomer but does not dissolve the polymer produced by polymerization. The average diameter of the polymer particles is 0.03 to 10 μm.
It is relatively easy to adjust the particle size to fall within this range, and the particle size distribution is also relatively narrow. In addition, precipitation polymerization is
Unlike emulsion polymerization or suspension polymerization, no emulsifier or suspension stabilizer is used, so one of the advantages is that there is no need to remove these additives after the polymerization reaction. Although it is not essential to add a crosslinking agent to the polymerization system, it is usually possible to actively polymerize by adding a polyfunctional monomer containing two or more polymerizable carbon-carbon double bonds in the molecule during polymerization. It is desirable to crosslink.
There are many polyfunctional monomers suitable for adding to the polymerization system for such purposes, to name a few: divinylbenzene, ethylene glycol dimethacrylate, N,N'-methylenebisacrylamide, and succinic acid. divinyl, diallyl succinate,
These include vinyl methacrylate, allyl methacrylate, triallyl cyanurate, triallyl isocyanurate, and the like. Further, crosslinking can also be introduced by utilizing the reactivity of the polymer produced after the polymerization reaction and reacting it with a polyfunctional compound. For example, a polymer can be crosslinked by reacting an epoxy group contained in the produced polymer with a diamine such as ethylenediamine. The shape of the particles is often spherical, but sphericity is not a necessary condition, and irregular shapes are also acceptable. The diameter of irregularly shaped particles is 1/2 of the sum of the maximum and minimum diameters. The average diameter is given by formula (1)
defined by and represented by . Here, d i is the diameter of the i-th particle, and N is the total number of particles. = N 〓 i=1 d i /N... (1) It has been empirically shown that the agglutination reaction is easy to judge when the average diameter is 0.1 μm or more and 10 μm or less. Further, for the purpose of cell labeling, the average diameter is preferably in the range of 0.03 μm or more and 5 μm or less. Furthermore, particles suitably colored with dyes or pigments are convenient for both aggregation reactions and cell labeling purposes. Particles with fluorescent light are also preferred for cell labeling. The immobilization reaction of immunologically active substances onto microparticles is carried out in an aqueous medium, with a pH of 7.0 to 9.0 and a temperature of 0 to 40 degrees Celsius.
A range of is appropriate. The concentration of the immunoactive substance in the reaction solution needs to be increased or decreased depending on the properties of the individual immunoactive substance and cannot be determined uniformly. In this case, as already mentioned, adding hydrophilic proteins such as serum albumin and gelatin to the reaction solution is often effective in improving the dispersion stability of the immunoactive substance-immobilized microparticles. Furthermore, treatment with amino acids such as glycine and alanine after the immobilization reaction often brings about similar favorable effects. The immunologically active substance used in the present invention is required to have an amino group, and most of the immunologically active substances are proteins or contain polypeptide moieties and therefore meet this condition. Here, the term "immune active substance" refers not only to antigens and antibodies, but also to substances that specifically bind to substances involved in humoral or cellular immune reactions, such as complement, Fc receptors, and C3 receptors. do. To give a few specific examples, Treponema pallidum antigen, B
Hepatitis surface antigen (HBs antigen), antibody against hepatitis B surface antigen (anti-HBs antibody), rubella antigen, toxoplasma antigen, streptolysin O, anti-streptolysin O antibody, mycoplasma antigen, human chorionic gonadotropin (HCG), anti- HCG antibody, heat-agglutinated human IgG, rheumatoid factor, nuclear protein, DNA, anti-
DNA antibody, C-reactive protein (CRP), anti-CRP antibody, anti-estrogen antibody, α-fetoprotein (α-FP), anti-α-FP antibody, carcinoembryonic antigen (CEA), anti-CEA antibody, C1q, anti-C1q antibody, C3,
Anti-C3 antibody, anti-C3b antibody, anti-C3bi antibody, C4, anti-C4
These include antibodies, protein-A, conglutinin, and immunoconglutinin. Example 1 Glycidyl methacrylate, 2-oxyethyl methacrylate, and ethylene glycol dimethacrylate were mixed in a molar ratio of 85.7:9.5:4.8, and 24 parts of the monomer mixture (weight, same hereinafter),
A mixture of 76 parts of ethyl propionate and 0.13 parts (4.7 mmol/) of 2,2'-azobis(2,4-dimethyl-4-methoxyvaleronitrile) was polymerized at 40 DEG C. for 3 hours under a nitrogen gas atmosphere. The cloudy polymerization mixture was poured into acetone and centrifuged at 1500×g for 10 minutes, and the precipitated particles were redispersed and washed with ethanol, followed by centrifugation again. It was dried under reduced pressure to obtain 11.3 parts of a particulate polymer. This particulate polymer was spherical and had a diameter between 1.8 μm and 4.2 μm, with an average of 3.3 μm and a standard deviation of 0.45 μm. TP to the polymer fine particles prepared as above.
The antigen was immobilized as described below. First, the syphilis pathogen Treponema pallidum (hereinafter abbreviated as TP)
Nichols strain cell components were added to phosphate-balanced saline (disodium hydrogen phosphate + monopotassium hydrogen phosphate 0.01 mol/, sodium chloride 0.14 mol/,
PH7.2, hereafter abbreviated as PBS) at a rate of 10 9 cells/ml was heated at 10K while cooling with ice water.
The cells were treated with Hz ultrasound for 20 minutes to destroy the bacterial cells, and this was used as a TP antigen solution. One volume of this TP antigen solution was mixed with three volumes of a solution of bovine serum glycoprotein dissolved in PBS at a concentration of 1 mg/ml, and 1 ml of this mixed solution was mixed with a dispersion of 50 mg of the polymer in 1 ml of PBS. The mixture was stirred at 30°C for 2 hours. Next, fine particles were separated by centrifugation, washed three times with PBS by centrifugation, and then washed with bovine serum albumin (hereinafter abbreviated as BSA).
It was dispersed in 20 ml of PBS to which 1% of was added. This TP antigen-immobilized microparticle dispersion was left in a refrigerator at 4°C for 3 days, and then the activity was assayed as follows. A diluted syphilis-positive serum solution with a TPHA titer of 640 was added to a polystyrene microtiter plate with a U-shaped tube bottom in a 2n dilution series starting from a dilution factor of 10 times, in an amount of 50 μm each. However, as a diluting solution, 1% BSA in PBS, 5% Reiter antigen solution for syphilis complement fixation reaction KW (Japan Lyophilization Research Institute),
A solution to which ammonium chloride was added at a concentration of 0.73 mol/ was used. A similar dilution of syphilis-negative serum was also added to a microtiter plate as a control. Next, 50μ of the TP antigen-immobilized microparticle dispersion was added to each well of the microtiter plate containing the serum dilution, shaken for 3 minutes to mix both solutions, and then allowed to stand at nitrogen temperature for 2 hours.
The degree of aggregation was determined by the sedimentation image. The results are shown in Table 1, and it can be seen that syphilis antibodies in serum can be detected with a sensitivity higher than that of TPHA.
【表】【table】
【表】
実施例 ２
１mg／mlの濃度のヒトIgG／PBS溶液0.4mlと
１mg／mlの濃度のウシ血清グリコプロテイン／
PBS溶液1.6mlとを混合し、その混合液に、実施
例１に使用したのと同じ重合体微粒子50mgを分散
させ、30℃で３時間撹拌した。この反応混合物を
４℃の冷蔵庫中で１夜放置した後遠沈により
PBSで洗浄し、BSAを１％添加したPBS4mlに微
粒子を再分散させ、30℃で２時間撹拌した後、４
℃の冷蔵庫に１夜放置した。このようにしてヒト
IgGを固定化した重合体微粒子と抗ヒトIgG抗体
とを次のようにして反応させた。すなわち顕微鏡
用スライドグラス上で抗ヒトIgG血清（ヤギ）
IgG分画のPBS溶液10μと上記ヒトIgG固定化
微粒子分散液10μとを混合し、３分後の凝集状
態を肉眼で判定した。その結果を表２に示す。抗
ヒトIgG抗体の検出限界は約10nｇ／mlであるこ
とがわかる。[Table] Example 2 Human IgG with a concentration of 1 mg/ml/0.4 ml of PBS solution and bovine serum glycoprotein with a concentration of 1 mg/ml/
1.6 ml of PBS solution was mixed, and 50 mg of the same polymer fine particles used in Example 1 were dispersed in the mixed solution, followed by stirring at 30° C. for 3 hours. The reaction mixture was left in a refrigerator at 4°C overnight, and then centrifuged.
After washing with PBS, the microparticles were redispersed in 4 ml of PBS containing 1% BSA, stirred at 30°C for 2 hours, and then
It was left in the refrigerator at ℃ overnight. In this way, humans
Polymer microparticles on which IgG was immobilized were reacted with an anti-human IgG antibody in the following manner. i.e. anti-human IgG serum (goat) on a microscope slide.
10μ of the PBS solution of the IgG fraction and 10μ of the human IgG-immobilized fine particle dispersion were mixed, and the state of agglutination after 3 minutes was visually determined. The results are shown in Table 2. It can be seen that the detection limit of anti-human IgG antibody is approximately 10 ng/ml.
【表】
実施例 ３
５mg／mlの濃度のBSA／PBS溶液２mlに実施
例１で使用したものと同じ重合体微粒子を分散さ
せ、30℃で７時間撹拌した後４℃の冷蔵庫に１夜
放置した。次いで遠沈によりPBSで洗浄し、ヒ
ト血清アルブミンを0.5％添加したPBS4mlに微粒
子を再分散し、30℃で１時間撹拌してから４℃の
冷蔵庫に１夜放置した。このようにしてPBSを
固定化した重合体微粒子と抗BSA抗血清（ウサ
ギ）とを実施例２と同様にしてスライドグラス上
で反応させた結果は表３の通りであつた。抗
BSA抗体検出限界は0.1μｇ／mlであることがわ
かる。[Table] Example 3 The same polymer particles used in Example 1 were dispersed in 2 ml of BSA/PBS solution with a concentration of 5 mg/ml, stirred at 30°C for 7 hours, and then left in a refrigerator at 4°C overnight. did. Next, the microparticles were washed with PBS by centrifugation, redispersed in 4 ml of PBS supplemented with 0.5% human serum albumin, stirred at 30°C for 1 hour, and then left in a refrigerator at 4°C overnight. The polymer particles on which PBS was immobilized in this manner were reacted with anti-BSA antiserum (rabbit) on a slide glass in the same manner as in Example 2. The results are shown in Table 3. anti
It can be seen that the BSA antibody detection limit is 0.1 μg/ml.
【表】
実施例 ４
グリシジルメタクリレート、２−オキシエチル
メタクリレートおよびエチレングリコールの３者
の混合比を71.4：23.8：4.8（モル比）に変えた以
外は実施例１と全く同様にして重合し、平均直径
1.0μｍの微粒子10.8部を得た。この重合体微粒子
を用いて実施例３の場合と全く同様にしてBSA
を固定化した。実施例３と同様にしてスライドグ
ラス上で抗BSA抗血清と反応させた結果は実施
例３の場合と全く同等で、抗BSA抗体検出限界
は0.1μｇ／mlであつた。
実施例 ５
グリシジルメタクリレート、２−オキシプロピ
ルメタクリレートおよびトリエチレングリコール
ジメタクリレートを47.6：47.6：4.8（モル比）の
比率で混合し、その単量体混合物24部、メチルｎ
−プロピルケトン76部および２，2′−アゾビス
（２，４−ジメチル−４−メトキシバレロニトリ
ル）0.13部（4.7ｍmol／）の混合物をアルゴン
雰囲気下40℃で３時間重合させた。白濁した重合
混合物を実施例１と同様に処理して重合体微粒子
8.4部を得た。平均直径は2.5μｍであつた。この
重合体微粒子を0.5％ヒト血清アルブミン水溶液
中に重合体含量が1.25％になるように分散し、実
施例３と同様にしてBSAを固定化した。そして
実施例３と同様にして抗BSA抗血清とスライド
グラス上で反応させた。その結果を表４に示す。[Table] Example 4 Polymerization was carried out in the same manner as in Example 1 except that the mixing ratio of glycidyl methacrylate, 2-oxyethyl methacrylate, and ethylene glycol was changed to 71.4:23.8:4.8 (molar ratio), and the average diameter
10.8 parts of fine particles of 1.0 μm were obtained. Using these polymer particles, BSA was prepared in exactly the same manner as in Example 3.
was fixed. The results of reaction with anti-BSA antiserum on a slide glass in the same manner as in Example 3 were exactly the same as in Example 3, and the anti-BSA antibody detection limit was 0.1 μg/ml. Example 5 Glycidyl methacrylate, 2-oxypropyl methacrylate and triethylene glycol dimethacrylate were mixed in a ratio of 47.6:47.6:4.8 (molar ratio), and 24 parts of the monomer mixture, methyl n
A mixture of 76 parts of -propylketone and 0.13 parts (4.7 mmol/) of 2,2'-azobis(2,4-dimethyl-4-methoxyvaleronitrile) was polymerized at 40 DEG C. for 3 hours under an argon atmosphere. The cloudy polymer mixture was treated in the same manner as in Example 1 to obtain fine polymer particles.
Got 8.4 copies. The average diameter was 2.5 μm. This polymer fine particle was dispersed in a 0.5% human serum albumin aqueous solution so that the polymer content was 1.25%, and BSA was immobilized in the same manner as in Example 3. Then, in the same manner as in Example 3, it was reacted with anti-BSA antiserum on a slide glass. The results are shown in Table 4.
【表】
実施例 ６
グリシジルメタクリレートの代りにグリシジル
アクリレートを使用しグリシジルアクリレート、
２−オキシエチルメタクリレートおよびトリエチ
レングリコールジメタクリレートの混合比を
23.8：71.4：4.8（モル比）にした以外は実施例５
と同様にして重合し、平均直径約1μｍの重合体
微粒子7.2部を得た。実施例５と全く同様にして
BSAを重合体微粒子に固定化し、実施例５と同
様にして抗BSA抗血清と反応させた。その結果
は実施例５の場合と同等で、抗BSA抗体検出感
度は約10μｇ／mlであつた。
実施例 ７
実施例１の２−オキシエチルメタクリレートの
代りに２−オキシエチルアクリレートを使用した
以外は実施例１と全く同様にして重合し、平均直
径約3μｍの重合体微粒子9.5部を得た。この重合
体微粒子に実施例１と同様にしてTP抗原の固定
化を行ない活性を検定した結果、血清中の梅毒抗
体検出感度は実施例１の場合と同等であつた。[Table] Example 6 Using glycidyl acrylate instead of glycidyl methacrylate, glycidyl acrylate,
The mixing ratio of 2-oxyethyl methacrylate and triethylene glycol dimethacrylate is
Example 5 except that the molar ratio was 23.8:71.4:4.8
Polymerization was carried out in the same manner as above to obtain 7.2 parts of polymer fine particles having an average diameter of about 1 μm. In exactly the same manner as in Example 5
BSA was immobilized on polymer fine particles and reacted with anti-BSA antiserum in the same manner as in Example 5. The results were similar to those in Example 5, and the anti-BSA antibody detection sensitivity was about 10 μg/ml. Example 7 Polymerization was carried out in exactly the same manner as in Example 1, except that 2-oxyethyl acrylate was used in place of 2-oxyethyl methacrylate in Example 1, to obtain 9.5 parts of polymer fine particles having an average diameter of about 3 μm. The TP antigen was immobilized on the polymer particles in the same manner as in Example 1, and the activity was assayed. As a result, the sensitivity for detecting syphilis antibodies in serum was the same as in Example 1.
Claims (1)
グリシジルメタクリレートと、(2)炭素炭素不飽和
二重結合を有する水溶性単量体との共重合体から
なる平均直径が0.03〜10μｍの微粒子に、アミノ
基を有する免疫活性物質が共有結合によつて固定
化されていることを特徴とする免疫活性微粒子。1 (1) Glycidyl acrylate and/or
An immunoactive substance having an amino group is covalently bonded to fine particles with an average diameter of 0.03 to 10 μm made of a copolymer of glycidyl methacrylate and (2) a water-soluble monomer having a carbon-carbon unsaturated double bond. Immunoactive microparticles characterized by being immobilized.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17256380A JPS5796260A (en) | 1980-12-09 | 1980-12-09 | Immunoactive particle |
| CA000391753A CA1177751A (en) | 1980-12-09 | 1981-12-08 | Immunoparticles and process for preparing same |
| EP81110260A EP0054249B2 (en) | 1980-12-09 | 1981-12-08 | Immunoparticles and process for preparing the same |
| DE8181110260T DE3171273D1 (en) | 1980-12-09 | 1981-12-08 | Immunoparticles and process for preparing the same |
| US06/757,761 US4656144A (en) | 1980-12-09 | 1985-07-22 | Immunoparticles and process for preparing same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17256380A JPS5796260A (en) | 1980-12-09 | 1980-12-09 | Immunoactive particle |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5796260A JPS5796260A (en) | 1982-06-15 |
| JPS6315553B2 true JPS6315553B2 (en) | 1988-04-05 |
Family
ID=15944161
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP17256380A Granted JPS5796260A (en) | 1980-12-09 | 1980-12-09 | Immunoactive particle |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5796260A (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5919856A (en) * | 1982-07-27 | 1984-02-01 | Toray Ind Inc | Fine carrier particle for immobilizing amynologically active material |
| US4737544A (en) * | 1982-08-12 | 1988-04-12 | Biospecific Technologies, Inc. | Biospecific polymers |
| JPS59204601A (en) * | 1983-05-09 | 1984-11-20 | Unitika Ltd | Manufacture of molded article having physiological activity |
| AU2003280667A1 (en) | 2002-10-31 | 2004-05-25 | Reverse Proteomics Research Institute Co., Ltd. | Method of immobilizing compound on solid phase support |
-
1980
- 1980-12-09 JP JP17256380A patent/JPS5796260A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5796260A (en) | 1982-06-15 |
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